Mónica Poverene

Wild helianthus species and wild-sunflower hybridization in Argentina

SUMMARY Two wild Helianthus species native to North America have been naturalized in Argentina, H. annuus ssp. annuus and H. petiolaris. They grow as adventitious overlapping about 50% of the crop area. Hybridization and introgression between these wild species and sunflower have important biological and practical consequences, the former including homoploid hybrid species formation, and the latter including a possibility of transgene spreading from genetically modified (GM) sunflower cultivars to wild or weedy populations. Wild populations were screened for isozyme and morphological variation. Intermediate plants were found in several locations and subjected to progeny tests. Variability among progeny of each one was compared with the variability in wild accessions of both species. Segregation for phenotypic traits, intermediate phenology and low fertility levels were found in most progenies, accounting for the hybrid origin of their maternal plants. Attempts to quantify gene flow included screening of progenies from H. petiolaris populations growing near sunflower crops. Hybrid plants were recognized by morphological traits and reduced fertility. Overall hybridization was about 1%. Hybrid progeny on wild H. annuus plants were identified by a crop isozyme marker. A mean frequency of 7% hybridization was found. These results confirm that gene flow occurs among crop and wild Helianthus species, and it concerns crop management and environmental impact if release of GM sunflower cultivars is to be authorized in Argentina. Resumen Dos especies de Helianthus nativas de América del Norte se han naturalizado en Argentina, H. annuus ssp. annuus y H. petiolaris y crecen coincidiendo en un 50% con el área cultivada. La hibridación e introgresión entre estas especies silvestres y el girasol cultivado tienen importancia biológica y consecuencias prácticas, en la formación de híbridos homoploides y en la posibilidad de escape de transgenes desde cultivos de girasol genéticamente modificados (GM) a las especies silvestres o malezas. Se evaluó la variación morfológica e isoenzimática de las poblaciones silvestres. En diferentes localidades se encontraron plantas intermedias y algunas se sometieron a pruebas de progenie. La variabilidad encontrada dentro de las progenies fue comparada con la variabilidad presente en poblaciones silvestres de ambas especies. La mayor parte de las progenies mostró segregación para rasgos fenotípicos, fenología intermedia y niveles bajos de fertilidad, demostrando el origen híbrido de las plantas madres. A fin de cuantificar el flujo génico, se estudiaron progenies de poblaciones de H. petiolaris que crecían cercanas a cultivos de girasol. Las plantas híbridas fueron reconocidas por rasgos morfológicos y reducida fertilidad. El total de híbridos encontrados fue de 1%. La progenie híbrida en plantas de H. annuus silvestre fue identificada por un marcador isoenzimático. Se encontró una frecuencia promedio de hibridación del 7%. Estos resultados confirman que ocurre el flujo génico entre el cultivo y las especies silvestres de Helianthus lo cual tiene importancia en el manejo del cultivo y el impacto ambiental, si se aprobara la liberación de cultivos de girasol genéticamente modificados en Argentina. RÉSUMÉ Deux espèces sauvages du genre Helianthus indigènes d’Amérique du Nord ont été naturalisées en Argentine, H. annuus ssp. annuus et H. petiolaris, et elles se répandent comme adventices en recouvrant environ 50% de l’aire cultivée. L’hybridation et l’introgression entre ces deux espèces sauvages et le tournesol cultivé ont une importance biologique et des conséquences pratiques dans la formation d’hybrides homoploïdes et dans la possibilité de dissémination de transgènes depuis des cultures de tournesol génétiquement modifiés (GM) vers les espèces sauvages ou envahissantes. Les populations sauvages ont été évaluées dans sa variabilité morphologique et des isozymes. Des plantes intermédiaires ont été trouvées dans plusieurs localités et on été soumises à des tests de descendance. La variabilité trouvée dans les descendances a été comparée avec la variabilité dans les populations sauvages des deux espèces. La ségrégation pour des caractères phénotypiques, la phénologie intermédiaire et des bas niveaux de fertilité ont été trouvés dans la plupart des descendances, en accord avec l’origine hybride des plantes mères. Avec le but de quantifier le flux de gènes ont a inclus l’étude des descendances de H. petiolaris qui se développaient proches des cultures de tournesol. Les plantes hybrides ont été reconnues par des caractères morphologiques et une fertilité réduite. La totalité d’hybrides trouvés a été de 1%. La descendance hybride dans les plantes H. annuus sauvages a été identifiée par la présence d’un isozyme spécifique du tournesol cultivé. On a trouvé une fréquence moyenne d’hybridation de 7%. Ces résultats confirment que le flux de gènes se produit entre les espèces cultivée et sauvages de Helianthus, ce qui est important pour la gestion des cultures et l’impact environnemental si on admet le lancement des cultivars de tournesol GM en Argentine.

