Breeding for Sclerotinia resistance in sunflower: A modified screening test and assessment of genetic variation in current germplasm

Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating pathogens of sunflower (Helianthus annuus), causing yield losses up to 100%. This study was conducted to (1) screen sunflower lines for their reaction to Sclerotinia using artificial infection by a modified leaf test, and (2) identify the most suitable trait for screening against Sclerotinia resistance. A set of 85 inbred lines was screened in three environments by infecting the leaves with mycelium and covering them with plastic bags containing water. Susceptibility was assessed by leaf lesion, petiole score, stem rating, stem lesion and diseased stems. The test proved to be effective and practicable, with an infection rate of up to 99%. All scoring traits were strongly correlated with each other and effective in differentiating among susceptible and resistant lines. Although petiole score had the highest heritability, its association with leaf length hampers its utilization as a scoring trait. Stem lesion was a good predictor of mid-stalk rot and can be recommended as a reliable trait for screening sunflower lines against Sclerotinia based on its high heritability estimate (h²= 0.59) and close correlations with other traits measured after artificial and natural infection.     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

Genetic control of in vitro-organogenesis in recombinant inbred lines of sunflower (Helianthus annuus L.)

Ninety-three recombinant inbred lines (F₈) of sunflower developed by the single-seed descent method from the cross ‘PAC-2 × RHA-266’ were used to screen their regenerability by organogenesis. The experiment was designed in randomized complete blocks with three replications. Each replication consisted of 10 Petri dishes with four explants. Cotyledons were excised from 2-day-old seedlings. Each cotyledon was divided into two pieces (four explants), which were incubated in solid regeneration medium consisting of full-strength Murashige and Skoog medium modified by adding hormones. A high genotypic variability for organogenesis parameters between genotypes was observed in this study. The difference between all recombinant F₈ lines and their parents was not significant, showing that the 93 inbred lines used in this experiment are representative of the total possible recombinant lines from the cross ‘PAC-2 × RHA-266′. These F₈ lines were not consciously selected for any trait; therefore, they represent a random set of lines segregating for the organogenesis parameters, as well as for other traits which could be important for breeding.     

Inheritance of resistance to race 330 of Plasmopara halstedii in three sunflower lines

Sunflower lines RHA‐274, HA‐61 and RHA‐325 were studied for their resistance to race 330 of downy mildew (Plasmopara halstedii). The same inbred line, with normal (HA‐89) or sterile cytoplasm (cmsHA‐89) was used in all the crosses as susceptible parent, and, in each cross, only one genotype of the resistant parent was studied. The resistant‐to‐susceptible ratios obtained in the BC₁ and F₂ progenies from the crosses of the lines RHA‐274 and HA‐61 to cmsHA‐89 and HA‐89, respectively, suggested that, in each resistant line, two dominant genes are responsible for resistance to this downy mildew race. One of the genes (A) is epistatic to the other (B), and the recessive allele b in homozygosity is also epistatic to aa, with plants carrying aabb genotypes being resistant. Resistance to race 330 seemed to be controlled by two complementary genes in the sunflower inbred line RHA‐325, the dominant allele of one of them being present in cmsHA‐89. In the genotypes HA‐89 or cmsHA‐89, the existence of genes that modify the expected segregations following the crosses with resistant parents is proposed. It is concluded that, although major genes have been described as responsible for monogenic resistance to downy mildew, other types of regulation of this character, such as complementarity and epistatic relationships, do occur.     

Inheritance of resistance to race F of broomrape in sunflower lines of different origins

A new race F of broomrape overcomes all known resistance genes in cultivated sunflower, but recently, sources of resistance against race F have been developed. The objective of the present research was to study the inheritance of resistance to race F in crosses between 12 resistant sunflower breeding lines, derived from three different sources of resistance, and the susceptible male‐sterile line P‐21. Parental lines and F₁, F₂, F₃ and BC₁ generations were evaluated for broomrape resistance. Segregations in the F₂ and BC₁ to resistant parent approached resistant to susceptible ratios of 1: 15 and 1: 3, respectively, in most of the crosses, suggesting a double dominant epistasis. However, segregations of 3: 13 and 1: 1 for F₂ and BC₁, respectively, indicating a dominant‐recessive epistasis, were also found. The F₃ data confirmed these results. Owing to the recessive nature of this resistance, it must be incorporated into both parental lines for developing resistant hybrid cultivars.     

