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.     

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.     

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.     

The role of peroxidase in the resistance of sunflower against Orobanche cumana in Russia

We present the results of a histochemical study comparing seedlings of races C and D of Orobanche cumana Wallr. (syn. O. cernua Loefl.) attacking sunflower (Helianthus annuus L.) in southern Russia. Three groups of O. cumana seedlings were distinguished according to the peroxidase content of the cells in the radicles: (1) those with neither extracellular nor intracellubr peroxidase and whose radicles have a smooth apex (these were classified as non-infective): (2) those with a high peroxidase content of the nuclei and the cytoplasm layer adjacent to the cell wall, as well as excretion of peroxidase from the apex of the radicles: (3) those with a similarly high peroxidase activity in the parasite cells, but without extracellular excretion. The apices of the radicles of the last two groups are swollen. It is suggested that these belong to O. cumana races C and D respectively. The extracellular peroxidase in O. cumana race C reacts with phenolic compounds, which are lignin precursors of the host, resulting in host resistance due to the formation of lignin layers in sunflowers possessing the Or3 gene for resistance. The absence of extracellular peroxidase in O. cumana race D prevents lignin formation and enables the parasite to attach to the host vascular system. Comparison of these data with the information on the earlier O. cumana races A and B, and older sunflower cultivars, points to a crucial role of peroxidase in the process of breeding new sunflower cultivars and the evolution of new O. cumana races.     

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.     

阴山北麓地区向日葵列当生理小种的鉴定及种子的单株纯化

向日葵列当是一种全寄生的种子植物,对向日葵生产造成严重的影响,而选育抗列当品种是目前防控向日葵列当最为有效的防控措施。由于向日葵列当种群存在生理小种分化现象,因此明确不同地区的向日葵列当生理小种的类型以及分布对于抗列当品种的布局非常重要。本研究利用国际通用的向日葵列当生理小种鉴别寄主,对阴山北麓地区的武川县和四子王旗共计7个向日葵地块中采集的列当种子的生理小种进行了鉴定,结果表明除了呼和浩特市武川县头号村采集的向日葵列当样本为E小种外,其他6个不同地点采集到的向日葵列当样本均鉴定为G小种。为了更好地对向日葵品种抗列当水平进行准确的评价,本研究选择了已经鉴定为E、F和G三个小种类型的向日葵列当的样本进行了单株纯化以及扩繁。扩繁得到的列当的种子将用于准确评价向日葵不同的育种材料和品种对列当不同生理小种的抗性水平。     

Cross‐resistance of Echinochloa species to acetolactate synthase inhibitor herbicides

This study was conducted to evaluate the cross‐resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac‐sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus‐galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross‐resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac‐sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus‐galli, respectively. An in vitro ALS enzyme assay with herbicides showed that the I ₅₀‐values of the resistant accessions were approximately two‐to‐three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac‐sodium. A whole plant assay with fenitrothion showed that the GR ₅₀‐values significantly decreased in both the resistant E. oryzicola and E. crus‐galli accessions when azimsulfuron, penoxsulam and bispyribac‐sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross‐resistance for azimsulfuron, penoxsulam and bispyribac‐sodium is related to enhanced herbicide metabolism.     

云南野生稻和地方稻资源抗白叶枯病分析

为鉴定云南稻种质资源对水稻白叶枯病的抗性情况,于孕穗期采用剪叶接种方法,用水稻白叶枯病强致病型代表菌株BD8438、CN9404和X1接种云南野生稻和地方稻种质资源,以病斑长度大于6 cm为感病分界线,对其抗感表现型进行调查分析。结果显示,共鉴定出来源于云南省不同种植生态区的186份地方稻抗性材料和22个野生稻抗病居群。野生稻对水稻白叶枯病的抗病能力较地方稻强,其中疣粒野生稻的抗性最强,抗病等级为0~2;药用野生稻次之,抗病等级为1~2;普通野生稻相对较差,抗病等级为1~5。地方稻抗性资源来自于云南省各个传统水稻种植区,抗性1级的材料占17%,抗性2级的占2%,抗性3级的占81%;按照稻种质资源亚种类型、粘糯性和水旱性分类,各类型地方稻抗性材料所占比例分别为粳稻占61%、籼稻占39%;粘稻占66%、糯稻占34%;水稻占83%、陆稻占17%。从利用抗白叶枯病基因培育新品种的角度评价,这些抗性资源具有潜在的发掘利用价值。     

