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

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.Financial support from the Spanish Ministry of Education and Science (AGL2010-17909), Junta de Andalucía (P12-AGR370 and P12-AGR1281) and Spanish National Research Council (20134R060).Peer Reviewe

Pathogenic and molecular diversity in highly virulent populations of the parasitic weed Orobanche cumana (sunflower broomrape) from Europe

The definitive version is available at www3.interscience.wiley.comThe 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.This research was supported by the Ramón Areces Spanish Foundation, Spanish National Institute for Agricultural Research (RTA04-048), Spanish National Research Council (CSIC) (PIE200940I120) and Spanish Ministry for Science and Education (HH2005-0017). The placement of S. Raranciuc was granted by the European Science Foundation (CA849). L. Molinero-Ruiz was supported by an I3P post-doctoral contract (CSIC & European Social Fund).Peer reviewe

Genetic and molecular approach to Verticillium dahliae infecting sunflower

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.Consejería de Agricultura y Pesca, Junta de Andalucía (Grant/Award Number: “TRA201300.3”) Spanish Ministry of Education and Science (Grant/Award Number: “AGL2010-17909”)Peer Reviewe

Aggressiveness of Cephalosporium maydis causing late wilt of maize in Spain

Late wilt of maize, caused by the vascular and soilborne pathogen Cephalosporium maydis, was identified in the Iberian Peninsula in 2008. During the last years the incidence and economical impact of the disease has importantly increased both in Portugal and Spain. Varieties of maize displaying tolerance to the pathogen are available, but the effectiveness can be dependent on the virulence of the fungus (i.e. ability to cause disease on a specific genotype). On the other hand, strains of crop pathogens from different geographic origins can differ with regard to the degree of disease caused on a specific genotype (i.e. aggressiveness). Our working hypothesis was that isolates of C. maydis from different maize growing areas may differ in aggressiveness towards maize plants. Seven fungal strains were isolated in 2009 from diseased plants collected in the most important maize growing regions of Spain and used to inoculate two susceptible maize varieties grown in shadehouse from March to July 2010. The experimental unit consisted of two 4-day-old seedlings planted in an 8-liter pot filled with sand/silt previously infested with 200 g of wheat grains colonized by the fungi. Non colonized wheat grains were used for the control treatments. Six replications (pots) were established for each variety/isolate combination according to a complete randomized 2 × 8 factorial design. The percentage of necrotic and dry aboveground tissues was recorded 14 weeks after inoculation and thereafter weekly until physiological senescence of the control plants. At the end of the experiment, weights of roots and aboveground parts of the plants were recorded. Initial occurrence of symptoms in the plants was significantly dependent on the isolate of C. maydis and on the maize variety. However, final severity of aboveground symptoms (leaf necroses and drying up) was only dependent on the fungal isolate. All the isolates significantly reduced the root weight of both varieties of maize. The highest root weight reductions were also associated to a significant low weight of aboveground parts. Considering all the symptoms analyzed and their progression in the maize plants, our results reveal that a diversity of aggressiveness exists among isolates of C. maydis. The need for a characterization of maize genotypes by their reaction against highly aggressive isolates of the fungus in the Iberian Peninsula is suggested. This study is a first step towards a recommendation of crop varieties that are tolerant to C. maydis in different areas of the Iberian Peninsula. Future research aims at studying the relationship between aggressiveness levels, molecular characteristics and geographical origin whithin C. maydis.Supported by MICINN, Spanish National Research Councul (CSIC), grant PIE200940/120.Peer Reviewe

First Report of the Highly Virulent Race 705 of Plasmopara halstedii (Downy Mildew of Sunflower) in Portugal and in Spain

Sunflower (Helianthus annuus L.) plants with symptoms of stunting and chlorotic leaves were observed in the summer of 2016 in two fields of sunflower under pivot irrigation in Fuentes de Andalucia (Spain) and Serpa (Portugal).Peer reviewe

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.Peer Reviewe

Updated Characterization of Races of Plasmopara halstedii and Entomopathogenic Fungi as Endophytes of Sunflower Plants in Axenic Culture

The management of downy mildew (Plasmopara halstedii) in sunflower, is heavily dependent on genetic resistance, whilst entomopathogenic fungi (EF) can reduce other sunflower diseases. In this work, we characterized P. halstedii from Spain and other countries collected in the past few years. Twenty-three races were identified (the most frequent in Spain being 310, 304, 705 and 715), with an increasing proportion of highly virulent races. Five isolates from countries other than Spain overcame the resistance in RHA-340. In addition, we assessed the efficacy of five EF against downy mildew and their effects on sunflower growth in axenic conditions. None of the entomopathogens reduced disease severity, nor did they have any effect on plant growth when applied together with P. halstedii. In contrast, three EF reduced some of the plant growth variables in the absence of the pathogen. Microbiological and molecular diagnostics suggest that the axenic system and the short experimental time used in this study did not favor the successful establishment of EF in the plants or their potential biocontrol effect. Our results show a shift in P. halstedii racial patterns and suggest that soil as a growth substrate and long infection times are needed for EF effectiveness against downy mildew.Spanish National Research Council project PIE201940E058 to L.M.-R., Spanish Ministry of Economy and Competitiveness project AGL2016-80483-R to E. Quesada-Moraga and Spanish Ministry of Education, Culture and Sports, grant number FPU16/03983 to P.M.-F

The late wilt of corn (Zea mays L.) caused by Cephalosporium maydis and other fungi associated at the Iberian Peninsula

