First Report of Cirsium arvense (Canada thistle) as a New Host of Orobanche cumana Wallr. in Xinjiang, China

Cirsium arvense (Canada thistle) is a perennial herb native to Eurasia that has been introduced to temperate regions of the world where it is considered one of the serious weeds for arable and pastoral agriculture (Schröder et al. 1993). C. arvense reproduces both clonally and sexually. The weed is highly competitive, causes yield reductions in crops such as wheat, alfalfa, sugarbeet, and can reduce forage availability and production (Wilson 1981). Canada thistle is also a harbour for plant pathogens such as plant-parasitic nematodes (Tenuta et al. 2014). Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant species with a restricted range of hosts both in the wild, where it mainly parasitizes a few species of the Asteraceae, and in agricultural fields, where it is exclusively found growing on sunflower (Fernández-Martínez et al. 2015). O. cumana infection can cause up to 80% of the yield loss in sunflower, which is a serious threat for sunflower production in Xinjiang and Inner Mongolia, China (Parker 2009). In July 2019, broomrape was observed parasitizing C. arvense in the greenhouse used for sunflower resistance identification (Shihezi, 86° 3' 36" E, 44° 18' 36" N, 500 m elevation) in Xinjiang, China. Fifty percent of the plants were parasitized by broomrape in the greenhouse and the host had an average of 1-2 broomrape shoots per plant. For molecular analysis, total genomic DNA was extracted from the flowers of broomrape and the rps2, rbcL, trnL-F genes, and ribosomal DNA internal transcribed spacer (ITS) region were amplified by PCR using the primer pairs rps2F/rps2R, rbcLF/rbcLR, C/F, ITS1/ITS4, respectively (Park et al. 2007; Manen et al. 2004; Taberlet et al. 1991; Anderson et al. 2004). The ITS (659bp), rps2 (451 bp), trnL-F (914 bp), and rbcL (961 bp) gene sequences of the broomrape were deposited in GenBank, the accession numbers are MT856745, MW809407, MW809408, and MW809409. The results of BLAST analysis showed that ITS sequence shared 100% similarity with O. cumana (659/659 nucleotide identity, MK567978), the rps2 sequence shared 99% similarity with O. cumana (449/451 nucleotide identity, KT387722), trnL-F sequence shared 99% similarity with O. cumana (907/911 nucleotide identity, MT027325), rbcL sequence shared 99% similarity with O. cumana (956/964 nucleotide identity, MK577840). The morphological characteristics such as stem, inflorescence, corolla, bracts, calyx, stamens, gynoecium are consistent with O. cumana described by Pujadas-Salvá and Velasco (2000). Morphological and molecular identification strongly support that the broomrape parasitic on C. arvense belonged to the O. cumana. Greenhouse pot experiments were carried out to assess the parasitic relationship between sunflower broomrape and C. arvense (Fernández-Martínez et al. 2000). In January 2020, C. arvense roots were harvested from an extant field of C. arvense in the greenhouse at Shihezi University (Supplementary Figure S1A). The soil was dug to 30-40 cm depth and C. arvense roots were removed and carefully washed in water. The healthy and living C. arvense roots were selected and cut into 10-11 cm pieces. Four C. arvense root pieces were grown (buried at a depth of 10-12 cm) in 8-L pots containing a mixture of sand-vermiculite-compost (1:1:1 v:v:v) and O. cumana seeds (50 mg of O. cumana seeds per 1 kg of the substrate) with 5 replicates. Three non-infected plants were grown and evaluated in parallel. Approximately 80 days after planting, at the flowering stage of the O. cumana, C. arvense plants were uprooted from the soil. Compared to non-infected plants, the hosts’ symptoms were slow growth, leaf wilting, and chlorosis, and similiar to the broomrape-infected C. arvense plants observed in the greenhouse field. The roots of C. arvense and broomrape were carefully washed in water and observed the parasitism of O. cumana. The infection was confirmed by observation of the attachment of the O. cumana to the C. arvense roots (Supplementary Figure S1D). To the best of our knowledge, this is the first report of O. cumana parasitizing C. arvense in Xinjiang, China. C. arvense as a new host of O. cumana indicates that sunflower broomrape can also propagate and survive in a host such as Canada thistle grown in sunflower fields. This finding suggests that it may be more difficult to control sunflower broomrape by rotation. 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Biological and Transcriptomic Characterization of Pre-Haustorial Resistance to Sunflower Broomrape (Orobanche cumana W.) in Sunflowers (Helianthus annuus)