向日葵上一种列当的发生分布及防治

从1979～1980年调查研究明确了吉林省白城地区寄生在向日葵上的恶性杂草——列当的种名是Orobanche coerulescens Steph(本文暂称白城列当)而不是向日葵列当(O.cumana)。白城列当在全地区8个县均有分布,发生密度较大的地块1平方米均有300棵左右,寄生株率96%以上,1株向日葵上最多寄生146棵。其垂直分布主要在土壤耕层里,是寄生于向日葵根部,以5～15厘米耕层寄生率最高达93%。在向日葵植株周围的水平分布以距向日葵5～20厘米寄生率达68.1%。被害的向日葵植株,表现株矮、茎细、盘小,甚至全株枯死。籽实减产38.3～70.3%,籽仁含油率降低3.3～17.9%,严重影响向日葵的生产,应引起各方面重视。提出因地制宜地采取选育抗性品种,合理轮作及提早铲除等综合防治措施,以便尽早地消灭在始发阶段。     

Pathogenic variability of Plasmopara halstedii infecting sunflower in the Czech Republic

Plasmopara halstedii was isolated from diseased sunflowers collected from eight locations in the Czech Republic from 2007 to 2014. Races of the pathogen were determined based on 84 isolates collected during the study. In total, eight races of P. halstedii were detected using a set of nine sunflower differential lines. Races 700, 704, 705, 710, 714 and 715 were proven by soil drench inoculation, and two additional races (730 and 770) proposed by the previously applied leaf disc inoculation method. Race 700 was the most dominant in the Czech P. halstedii populations, with race 710 being the second most frequent. Races 704 and 714 were found over three seasons, while other races were recorded only in one growing season (race 730 in 2010, and the new races 705 and 715 in 2014). A comprehensive study was further conducted for isolates collected in 2013–14 using an extended differential set consisting of 15 sunflower lines. According to the latter methodology which marks races with five‐digit virulence codes, races 70060, 70471, 70571, 71060, 71461 and 71571 were recorded. The growing complexity of P. halstedii pathogenicity exhibited by the ability to infect higher numbers of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Although the limited number of isolates studied cannot characterize the entire pathogen diversity in the Czech Republic, the trend towards more diverse virulence in P. halstedii populations is clearly demonstrated by the new records of races 704, 705, 714 and 715, all capable of overcoming the resistance gene Pl₆.     

Resistance to Taiwanese race 1 strains of Ralstonia solanacearum in wild tomato germplasm

A total of 252 wild Solanum accessions and one population of 49 introgression lines of LA716 were screened for resistance to a race 1/biovar 4/phylotype I strain Pss186 of Ralstonia solanacearum. Most wild tomato accessions were highly susceptible. However, five accessions of S. pennellii, i.e. LA1943, LA716, LA1656, LA1732 and TL01845 were resistant to strain Pss186. These accessions were then challenged against two other race 1/phylotpye I strains Pss4 and Pss190, which were more aggressive. All the five S. pennellii accessions were susceptible to Pss4, but displayed high to moderate resistance to Pss190 with a percentage of wilted plants ranging from 0% to 60%. Pss190 is an aggressive strain that made a resistant tomato line Hawaii 7996 susceptible. Thus, the results found in this study provide evidence of the presence of strain-specific resistance. LA3501, which has an introgression segment on chromosome 6, was found to be resistant to Pss186 among the screened introgression lines. This confirms the importance of resistance trait loci on chromosome 6 that have been identified by other studies. This is the first report of S. pennellii being resistant to bacterial wilt. These new resistant sources will provide breeders with more resources to breed for stable resistance to bacterial wilt of tomato.     

A detailed evaluation method to identify sources of quantitative resistance to Fusarium oxysporum f. sp. pisi race 2 within a Pisum spp. germplasm collection

Fusarium oxysporum f. sp. pisi (Fop) is an important pathogen of field pea (Pisum sativum) worldwide. The constant evolution of the pathogen drives the necessity to broaden the genetic basis of resistance to Fop. To achieve this, it is important to have a large germplasm collection available and an accurate and efficient method for disease assessment. Here, a detailed evaluation method coupling disease incidence, disease rating over time and its related area under the disease progression curve (AUDPC) was established and used to screen a Pisum spp. germplasm collection against one isolate of Fop race 2. A large variation in the disease response of specific pea accessions ranging from highly resistant to susceptible was observed within the collection, indicating the quantitative expression of the resistance. The repetition of the inoculation experiments on a subset of 19 accessions, including two susceptible accessions, indicated that the scoring method was robust and reproducible and confirmed the highly resistant phenotypes of 11 accessions. To initiate the characterization of resistance mechanisms within these accessions, the external and internal stem symptoms were compared between these selected pea accessions, together with the extent of fungal colonization within plants. All these tests indicated that, in all resistant accessions, the resistance mechanisms efficiently stopped pathogen progression at the crown. Incorporation of these sources of resistance to breeding programmes will contribute to improved Fop resistance in pea cultivars.     