[ES] Las especies de hongos de suelo asociadas a Cephalosporium maydis como agente causal de la marchitez tardía del maíz en la Península Ibérica se identificaron muestreando 19 campos con síntomas de marchitez en las principales zonas de cultivo entre 2011 y 2012. En el 47% de los campos no se identificó C. maydis, pero sí Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani y Trichoderma harzianum infectando las plantas de maíz. 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 fúngicas en plantas de maíz se confirmó 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 inserción entre la raíz y tallo y a 10 cm de altura en el tallo de las plantas. El efecto de la infección por C. maydis sobre la producción de las plantas de maíz se cuantificó en macetas y condiciones seminaturales en el 2011. En plantas inoculadas se obtuvo una reducción del peso de las mazorcas del 54%, además de pesos de raíz y de parte aérea (tallo y hojas) significativamente menores en comparación con el control no inoculado, lo que sugiere el gran impacto económico que puede tener la marchitez tardía en condiciones naturales. Asimismo este trabajo pone de manifiesto el grado de complejidad de la etiología de la marchitez tardía, que debería ser estudiado mediante la confirmación de la patogenicidad de los hongos de suelo identificados en maíz, 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 síntomas.[PT] As espécies de fungos de solo associadas ao Cephalosporium maydis como agente causal da murcha tardia do milho na Península Ibérica, foram identificadas rastreando 19 campos com sintomas de murcha nas principais zonas de cultivo entre 2011 e 2012. Em 47% dos campos não foi identificado C. maydis, mas sim Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani e Trichoderma harzianum que infetaram as plantas de milho. Nos restantes campos, além do C. maydis foram identificados outros fungos de solo em percentagens apreciáveis: F. verticillioides (19%), F. proliferatum (19%), F.equiseti (9%), F. oxysporum (9%) e Pythium oligandrum (9%). O crescimento vascular do C. maydis e de outras espécies fúngicas em plantas de milho foi confirmado através de análises de plantas com murcha procedente de três campos diferentes. Tanto C. maydis como F. graminearum, F. equiseti, F. proliferatum y T. harzianum foram isolados na inserção entre a raíz e o caule e a 10 cm de altura no caule das plantas. O efeito da infeção por C. maydis sobre a produção das plantas de milho, quantificou-se em vasos e condições semi-naturais em 2011. Em plantas inoculadas, obteve-se uma redução do peso das maçarocas de 54%, além da redução dos pesos da raíz e parte aérea (folhas e caule) que foram significativamente menores em comparação com o controle não inoculado, o que sugere o grande impacto económico que pode ter o emurchecimento tardio em condições naturais. De igual forma , este trabalho realça o grau de complexidade da etiología do emurchecimento tardio, que deveria ser estudado mediante a confirmação da patogenecidade dos fungos de solo identificados no milho, com o objetivo de determinar o papel de cada uma destas espécies no desenvolvimento da doença e/ou severidade dos sintomas.[EN] 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.Trabajo parcialmente financiado por el proyecto PIE2009401120 (CSIC, MICINN).Peer reviewe

Pathogenic comparison of highly virulent O. cumana affecting sunflower in Moldova, the South of Russian Federation, Serbia and Spain

Trabajo presentado en el Third Internacional Symposium on broomrape (Orobanche spp.) in Sunflower, celebrado en Córdoba (España) del 3 al 6 de junio de 2014.The most effective method for controlling the parasitic weed Orobanche cumana (sunflower broomrape) is the incorporation of genetic resistance into cultivated sunflower. According to different genes of resistance that are overcome, several races of the parasite are pathogenically identified. Race G is the most virulent one because it infects the universal resistant inbred line P96. Besides, populations of O. cumana are molecularly clustered according to geographical origin, and pathogenic traits are secondary criteria for genetic differentiation. In this work highly virulent parasite accessions collected in Moldova, the South of Russian Federation, Serbia and Spain were pathogenically compared. Six accessions were inoculated onto differentials of highly virulent races of the parasite: NR5, L86 and P96. Plants were grown under shadehouse and glasshouse for ten weeks following a factorial on a completely randomised design. At the end of the experiment none of the accessions infected P96. According to the reactions of the inbred lines NR5 and L86, accessions from Moldova and Serbia were identified as race E, and accessions from Spain were identified as race F. Accessions from the Russian Federation showed intermediate pathogenic behaviour onto NR5 and L86. Since a highly virulent biotype of O. cumana is present in Rostov oblast and seed used in this work comes from individual broomrape plants, the presence of race F in this area might be confirmed through the study of genetically heterogeneous parasite accessions from the area. Our results show that, in order to efficiently manage the genetic resistance into cultivated sunflower, there is a need for a worldwide study of highly virulent populations of O. cumana.Financial support from the Spanish Ministry of Education and Science (research project AGL2010-17909). Broomrape collection in Serbia is a part of project TR31025 supported by Ministry of Education, Science and Technological Development of Republic of Serbia.N

First report of stalk rot of maize caused by phaeocytostroma ambiguum in the Iberian Peninsula

Maize (Zea mays L.) with symptoms of premature leaf senescence and wilting was observed in two commercial fields, in Santarem (Portugal) and Almodovar del Río (Spain), in the summer of 2014. Similar symptoms including brown lesions of lower internodes and disintegrated stalk pith tissues occurred again in two commercial fields in Almacelles and Biota (Spain) in the summers of 2016 and 2017, respectively. Depending on the field, estimated disease incidence varied between 10 and 30%. Lodging was observed on 10 to 20% of maize plants in the two commercial fields following rain and strong wind