Infestations with sunflower broomrape (Orobanche cumana Wallr.), an obligatory root parasite, constitute a major limitation to sunflower production in many regions around the world. Breeding for resistance is the most effective approach to reduce sunflower broomrape infestation, yet resistance mechanisms are often broken by new races of the pathogen. Elucidating the mechanisms controlling resistance to broomrape at the molecular level is, thus, a desirable way to obtain long-lasting resistance. In this study, we investigated broomrape resistance in a confectionery sunflower cultivar with a robust and long-lasting resistance to sunflower broomrape. Visual screening and histological examination of sunflower roots revealed that penetration of the broomrape haustorium into the sunflower roots was blocked at the cortex, indicating a pre-haustorial mechanism of resistance. A comparative RNA sequencing between broomrape-resistant and -susceptible accessions allowed the identification of genes that were significantly differentially expressed upon broomrape infestation. Among these genes were β-1,3-endoglucanase, β-glucanase, and ethylene-responsive transcription factor 4 (ERF4). These genes were previously reported to be pathogenesis-related in other plant species. This transcriptomic investigation, together with the histological examinations, led us to conclude that the resistance mechanism involves the identification of the broomrape and the consequent formation of a physical barrier that prevents the establishment of the broomrape into the sunflower roots.

Costunolide Influences Germ Tube Orientation in Sunflower Broomrape – A First Step Toward Understanding Chemotropism

Orobanche cumana WALLR. is a host-specific root parasite of cultivated sunflowers with increasing economic importance in Europe, North Africa, and parts of Asia. While sesquiterpene lactones (STLs) released from sunflower roots were identified as natural germination stimulants of O. cumana seeds in the soil, the chemical nature of the signals guiding the emerging germ tube toward the host root has remained unknown hitherto. Thus, we designed a bioassay that allowed the observation of broomrape germination and subsequent germ tube development in the presence of substances with putative chemotropic activity. Root exudates and sunflower oil extracts, both containing STLs in micromolar concentrations, caused the positive chemotropic orientation of germ tubes. A similar positive chemotropic effect was achieved with costunolide, one of the four STLs of sunflower present in the exudate and oil extracts. In contrast, GR24, a synthetic strigolactone (SL) with germination-inducing activity on O. cumana seeds, showed no effect on the germ tube orientation. The effect of costunolide was concentration-dependent and within the range of its natural micromolar occurrence in roots. We assume that an STL gradient is responsible for the stronger inhibition of elongation growth on the host-facing flank of the germ tube compared with the far side flank. This would confer a double role of STLs from sunflower root exudates in the sunflower–broomrape interaction, namely, as germination stimulants and as chemotropic signals.

Orobanche cumana (sunflower broomrape).

O. cumana is an obligatory, non-photosynthetic root parasite. It is believed to have evolved relatively recently from forms of O. cernua parasitizing wild Asteraceae, in particular species of Artemisia, and transferring to cultivated Helianthus annuus (sunflower). O. cumana is thought to be native to Eastern Europe (Russia) and has subsequently spread to most other sunflower growing regions of central and western Europe and Asia. The absence of O. cumana in sunflower growing regions of South America (for example Argentina) is believed to be associated with warmer winter temperatures not suitable for this species, rather than the seeds not being present. O. cumana can cause immense damage to cultivated sunflowers resulting in a significant decrease in yield. Despite resistant sunflower varieties being developed more virulent races of O. cumana have repeatedly evolved, or been selected, to overcome resistance. Thus, in spite of constant breeding efforts, losses continue in established sunflower growing areas and there is potential for it to invade new areas, wherever sunflower is grown.

Results of Studying the Efficiency of Chemical Measures Against Weed Vegetation in Sunflower Crops

This study identified the most effective methods for protecting sunflowers from weeds through the treatment of crops with the herbicides Euro Lightning, Euro Lightning Plus and Express. These options provided substantial protection of sunflowers from weeds such as the cursed thistle, field bindweed, yellow bristle grass, field mustard, frost blite and redroot pigweed. In the fight against ragweed, the herbicides Euro Lightning and Euro Lightning Plus provided excellent action and the herbicide Express provided good results. Variants with the use of the soil herbicides Gezagard and Gardo Gold suppressed yellow bristle grass, field mustard and frost blite, and satisfactorily protected against redroot pigweed. However, they were ineffective against perennial species of weeds (cursed thistle and field bindweed) and ragweed growing in the experimental area. The application of soil herbicides did not influence the growth of sunflower broomrape and plots. The use of the herbicides Euro Lightning and Express helped to reduce the contamination of crops with this type of weed. When processing crops with the herbicide Euro-Lightning Plus, this type of weed was completely absent. Statistical processing of crop data showed a difference in yield in the studied variants. Carrying out weed control measures significantly increased the yield of sunflowers in comparison to control options (without treatment). There was a significant difference in the yield of oilseeds when herbicides intended for application on vegetating plants (Euro Lightning and Express) were used, compared to when the variants of soil herbicides Gezagard and Gardo Gold were used. Keywords: sunflower, weed plants, herbicides, oilseed productivity

Biological and Transcriptomic Characterization of Pre-haustorial Resistance to Sunflower Broomrape (Orobanche cumana W.)