Development of co-dominant amplified polymorphic sequence markers for resistance of sunflower to downy mildew race 730

The inheritance of the reaction of sunflower to downy mildew was investigated using resistant and susceptible near isogenic lines (NILs) and their F₃ families. Resistance to race 730 was evaluated using the whole seedling inoculation technique. Seventy-three F₃ families were inoculated, among which 54 families were resistant and 19 susceptible, fitting a 3 : 1 segregation ratio. F₃ families were also studied using several PCR markers. Ten markers at the Pl6 locus, specific for the resistant line, also segregated in F₃ families with a 3 : 1 ratio. The same segregation ratio occurred for microsatellite haplotypes that resembled the resistant parent, and were amplified with ORS 166 and ORS 1043. The only common fragment that was observed between resistant and susceptible parental lines was one of the TIR-NBS-LRR resistance gene analogue markers, having a restriction site. Two co-dominant cleaved amplified polymorphic sequence (CAPS) markers were obtained. The mapping data indicate that several dominant markers and two CAPS markers, developed here, completely co-segregate with the Pl6 gene conferring resistance to race 730. CAPS markers will facilitate efficient marker-assisted selection for sunflower resistance to downy mildew race 730.     

Resistance of sunflower inbred lines to various forms of attack by Sclerotinia sclerotiorum and relations with some morphological characters

Summary Twenty sunflower inbred lines were studied for their reactions to 7 Sclerotinia sclerotiorum tests on different plant parts. A principal component analysis (P.C.A.) and t-tests on the means of resistant and susceptible groups indicated that reactions of inbred lines to infection of capitula by ascospores are independent from those to ascospore infections of terminal buds. They are even more contrasted with the results of any test measuring mycelial extension. However, for the last, there is a close association between the reactions of roots, leaves and capitula. Of the inbred lines, some showed good levels of resistance to most forms of attack, others were generally susceptible and there were some with good resistance to one particular form of attack. A P.C.A. of 18 morphological and field characters showed no general association between these characters and Sclerotinia sclerotiorum test results, although it was found that lines resistant to mycelial extension on capitula were generally earlier than those that were more susceptible. It is proposed that breeding programmes for general resistance to Sclerotinia sclerotiorum should include a combination of two or three tests.     

Inheritance of resistance to sunflower downy mildew races 1, 2 and 3 in cultivated sunflower

Sunflower downy mildew (SDM) caused by Plasmopara halstedii, is a major disease of sunflower. Eleven resistance genes have been identified, but allelic relationships among these genes are not clear. This study examined the inheritance and allelic relationships of genes conferring resistance to SDM races 1, 2 and 3 (virulence phenotypes 100, 300 and 700, respectively) and confirmed a twelfth resistance gene. Three USDA Plant Introductions, AMES 3235, PI 497250, and PI 497938, and three released lines, RHA 266, RHA 274 and DM‐2 were studied. RHA 266 has only the Pl₁ gene for race 1 resistance. Digenic inheritance of resistance was found in AMES 3235, PI 497250, and RHA 274. These lines have the Pl₁ and Pl₁₂ genes, conferring resistance to race 1, and the Pl₂ and Pl₁₁ genes, conferring resistance to race 2. DM‐2 and PI 497938 have Pl₁₂ (but not Pl₁ for resistance to race 1, the Pl₁₂ gene (but not the Pl₂) for resistance to race 2, and Pl₅ for resistance to race 3. These resistance genes will serve as a foundation for future gene designations and genetic diversity studies of resistance to SDM.     