Orobanche cumana race F: performance of resistant sunflower hybrids and aggressiveness of populations of the parasitic weed

The production of sunflower is threatened by Orobanche cumana (broomrape) infections in many countries of southern and eastern Europe. The most virulent race of this parasitic weed, race F, is widespread in Spain. Most of the current commercial hybrids of resistant sunflower do not completely prevent broomrape emergence in the field. The performance (broomrape incidence and severity, final yields and oil contents) of four resistant and two susceptible sunflower hybrids was studied in different years in several O. cumana – infested locations. O. cumana – sunflower interactions were characterised by low severity and intermediate incidence in the resistant hybrids; both depended on the environment. An increase in sunflower yield because of the use of resistant hybrids was most noticeable under heavy field infestations and under drought. Characterisation of 20 populations of O. cumana classified as race F in terms of the standardised area under the degree of attack progress curve (SAUDC) and O. cumana incidence (BI) suggested differential intrinsic pathogenicities. These results highlight the variability within race F of O. cumana , mirroring the environmentally dependent horizontal resistance in sunflower.     

Resistance to Pseudomonas syringae in a collection of pea germplasm under field and controlled conditions

A total of 242 Pisum accessions were screened for resistance to Pseudomonas syringae pv. pisi under controlled conditions. Resistance was found to all races, including race 6 and the recently described race 8. Fifty‐eight accessions were further tested for resistance to P. syringae pv. syringae under controlled conditions, with some highly resistant accessions identified. Finally, a set of 41 accessions were evaluated for resistance to P. syringae pv. pisi and pv. syringae under spring‐ and winter‐sowing field conditions. R2, R3 and R4 race‐specific resistance genes to P. syringae pv. pisi protected pea plants in the field. Resistance sources to race 6 identified under controlled conditions were ineffective in the field. Frost effects were also evaluated in relation to disease response. Results strongly suggest that frost tolerance is effective in lowering the disease effects caused by P. syringae pv. pisi and pv. syringae under frost‐stress conditions, even in the absence of disease resistance genes, although the highest degree of this protection is reached when frost tolerance and disease‐resistance genes are combined in the same genetic background.     

Resistance levels of sulfonylurea‐resistant Schoenoplectus juncoides (Roxb.) Palla with various Pro197 mutations in acetolactate synthase to imazosulfuron,bensulfuron‐methyl,metsulfuron‐methyl and imazaquin‐ammonium

An investigation, using herbicidal pot tests in a greenhouse condition, was conducted to determine the whole‐plant dose–response relationships to several acetolactate synthase (ALS)‐inhibiting herbicides of sulfonylurea (SU)‐resistant Schoenoplectus juncoides with various Pro₁₉₇ mutations in ALS that was collected from Japanese rice paddy fields. All the tested SU‐resistant accessions with a Pro₁₉₇ mutation were highly resistant to two commonly used SU herbicides (imazosulfuron and bensulfuron‐methyl), but were much less resistant to another SU herbicide, metsulfuron‐methyl, and were substantially not resistant to imazaquin‐ammonium. These cross‐resistance patterns have been known previously in fragments of S. juncoides and other weed species and were comprehensively confirmed in this study with a whole set of Pro₁₉₇ mutations. The analyses of resistance levels, based on ED₉₀ values, newly showed that different accessions with a common amino acid substitution in ALS1 showed similar responses to these herbicides (confirmed with four amino acid substitutions), that the rankings of resistance levels that were conferred by various Pro₁₉₇ mutations in ALS1 differed among the SU herbicides and that the resistance levels of the ALS2‐mutated accessions were higher than, lower than or similar to those of the corresponding ALS1‐mutated accessions, depending on the compared pair, but the deviation patterns were generally similar among the SU herbicides in each compared pair. The final finding might suggest that the abundance of ALS2 is not as stable as that of ALS1. In addition, as a result of these new findings, together with expected further research, a suggested possibility is that substituting amino acids at Pro₁₉₇ generally could be estimated by plotting each accession's ED₉₀ values of imazosulfuron and bensulfuron‐methyl in a two‐dimensional graph.     