Isozyme variation in Helianthus petiolaris and sunflower, H. annuus

SummaryHelianthus petiolaris is a wild species used in genetic improvement of sunflower, as a donor of cytoplasmic male sterility and of genetic resistance to diseases. Isozyme variation for ADH, ACP, EST, GDH, LAP, PGI, PGD and SKDH in this species was studied using starch gel electrophoresis. The patterns thus obtained were compared with zymograms of inbred lines, hybrids and open pollinated varieties of H. annuus. The same alleles for EST and SKDH isozymes were found in both species, while ACP showed an allele that has not been found in sunflower. The rest of the isozyme systems showed both common alleles and characteristic ones for each species. ACP, GDH and PGD were monomorphic in H. petiolaris, while ADH and LAP were monomorphic in H. annuus. The isozyme markers obtained here could be useful in breeding programs involving interspecific crosses, and studies on introgression and on genetic variation in other populations of this wild species.

Variability among inbred lines and RFLP mapping of sunflower isozymes

Eight isozyme systems were used in this study: acid phosphatase (ACP), alcohol dehydrogenase (ADH), esterase (EST), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH), phosphoglucoisomerase (PGI), 6-phosphogluconate dehydrogenase (PGD), and phosphoglucomutase (PGM). The polymorphism of these enzyme systems was studied in 25 elite inbred lines. A total of 19 loci were identified, but only eight of them were polymorphic in the germplasm tested. The polymorphic index for the eight informative markers ranged from 0.08 to 0.57, with a mean value of 0.36. Five isozyme loci were mapped in F2:3 populations with existing RFLP data. Est-1, Gdh-2 and Pgi-2 were mapped to linkage groups 3, 14 and 9, respectively. As in previous reports, an ACP locus and a PGD locus were found to be linked, both located in linkage group 2 of the public sunflower map.

Transgenic glyphosate-resistant oilseed rape ( Brassica napus ) as an invasive weed in Argentina: detection, characterization, and control alternatives

The presence of glyphosate-resistant oilseed rape populations in Argentina was detected and characterized. The resistant plants were found as weeds in RR soybeans and other fields. The immunological and molecular analysis showed that the accessions presented the GT73 transgenic event. The origin of this event was uncertain, as the cultivation of transgenic oilseed rape cultivars is prohibited in Argentina. This finding might suggest that glyphosate resistance could come from unauthorized transgenic oilseed rape crops cultivated in the country or as seed contaminants in imported oilseed rape cultivars or other seed imports. Experimentation showed that there are alternative herbicides for controlling resistant Brassica napus populations in various situations and crops. AHAS-inhibiting herbicides (imazethapyr, chlorimuron and diclosulam), glufosinate, 2,4-D, fluroxypyr and saflufenacil proved to be very effective in controlling these plants. Herbicides evaluated in this research were employed by farmers in one of the fields invaded with this biotype and monitoring of this field showed no evidence of its presence in the following years.

Limited occurrence of resistant radish (Raphanus sativus) to AHAS-inhibiting herbicides in Argentina

Radish has developed feral and weedy biotypes, which is a concern for agriculture around the world. In Argentina, it is one of the most widespread and troublesome crop weeds. In Brazil, this species has developed herbicide-resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides. The objective of this study was to record the presence of herbicide-resistant weedy radish plants in Argentina. In spring 2008, we found a small population of radish at the end of the flowering stage in an imidazolinone-tolerant canola field treated with imazethapyr. Screening and dose-response tests were conducted to two successive generations. They proved the biotype resistant status, and showed extensive survival (between 50 and 80% of control) to the application of a double dose of four AHAS‑inhibiting herbicides from two different chemical families (imidazolinones and sulfonylureas). Dose-response assays exhibited very high resistance for imazethapyr (LD50 = 2452.5 g a.i. ha-1, GR50 = 2926.9 g a.i. ha-1) and intermediate for metsulfuron (LD50 = 3.0 g a.i. ha-1, GR50 = 43.2 g a.i. ha-1). The acquisition of cross-resistance to different herbicide families would confer an adaptive and invasive advantage in agricultural environments to this biotype. Due to the herbicide rotation conducted in the field, the dispersion of this biotype was restricted. This is the first report of resistance in weedy radish in Argentina.

