Indigenous highly virulent accessions of the sunflower root parasitic weed Orobanche cumana

Orobanche cumana (broomrape) is a severe constraint to sunflower production in southern and eastern Europe and the Middle East. Races A to E of this parasitic weed controlled by genes Or₁ to Or₅ have been described. A study of 38 seed accessions of O. cumana collected from different locations in Spain between 1983 and 2003 investigated the effect of long‐term storage in the laboratory on germination and infectivity and assessed their virulence on a number of sunflower cultivars. Only 18 infected the susceptible cultivar B117. Infectivity was maintained for up to 17 years of storage, but with a greatly decreased vigour as compared with that of recently collected seed. The 12 oldest viable accessions overcame the resistance of the gene Or₅ (in resistant line NR5). Seven out of them, in particular those collected in 1988 and 1989, were identified as race F. Three accessions were identified as race E allegedly holding components of higher virulence. Our results show evidence of the occurrence of race F prior to the use of sunflower hybrids resistant to race E, suggesting the former as indigenous to the country. This finding suggests the necessity of a continuous breeding of sunflower for resistance to O. cumana. The effectiveness and sustainability of genetic resistance must rely on the knowledge of the diverse virulence characteristics of O. cumana accessions.     

Pathogenic diversity within field populations of Orobanche cumana and different reactions on sunflower genotypes

Different races of the parasitic Orobanche cumana (sunflower broomrape) have been reported in Spain, race F being the most virulent. Full resistance in sunflower to races A–E is achieved with each of the single major genes Or₁ to Or₅ respectively. However, parasitised hybrids allegedly resistant to race F were observed in early 2002. The purpose of this study was to verify broomrape incidences (BI) on resistant sunflower genotypes, to assess the mixture of races within field populations and to test for partial resistance to race F in the sunflower hybrids showing a low degree of attack (DA) by the weed. Tests were conducted under field conditions in two locations of southern Spain. While no significant differences were found for yield and BI between locations, the DA on the cultivars depended on the location. With high infection levels and significantly lower yield in susceptible controls, marked differences in BI and DA were found within resistant cultivars, but all of them showed similar crop yield. When artificially inoculated with several populations of race F, line P96 and mainly line L86, were consistently slightly infected, suggesting they were inbred lines responsible for horizontal resistance in infested fields. L86 was extremely susceptible to race E populations, which is unusual as sunflower resistance to one race provided resistance to all the previously described races of O. cumana. No different virulences were detected within two groups of subpopulations (races E and F) inoculated onto resistant sunflower genotypes. However, race F subpopulations showed significant differences in aggressiveness, which seems to be related to horizontal (multigenic) resistance of the crop to the parasitic weed.     

Molecular analysis of resistance mechanisms to Orobanche cumana in sunflower

Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250–300 ng g⁻¹ FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1 .     

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

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.     

Identification,characterisation and discriminatory power of microsatellite markers in the parasitic weed Orobanche cumana

Orobanche cumana is an obligate root parasite of sunflower. It represents a major agricultural problem in many countries of southern and eastern Europe. Information on O. cumana population genetics, structure and dynamics is scarce, particularly due to the lack of suitable molecular markers for such studies. The objective of this study was to identify and characterise simple sequence repeat (SSR) markers for O. cumana. Four thousand two hundred SSR‐containing candidate sequences were obtained from O. cumana using next‐generation sequencing, from which 298 SSR primer pairs were designed and 217 of them used for validation. Seventy nine SSR primers produced reproducible, high quality amplicons of the expected size that were polymorphic among 18 O. cumana populations from different geographical locations and hosts (sunflower, wild hosts from the Compositae family). The number of alleles per locus ranged from 2 to 10, with an average polymorphism information content value of 0.37. The O. cumana SSR markers were highly transferable to the closely related species Orobanche cernua. SSR markers showed high resolving power; UPGMA cluster analysis allowed proper classification of Orobanche spp. samples into species (O. cumana and O. cernua), geographical origin and host. The functional SSR markers reported in this study constitute a valuable tool for genetic analyses in O. cumana and related species and will contribute insights into the biology and genetics of this parasitic weed.     