广西野生稻资源抗白叶枯病研究

1981年来,作者对广西保存并已列入全国统一编目的广西野生稻资源进行白叶枯病的抗性鉴定结果表明:在普通野生稻(Oryza rufipogon Griff)1553个编号中,筛选出46个属高抗至抗病类型,占总数2.96%;在药用野生稻(O.officinalis Wall)199个编号中,绝大多数属抗至中抗褐变类型。这些材料均可供抗性育种利用。此外,还发现普通野生稻抗性与原孳生地、生长习性类型、菌株致病力等关系密切。     

四川58个小麦品种苗期抗条锈基因推导及成株期抗性表现

为了解四川小麦品种所含抗条锈基因及其在田间的抗性表现,将29个毒性谱各异的小麦条锈菌鉴别菌株于苗期接种35个已知抗条锈基因载体品系和58份四川小麦品种（系）,通过抗病谱对比分析和各品种（系）系谱分析,推导了四川小麦品种（系）的抗条锈基因型。在田间,选用中国小麦条锈菌优势小种CY32、CY33、Su-4和对Yr10、Yr24和Yr26致病的贵农22致病类型田间突变株进行混合接种,在乳熟期对各品种（系）的抗条锈性进行了鉴定。结果显示,Yr2、Yr5、Yr7、Yr9、Yr10、Yr24、Yr27、YrSpp、YrAlba等9个基因以单基因或基因组合的形式存在于44个四川小麦品种（系）中。有6个品种（系）含有Yr9基因,10个品种（系）含有Yr10基因,11个品种（系）含有Yr24基因,其中1个同时含有Yr10和Yr24基因。23个品种（系）含有未知基因及其组合。共有19个小麦品种（系）成株期对包含上述混合菌种在内的田间流行菌系表现抗病,其中5个品种抗性受成株抗性基因控制。     

Identification of a new pathotype of Puccinia hordei with virulence for the resistance gene Rph7

Barley leaf rust resistance gene Rph7, derived from barley accession Cebada Capa, is the most effective R-gene for resistance to Puccinia hordei. Virulence for this gene was known in the USA, Israel and Morocco but not yet in Europe. We found an unexpected leaf rust infection in the field at Córdoba, Spain in 2004 on Rph7 carrying lines. This virulence for Rph7 was confirmed in growth chamber experiments, being the first report of Rph7 virulence in European populations of P. hordei. A collection of 680 barley accessions was screened for resistance against this new isolate. Twelve accessions showed segregation with individual plants showing resistance based on hypersensitivity (low infection type). These individual resistant plants were selected and grown in the greenhouse to obtain seeds.     

Genetics of resistance to race TTKSK of Puccinia graminis f. sp. tritici in Triticum monococcum

Race TTKSK (or Ug99) of Puccinia graminis f. sp. tritici possesses virulence to several stem rust resistance genes commonly present in wheat cultivars grown worldwide. New variants detected in the race TTKSK lineage further broadened the virulence spectrum. The identification of sources of genetic resistance to race TTKSK and its relatives is necessary to enable the development and deployment of resistant varieties. Accessions of Triticum monococcum, an A-genome diploid wild and cultivated wheat, have previously been characterized as resistant to stem rust. Three resistance genes were identified and introgressed into hexaploid wheat: Sr21, Sr22, and Sr35. The objective of this study was to determine the genetic control and allelic relationships of resistance to race TTKSK in T. monococcum accessions identified through evaluations at the seedling stage. Generation F(2) progeny of 8 crosses between resistant and susceptible accessions and 13 crosses between resistant accessions of T. monococcum were evaluated with race TTKSK and often with North American races, including races QFCSC, TTTTF, and MCCFC. For a selected population segregating for three genes conferring resistance to race TTKSK, F(2:3) progeny were evaluated with races TTKSK, QFCSC, and TTTTF. In that population, we detected two genes conferring resistance to race TTKSK that are different from Sr21, Sr22, and Sr35. One of the new genes was effective to all races tested. The identification of these genes will facilitate the development of varieties with new resistance to race TTKSK.