The inheritance and biochemistry of resistance to Sclerotinia sclerotiorum leaf infections in sunflower (Helianthus annuus L.)

Summary Resistance of sunflower leaves to attack by Sclerotinia sclerotiorum was studied by infecting them, in the field, with agar disks containing Sclerotinia mycelium. Resistance levels were determined by the length of lesions after a given period. There were significant differences in reaction between both sunflower hybrids and inbred lines. Different Sclerotinia isolates gave the same classification of sunflower genotypes. The results of trials repeated in one year or different years were significantly correlated. The general combining ability variance/specific combining ability variance ratio was 1.35. Strict sense heritability was 0.61±0.03. The midparent-offspring correlation coefficient was significant, but the relation between per se values of the male parents used with the values of their hybrids was much closer than that for the female parents. Mean heterosis for resistance was 28.9%, compared with the midparent. The results of the leaf resistance test are frequently correlated with levels of resistance to root attack by Sclerotinia. Possible use of this test in breeding both directly for leaf resistance and indirectly for root resistance are discussed. H.P.L. Chromatography studies of the phenols present in healthy and infected leaves distinguished 19 compounds, all of the inhibitin type. There was a large increase in phenol content in leaves infected by Sclerotinia, for all genotypes. However, more especially in uninfected leaves, the contents of 3 chromatogram peaks, numbered 4, 6 and 9, showed a close relation with levels of Sclerotinia resistance. It is proposed that these compounds could be used as markers of certain types of resistance.     

R-41, a sunflower restorer inbred line, carrying two genes for resistance against a highly virulent Spanish population of Orobanche cernua

The inheritance of resistance to a highly virulent population of sunflower broomrape (Orobanche cernua) has been studied in R-41, a Spanish sunflower restorer inbred line. Using the cytoplasmic male-sterile inbred line HA-89 (cms; very susceptible to this population of broomrape) as a female parent, progenies of the cross with R-41, i.e. Fl, F2 and BC1 to both parents, as well as the parental lines, were analysed for their reaction to the broomrape population EC-94. The goodness of fit of the observed vs expected segregation ratios indicated that the inheritance of resistance to broomrape in line R-41 is conferred by two independent dominant genes.     

Screening for resistance to broomrape (Orobanche cernua) in cultivated sunflower

Racial evolution of sunflower broomrape (Orobanche cernua) has been very rapid in Spain during recent years, in which resistance has been overcome several times and there has been an important increase in areas infested with this parasitic angiosperm. In order to find resistance to a highly virulent population of sunflower broomrape that could be used directly in breeding programmes, three different sets of cultivated plant material composed of 429 entries were tested by artificial inoculation. All evaluated inbred lines from Moden, Canada, were fully susceptible. Out of the 240 P.I. accessions tested, only 10 segregated for resistance to broomrape, the rest being susceptible. From the 160 USDA breeding lines evaluated, 5% were resistant and 19% segregated for resistance to O. cernua. These lines traced back mainly to crosses of RHA 274 and RHA 801 with Russian, Turkish and Romanian hybrids. The origin of P.I. accessions that segregated for resistance were primarily derived from the former USSR and from Romania.     

Fungitoxic effect of scopolin and related coumarins on Sclerotinia sclerotiorum. A way to overcome sunflower head rot

Summary The content of coumarins, as probable phytoalexins, was analysed in four sunflower genotypes that ranged in responses to head rot from highly susceptible to highly resistant. Low levels of all coumarins (scopolin, scopoletin and ayapin) were detected in the three most susceptible genotypes irrespective of time after inoculation. However, in the resistant genotype there was a clear time-dependent disease-induced increase of all coumarins that reached a maximum after 10–14 days. Detailed comparison of the most susceptible and the resistant genotype showed that in the resistant but not the susceptible, scopoletin peroxidase activity increased during the course of the experiment. Results confirmed a clear negative correlation between coumarin content and disease symptoms and in particular for scopolin. Furthermore we show for the first time that scopolin is inhibitory to Sclerotinia at similar doses to scopoletin. As scopolin is known to be less phytotoxic than ayapin and scopoletin, its accumulation may well confer head rot resistance with minimal plant damage and might be one of the bases for resistance to Sclerotinia.     