Genetic diversity of Orobanche cumana populations from Spain assessed using SSR markers

Orobanche cumana (sunflower broomrape) is found in Spain as an allochthonous species parasitising exclusively sunflower. For many years, it was distributed in the Guadalquivir Valley and Cuenca province, but in recent years, it has spread to new areas. The objective of this research was to study genetic diversity of O. cumana populations from Spain using robust co‐dominant molecular markers. Cluster analysis on a set of 50 populations using 15 microsatellite markers revealed the existence of two distant gene pools, one in Cuenca province and another one in the Guadalquivir Valley. Within each gene pool, both inter‐ and intrapopulation variability were extremely low. This population structure probably reflects a founder effect, with the two genetically distant gene pools deriving from separate introduction events. Different races occurred within the same gene pool, suggesting that current races might have evolved through mutation from a common genetic background. Most of the populations from new areas were identical to the populations from the Guadalquivir Valley. Only a few populations showed larger intrapopulation variation. In these cases, our results suggested the co‐existence of both gene pools within the same population, as well as the occurrence of genetic recombination between them. Genetic recombination between distant gene pools is an important mechanism for creating new variation, which might also have an effect on race evolution. These results will contribute to the establishment of improved crop breeding and management strategies for O. cumana control.     

向日葵新品种抗列当水平的田间鉴定

向日葵列当是一种寄生性杂草,严重危害向日葵产业的发展。为有效鉴定向日葵登记品种列当抗性、加快优良品种推广应用,本研究选取国内具有代表性的36个食用向日葵登记品种,在内蒙古向日葵主产区乌拉特前旗芦管壕村和四子王旗高油房村进行了品种列当抗性田间鉴定。结果表明,乌拉特前旗芦管壕村试验点列当生理小种为F小种,‘同欣2号’‘诚牌C93’等20份品种在该点表现为免疫水平;‘三瑞3号’‘天葵16’等11份品种呈现出高抗水平;其余5份品种田间表现为易感或者高感水平。四子王旗高油房村试验点列当生理小种为G小种,‘同欣2号’‘圣地壹号’等14份品种在该点表现为免疫;‘诚牌C93’‘三瑞3号’等16份品种表现为高抗;‘龙食葵5号’表现为中抗;其余5份品种表现为易感或者高感。其中,在两个地区均表现为免疫的品种共有13份,分别是‘同欣2号’‘圣地壹号’‘龙葵27’‘圣地777’‘启源6号’‘JR5511’‘益民931’‘益民303’‘益民966’‘益民969’‘金谷葵33’‘S2009’‘新农101’;呈现高抗列当的品种有9份,分别是‘三瑞3号’‘天葵16’‘正博鼎盛’‘双星6号’‘中食葵90’‘益民968’‘...     

Prohexadione-calcium induces sunflower (Helianthus annuus) resistance against the root parasitic weed Orobanche cumana

Prohexadione-calcium (PHDC) is a plant growth retardant that inhibits gibberellin biosynthesis and transiently alters the spectrum of flavonoids and their phenolic precursors. In this study, root chamber and pot experiments were conducted to evaluate the effects of PHDC on Orobanche cumana seed germination and induction of sunflower resistance to this root parasitic weed. Root chamber experiments indicated that PHDC did not interfere with the progress of Orobanche seed germination, but retarded O. cumana tubercle formation and development. In pot experiments, control of O. cumana by PHDC depended on its dose, method of application and sunflower cultivar. In sunflower cv. HA89, PHDC at 10 mg LS⁻¹ (litre soil) as two drenches or at 1 m m as two foliar sprays significantly reduced the total number and dry matter of O. cumana without influencing flower, leaf and root development. In sunflower cv. Albena, PHDC at 20 and 30 mg LS⁻¹, but not at 10 mg LS⁻¹, as two drenches significantly reduced O. cumana infection. Sunflower cv. HA89 was less susceptible to O. cumana and more inducible by PHDC than sunflower cv. Albena. The biochemical analysis of root extracts revealed a significantly higher accumulation of free phenolics in sunflower cv. HA89 after PHDC soil drench, but not in Albena. No significant lignification was observed after PHDC treatments. The results suggest that PHDC reduces O. cumana infection in sunflower by inducing host resistance, not by directly interfering with the parasite. Free phenolics play an important role in sunflower response to the parasite.     

A dominant avirulence gene in Orobanche cumana triggers Or5 resistance in sunflower

Orobanche cumana is a weed that grows as a root parasite on sunflower. In general, the O. cumana–sunflower parasitic system is regarded to follow the gene‐for‐gene model, although this has never been demonstrated at the genetic level in O. cumana. The Or5 dominant gene in sunflower confers resistance to O. cumana race E, but not to race F. The objective of this research was to study the inheritance of avirulence/virulence in crosses between plants of O. cumana lines classified as races E and F. Four race E and three race F lines were developed, from which four race E × race F cross‐combinations were made, in three cases including reciprocals. In all cases, F₁ seeds did not have the ability to parasitise sunflower line P‐1380 carrying the Or5 gene, indicating dominance of race E avirulence allele(s). Five F₂ populations comprising a total of 387 F_2:3 families were evaluated on sunflower line P‐1380. In all cases, one‐fourth of the F_2:3 families did not possess the ability to parasitise P‐1380 plants, suggesting that race E avirulence and race F virulence on P‐1380 are allelic and controlled by a single locus. This study demonstrated the gene‐for‐gene interaction in the O. cumana–sunflower parasite system and provided useful information to identify genes involved in O. cumana virulence. The approach followed in this research can contribute to define precisely races of the parasite on the basis of the presence of avirulence genes.     