Gene flow in Argentinian sunflowers as revealed by genotyping-by-sequencing data

Gene flow can have several different applied consequences, ranging from extinction to the escape of transgenes to the evolution of weedy or invasive lineages. Here, we describe patterns of hybridization and gene flow involving domesticated and wild sunflowers in Argentina. To address the risks of introgression of variants from the cultivated sunflower into invasive wild Helianthus, we used genotyping‐by‐sequencing (GBS) to genotype 182 samples from 11 sites in Argentina, along with previously published data from samples from the native range (North America), to determine the native source populations of the Argentinian samples and to detect admixture. We unexpectedly discovered two distinctive forms of H. petiolaris in Argentina, one from H. petiolaris subsp. petiolaris as expected, but the other from an unknown source. Extensive admixture was observed among Argentinian sunflowers, largely confirming phenotypic predictions. While many hybrids are F1s, there were signals consistent with introgression from the domesticated sunflower into H. petiolaris. Whether this introgression is incidental or a causal driver of invasiveness is not yet clear, but it seems likely that genes found in the domesticated sunflower genome (whether engineered or not) will quickly find their way into wild Argentinian sunflower populations.

IMI resistance associated to crop-weed hybridization in a natural Brassica rapa population: characterization and fate

Wild turnip (Brassica rapa) is a common weed and a close relative to oilseed rape (Brassica napus). The Clearfield® production system is a highly adopted tool which provides an alternative solution for weed management, but its efficiency is threatened by gene transfer from crop to weed relatives. Crop-weed hybrids with herbicide resistance were found in the progeny of a B. rapa population gathered from a weedy stand on the borders of an oilseed rape (B. napus) imidazolinone (IMI)-resistant crop. Interspecific hybrids were confirmed by morphological traits in the greenhouse and experimental field, survival after imazethapyr applications, DNA content through flow cytometry, and pollen viability. The transference of herbicide resistance was demonstrated even in a particular situation of pollen competition between both an herbicide-resistant crop and a non-resistant crop. However, IMI resistance was not found in further generations collected at the same location. These results verify gene transmission from oilseed rape to B. rapa in the main crop area in Argentina where resistant and susceptible varieties are found and seed loss and crop volunteers are common. Hybridization, introgression, and herbicide selection would be associated with the loss of effectiveness of IMI technology.

The role of domestication and maternal effects on seed traits of crop–wild sunflower hybrids (Helianthus annuus)

Hybridisation between crops and their wild relatives may promote the evolution of weeds. Seed germination and dormancy are the earliest life-history traits and are highly influenced by the maternal parent. However, the ecological role of the maternal effect on seed traits in the evolution of crop–wild hybrids has received little attention. In this study, we test the relative importance of maternal and hybridisation effects on seed traits of the first generation of crop–wild sunflower hybrids (Helianthus annuus). Seed germination was tested in two wild populations with contrasting dormancy, two cultivated materials and their reciprocal crosses at four different times after harvest and three different temperatures. Seed germination at each of the four times, after ripening response and secondary dormancy were recorded along with four morphological traits. Additionally, the pericarp anatomy was analysed with light and scanning electron microscopy. We observed strong maternal effects on all seed traits. Seed germination, morphology and pericarp anatomy differed largely between the crop and wild seeds and these traits in the crop–wild hybrids resembled their female parent. Slight but significant hybridisation effects were observed in germination, mainly in seeds produced on wild plants. Crop hybridisation changed seed germination, the after ripening response and secondary dormancy in the crop direction. Morphological and anatomical traits associated with domestication strongly correlated with the observed differences in seed germination and dormancy in crop–wild sunflower hybrids. The large maternal effects along with the evolutionary divergence in seed traits were responsible for the large phenotypic differences observed in crop–wild hybrids with the same genetic composition. Wild-like seed traits of hybrids suggest that there are no barriers to crop gene introgression at the seed level whereas crop-like seed traits could be strongly selected against, conditioning the selection of traits expressed later in the life cycle and in the next generations.

Cold Stress Tolerance during Early Growth Stages of Naturalized Helianthus petiolaris Populations

Abstract In the last 10 years the sunflower crop area of Argentina has shifted towards the Southwest as a result of the expansion of soybean cultivation. Early sowings require greater tolerance to low temperatures in order to optimize the use of water resources by the crop, especially in the seedling stage, when cold stress limits growth and productivity. Helianthus petiolaris is a wild genetic resource that has contributed to the production of sunflower cultivars. The goal of this study was to evaluate the tolerance to low temperatures in H. petiolaris plants. Morphological traits (plant height, leaf width and leaf length) and physiological traits (electrolyte leakage, chlorophyll content, and glucose content) were compared in eight accessions having an early germination in the field and one with normal life cycle. Acclimated and non-acclimated plants were subjected to cold stress followed by a recovery period. Physiological traits during early growth stages showed differences in cold tolerance and for acclimation ability between accessions, suggesting the existence of heritable components. Four accessions were the most cold-tolerant among the studied populations, suggesting a natural adaptation related to the geographical site. Results point out that H. petiolaris is a useful genetic resource to contribute to sunflower breeding in the search of cold tolerant cultivars.