Infestations with sunflower broomrape (Orobanche cumana Wallr.), an obligatory root parasite, constitute a major limitation to sunflower production in many regions around the world. Breeding for resistance is the most effective approach to reduce sunflower broomrape infestation, yet resistance mechanisms are often overcome by new races of the pathogen. Elucidating the mechanisms controlling the resistance to broomrape at the molecular level is thus the most desirable pathway to obtaining long-lasting resistance and reducing yield loss in sunflower. In this study, we investigated broomrape resistance in a confectionery sunflower hybrid with a robust and long-lasting resistance to sunflower broomrape. Visual screening and histological examination of sunflower roots revealed that penetration of the intrusive broomrape cells into the host root endodermis is blocked at the host cortex, indicating a pre-haustorial mechanism of resistance. A comparative RNA-Seq experiment conducted between roots obtained from the resistant cultivar, a bulk of five broomrape resistant lines and a bulk of five broomrape susceptible lines allowed the identification of genes that were significantly differentially expressed upon broomrape infestation. Among these differentially expressed genes, β-1,3-endoglucanase, β-glucanase and ethylene-responsive transcription factor4 (ERF4) genes were identified. These genes were previously reported to be pathogenesis-related genes in other plant species. This genetics investigation together with the histological examinations led us to conclude that the resistance mechanism involves the identification of the broomrape and the consequent formation of a physical barrier that prevents the penetration of the broomrape into the sunflower roots.

Dynamics of change in broomrape populations (Orobanche cumana Wallr.) in Romania and Russia (Black Sea area)

The emergence of new broomrape populations (races) has been observed in the past 20 years in several countries (Romania, Moldova, Ukraine, Russia, Turkey, Bulgaria, Spain, Serbia, and China) where sunflower is frequently grown in the same sites without applying traditional crop rotations. Differential lines for sunflower broomrape races A to F have been secured. The new broomrape races have been identified by researchers as races G and H. The question of whether the same broomrape mutations can occur in one year and affect the same countries remains unsolved. Several results of new broоmrape population emergences in some of the affected will be presented in the paper. A total of 390 genotypes were studied at four Romanian localities (Cuza Voda, Crucea-Stupina, Braila-Valea Canepii, and Tulcea-Agighiol) in 2014. At all four localities, a certain degree of sunflower broomrape infestation was observed in control hybrids and lines (Performer, LC-1093, LG-5661, and PR64LE20), which indicated the emergence of new populations higher than race H. The 390 studied genotypes had different reactions in all four localities. In 2015, 10 hybrids and controls were studied at five Romanian localities (Ciresu-Braila, Iazu-Ialomita, Stupina-Constanta, Topolog-Tulcea, and Viziru-Braila) and, according to the results, only hybrid Hy-7 was resistant in all localities. The results obtained from the three studied localities showed the emergence of new sunflower broomrape populations not controlled by gene for race H. Self-fertilization of hybrid Hy-7 produced the F2 generation in 2016. In 2017, broomrape resistance was studied at the infested (contaminated) plot at the All-Russian Research Institute of Oil Crops by the name of Pustovoit V.S. – VNIIMK in Rostov on Don. The plot was found to be infested by new broomrape populations originating from Russia, Ukraine, Romania, Turkey, and Spain. The obtained results showed an infestation degree in 17.1% plants of hybrid Hy-7, 35% in the F2 generation of Hy-7, control hybrids PR64LE25, LG-5580 and Donskoy-22 showed 19.4, 23, and 100% broomrape infestation, respectively. In conclusion, the plot contained broomrape populations which cannot be controlled by race H gene. According to the obtained results, a permanent change in variability of broomrape populations can be confirmed practically year after year. At present, new broomrape populations found at several localities are locally dispersed. Geneticists and breeders have to make joint efforts in further detailed studies of broomrape variability.

Assessing the Performance of Maize (Zea mays L.) as Trap Crops for the Management of Sunflower Broomrape (Orobanche cumana Wallr.)

Sunflower broomrape (Orobanche cumana Wallr.) is a weedy root parasite that causes huge damage to sunflower (Helianthus annuus L.). Trap crop can stimulate parasitic seed germination without attachment in trap crop, and it was considered as economic methods for controlling the parasite. In this study, co-culture and pot experiments were conducted to assess the allelopathic activity of maize (Zea mays L.) to stimulate broomrape seeds germination and to evaluate the performance of maize rotation for reducing sunflower broomrape infection. All the tested maize cultivars could induce sunflower broomrape germination, and the most active maize cultivar was N314, which induced approximately 40% broomrape seeds germination. Rotation of maize significantly reduced broomrape infection on subsequent sunflower and increased sunflower biomass. After cultivated N314 for 3 years, broomrape attachment on sunflower was the lowest (0.8 attachment plant−1), and the dry mass of sunflower shoot (28.7 g plant−1) was approximately twice higher than the sunflower without previous crop. The effectiveness of broomrape management was significantly different among various maize cultivars. Meanwhile, the differences among cultivars were decreased with the years of maize cultivated prolonged. Our results confirmed that maize was significantly effective in reducing the sunflower damage caused by sunflower broomrape and suggested that maize rotation could be included in the integrated management of sunflower broomrape.