向日葵抗列当品种(系)筛选及其应用评价

为了全面评价不同向日葵品种(系)对列当的抗寄生性和其在生产中的应用价值,对‘TP3313’等50个食葵品种(系)、‘内蒙杂4号’等6个油葵品种(系)进行了田间自然寄生试验,结果表明:食葵‘TP3313’为对列当免疫的品种,且病虫害发生较轻,产量相对较高(261.11kg/667m~2),因而具有极高的推广应用价值;‘TP3314’为高抗列当品种,但是受向日葵菌核病危害较重,产量为219.64kg/667m~2;‘SH361-L’、‘SH338-M’等8个品种表现为中感,属耐列当品种(系),并且产量相对较高,在生产中具有一定的应用价值。     

向日葵抗列当品种的田间鉴定与筛选

寄生性植物向日葵列当Orobanche cumana Wallr.已经成为我国向日葵产业发展的重要限制因素。种植抗性品种是目前控制向日葵列当最经济有效的途径。本研究在2012年－2017年以田间自然鉴定的方式对124个品种(食葵85个, 油葵39个)进行了188批次的抗列当田间鉴定试验, 参试品种包括了近10年来向日葵产区内的绝大部分市场销售品种以及部分新杂交组合。本项研究明确了上述品种的抗列当等级。筛选出免疫或高抗列当的向日葵食葵品种26个, 油葵品种25个, 其中部分商品性较好的免疫品种在向日葵列当综合防治技术集成中发挥了重要作用。结合田间试验鉴定的结果, 本文还对田间自然鉴定法存在的问题以及抗性等级的划分标准进行了讨论。     

Quantitative trait loci associated with isolate specific and isolate nonspecific partial resistance to Phoma macdonaldii in sunflower

Black stem, caused by Phoma macdonaldii , is one of the most important diseases of sunflower in the world. Quantitative trait loci (QTLs) implicated in partial resistance to two single pycnidiospore isolates of P. macdonaldii (MP8 and MP10) were investigated using 99 recombinant inbred lines (RILs) from the cross between sunflower parental lines PAC2 and RHA266. The experimental design was a randomized complete block with three replications. High genetic variability and transgressive segregation were observed among RILs for partial resistance to P. macdonaldii isolates. QTL-mapping was performed using a recently developed high-density SSR/AFLP sunflower linkage map. A total of 10 QTLs were detected for black stem resistance. The phenotypic variance explained by each QTL (R²) was moderate, ranging from 6 to 20%. Four QTLs were common between two isolates on linkage group 5 and 15 whereas the others were specific for each isolate. Regarding isolate-specific and isolate-nonspecific QTLs detected for partial resistance, it is evident that both genetic effects control partial resistance to the disease isolates. This confirms the need to consider different isolates in the black stem resistance breeding programmes. The four SSR markers HA3700, SSU25, ORS1097 and ORS523_1 encompassing the QTLs for partial resistance to black stem isolates could be good candidates for marker assisted selection.     

Chlamydospores of Fusarium Oxysporum Schlecht f.sp. orthoceras (Appel & Wollenw.) Bilai as Inoculum for Wheat-flour—Kaolin Granules to be Used for the Biological Control of Orobanche cumana Wallr.

The formation of chlamydospores of Fusarium oxysporum Schlecht. f.sp. orthoceras (Appel & Wollenw.) Bilai, a potential mycoherbicide for the parasitic weed Orobanche cumana Wallr., was optimised regarding the composition of the liquid growth medium. After 16 days of incubation, highest chlamydospore counts (approximately 2 × 10⁷ml^–1) were determined in an aqueous solution amended with 1.5% (w/v) finely ground sorghum stover and 30% (v/v) wheat-based stillage, a liquid by-product of ethanol production. The produced fungal biomass retained viability after being air-dried and stored. Granules made from wheat-flour and kaolin (Pesta) containing microconidia or air-dried chlamydospore-rich biomass were prepared and showed high efficacy in controlling O. cumana in the greenhouse. No differences in efficacy were observed between the different types of inoculum used.     