EXPLORING ENVIRONMENTAL DETERMINANTS OF THE GEOGRAPHIC DISTRIBUTION OF BROOMRAPE (Orobanche cumana Wallr.) / EXPLORANDO LOS DETERMINANTES AMBIENTALES DE LA DISTRIBUCIÓN GEOGRÁFICA DEL JOPO (Orobanche cumana Wallr.)

SUMMARY Broomrape is one of the most critical biotic constraints to sunflower crop production. In the most extended sunflower crop area of the world, distributed across Black sea region, this parasitic weed shows a high occurrence. The weed, originally from the Caucasus region, has progressively migrated to the South of the Eurasia continent. The actual invaded area reaches Spain, Israel, Kazakhstan, China, Mongolia and Iran. Due to the usual seed exchange between macro-regions it is not clear why this weed is absent in other extended sunflower crop area located in South America. It is possible that the invasive process could be limited by abiotic restrictions. With the aim to estimate the influence of environmental factors on the actual broomrape geographic distribution, we evaluated 14 sunflower habitats of Serbia with different levels of natural infection and nine natural habitats of wild sunflower in Argentina. The altitude, latitude, longitude, mean hottest month temperature, mean coolest month temperature, and average rainfall of 10 habitats from Serbia showed no differences between infected and non-infected soils. The natural initial infection of four soils from Serbia did not influence the Orobanche attack intensity in a greenhouse experiment with artificial inoculation. Soil texture, total N, humus and calcareous content, as well as pH, had no effect on Orobanche attack intensity. In the greenhouse experiment, Principal Components Analysis showed that Orobanche attack intensity (expressed as parasite plant number per sunflower plant) was inversely associated only with P availability (ALP2O5), but the linear relationship between both variables was not significant. When Argentinean habitats were included in the database, strong differences between invaded and not invaded areas were found regarding geographic localization, as expected. The invaded habitats differed by the coolest month mean temperature, but no significant differences were found for the remaining climatic parameters. Soil fertility and the texture of 13 soils with different natural Orobanche infection showed no differences between invaded and non-invaded habitats. A better understanding of the abiotic determinants of Orobanche geographic distribution could help to design management tools to prevent the continuous increase of the invaded area and to limit damage to sunflower in the regions presently affected by the weed. RESUMEN El jopo es una maleza parásita que constituye una de las principales limitantes bióticas de la producción mundial de girasol. Esta maleza presenta una elevada ocurrencia en la extendida región cultivada con girasol en la zona del Mar Negro. Nativa del Cáucaso, la maleza ha migrado progresivamente hacia el sur de Eurasia. Actualmente el área invadida alcanza a España, Israel, Kazakhastan, China, Mongolia e Irán. No está claro porque, a pesar del intenso intercambio de semillas, la maleza está ausente en importantes áreas de girasol de Sud América. Es posible que el proceso invasivo haya estado limitado por restricciones abióticas. Con el propósito de estimar la influencia de factores ambientales sobre la distribución actual del jopo evaluamos 14 hábitats de girasol de Serbia con diferentes niveles de infección natural y nueve hábitats de girasol silvestre de Argentina. En 10 hábitats de Serbia no se observaron diferencias entre suelos infectados y libres de la maleza en la latitud, longitud, temperatura media del mes más cálido, temperatura media del mes más frío y promedio anual de lluvias. En un experimento con inoculación artificial en invernadero, la infección natural de cuatro suelos de Serbia no afectó la intensidad de ataque de Orobanche. La textura del suelo, contenido de N, de humus, caliza y el pH no tuvieron efectos sobre la intensidad de ataque de Orobanche. En el experimento de invernadero el Análisis de Componentes Principales mostró que la intensidad de ataque de Orobanche (expresado como el número de plantas parásitas por planta de girasol) estuvo inversamente relacionada con la disponibilidad de P (AL-P2O5) pero la regresión linear entre ambas variables no fue significativa. Cuando en la base de datos se incluyeron los hábitats de Argentina, las áreas invadidas se diferenciaron de las libres en aspectos de localización geográfica, como era esperable. Los hábitats invadidos difirieron en la temperatura media del mes más frío, pero no se encontraron diferencias en el resto de las variables climáticas. La fertilidad química y la textura de 13 suelos con diferentes niveles naturales de Orobanche no mostraron diferencias entre áreas invadidas y no invadidas. Una mejora en la comprensión de los determinantes abióticos que modulan la distribución geográfica de Orobanche podría ayudar a diseñar herramientas de manejo para prevenir el continuo incremento del área invadida y limitar el daño al girasol en las regiones actualmente afectadas por la maleza.