Genetic diversity of Moroccan populations of Orobanche foetida: evolving from parasitising wild hosts to crop plants

Orobanche foetida is widely distributed in western Mediterranean countries infecting wild legumes. Recently it has been reported in Morocco infecting common vetch, presenting a further constraint for legume production in this area. Comparative studies between Orobanche populations attacking wild species and Orobanche populations growing on crops from the same region may help to clarify whether a host specialisation process occurred. In the present study, the genetic variability of five O. foetida populations, four infecting wild plants and one infecting cultivated vetch in Morocco was characterised using amplified fragment‐length polymorphism (AFLP) markers to elucidate the existing genetic relationship between populations and to suggest a potential origin for the recently detected vetch‐infecting population. Analysis of molecular variance suggested the existence of genotypic differentiation among populations. The most genetic divergent population by cluster analysis was the population collected on Ornithopus sativus. The vetch‐infecting O. foetida population was closer to the three populations infecting Scorpiurus muricatus. The possibility of the presence of a reservoir of diversity is balanced against direct genetic adaptation and completely new introduction. The potential for this Orobanche species to shift host and become a threat to agriculture exists and has to be taken into account for future legume breeding for these areas.     

Germination stimulatory activity of bacterial butenolide hormones from Streptomyces albus J1074 on seeds of the root parasitic weed Orobanche minor

Damage caused by Orobanchaceae root parasitic weeds is a substantial agricultural problem for global food security. Many studies have been conducted to establish practical methods of control, but efforts are still required for successful management. Seed germination of root parasitic weeds requires host-derived germination stimulants including strigolactones (SLs). Studies on SLs have revealed that a butenolide ring is the essential moiety for SL activity as a germination stimulant. Interestingly, recent studies have revealed that butenolide hormones regulate the biosynthesis of secondary metabolites and mediate communication in actinomycete bacteria. Because of the structural similarity between SLs and the bacterial butenolides, we evaluated the germination stimulatory activity of butenolides isolated from Streptomyces albus J1074 on root parasitic weeds. These butenolides were found to specifically induce seed germination of Orobanche minor. Our findings contribute to understanding the molecular mechanisms of germination stimulant perception and to the development of a method for their biological control.     

Methodology of virulence screening and race characterization of Plasmopara halstedii,and resistance evaluation in sunflower – a review

Sunflower downy mildew is a disease of high global economic impact as well as a causal agent that is extremely difficult to eradicate. During the past decades, several approaches for the determination of Plasmopara halstedii (Ph) races have been used worldwide and are discussed in this review. Procedures of isolation, cultivation and maintenance of Ph isolates, as well as the screening of sunflower for resistance, are also critically reviewed. The predominant, globally used resistance screening protocol is a ‘whole seedling immersion’ inoculation. ‘Soil drench’ inoculation allows more precise control of the number of Ph zoosporangia applied to a single sunflower seedling. A detached leaf assay has been described, but it has been used mainly for Ph subcultivation and fungicide tests. For race determination, a differential set consisting of nine sunflower genotypes has been used since 1988, coupled with a numerical triplet code for virulence phenotyping of Ph. The increasing variability in global Ph populations has demonstrated the inadequacy of the current set of differentials, and several researchers have proposed additional public lines as new differentials. Furthermore, bulk isolates may show different results in repeated tests, as Ph may contain genetically distinct zoospores within a single zoosporangium. For precise race determination, single zoosporangia or single zoospore isolates are advisable. However, due to low success of isolation, approximately 1–2%, this method cannot be applied in routine Ph race screening. Methods surveyed in this review have a broad spectrum of applications, including taxonomic studies.     

室内条件下向日葵列当和弯管列当在三种不同寄主上寄生能力的比较

以向日葵列当Orobanche cumana Wallr.和弯管列当O.cernua Loefl.为研究对象,利用已有的室内寄生体系来研究两种列当在向日葵、烟草、番茄3种寄主上的寄生能力。结果表明,向日葵列当在室内条件下不仅能够寄生在向日葵上,同时也可以在烟草与番茄上寄生;但其寄生效率在上述不同的寄主之间存在显著差异,而在相同寄主的不同品系间没有差异。交互侵染的结果表明,在寄主番茄上,弯管列当平均每株寄生的瘤结数量为16.3个,向日葵列当只有2.5个;在寄主向日葵上,向日葵列当的平均寄生瘤结数量为25.2个,弯管列当为27.6个,预示着弯管列当在番茄上寄生效率均显著高于向日葵列当,而在寄主向日葵上,二者寄生效率差异不显著,均能高效寄生在向日葵根系上。上述研究结果将为向日葵列当和弯管列当发生严重地区作物轮作对象的选择提供理论依据。