Accumulation of soluble phenolic compounds in sunflower capitula correlates with resistance to Sclerotinia sclerotiorum

Disease symptoms and total soluble phenolics content have been analysed in four sunflower (Helianthus annuus L.)lines with different resistance levels(from highly susceptible to resistant) to head rot caused by Sclerotinia sclerotiorum (Lib.) de Bary. At the beginning of the flowering stage, capitula were inoculated by spraying with a water suspension of ascospores, and disease symptoms were evaluated from day 6 to day14 after inoculation. The most susceptible genotypes showed all their ovaries to be necrosed and abundant lesions in corollas, bracts and receptacle. In the resistant line, the ovary and corolla were only partially necrosed with no symptoms in the bracts or the receptacle. Total soluble phenolics were extracted and quantified from different parts of the capitulum in both inoculated and non-inoculated plants. The amount of phenolic compounds depended on the sunflower line, the time after inoculation, and the tissue. Higher constitutive and induced phenolic content as well as phenylalanine ammonia-lyase activity were present in the most resistant line, these differences correlated with the absence/presence of disease symptoms. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of resistance to phomopsis (Diaporthe helianthi) in sunflower

The inheritance of resistance of sunflower to phomopsis (Diaporthe helianthi) was studied on 10 inbred lines representing a range of resistance and susceptibility levels, and 25 hybrids from a factorial cross of these lines. Analyses of observations over a 3-year period, of semi-natural infection (presence of stem lesions greater than 5 cm) on the hybrids indicated that additive gene control was predominant, with no significant interactions between parental effects. Correlations between infection of parental lines and the mean of their hybrids were not always significant, with certain inbreds which appeared quite susceptible giving hybrids with good levels of resistance. In order to predict hybrid values, it appears necessary to determine the general combining abilities of parental lines. Some inbreds gave very high levels of resistance, others gave very high levels of susceptibility. A test measuring the rate of extension of D. helianthi mycelium on leaves was significantly correlated with the results of natural infections. In particular, it permitted distinction of the most susceptible genotypes, and thus could be used in first generations of breeding to eliminate the most susceptible plants.     

Inheritance of resistance to broomrape (Orobanche cumana Wallr.) race F in a sunflower line derived from wild sunflower species

Genetic resistance to broomrape (Orobanche cumana Wallr.) race F in sunflower line J1, derived from the wild perennial species Helianthusgrosseserratus Martens and Helianthus divaricatus L., has been reported to be controlled by dominant alleles at a single locus, Or6. However, deviations from this monogenic inheritance have been observed. The objective of the present study was to gain insight into the inheritance of resistance to broomrape race F in the sunflower line J1. F₁, F₂, F₃ and BC generations from crosses between J1 and three susceptible lines, P21, NR5 and HA821 were evaluated. F₁ hybrids showed both resistant (R) and moderately resistant (MR) plants, the latter having a maximum of five broomrape stalks per plant compared with >10 in the susceptible parents. This indicated incomplete dominance of the Or6 alleles. F₂ plants were classified as R, MR or susceptible (more than five broomrape stalks per plant). Three different segregation ratios were observed: 3 : 1, 13 : 3 and 15 : 1 (R + MR : S), suggesting the presence of a second gene, Or7, whose expression was influenced by the environment. A digenic model was confirmed, based on the evaluation of F_2:3 families.     

Effect of Induced Autotetraploidy on Response to Sclerotinia Clover Rot in Trifolium pratense L.

The effect of induced autotetraploidy on the response of red clover to Sclerotinia clover rot (SCR) was studied under laboratory, glasshouse and field conditions. To assess the response in the laboratory, the detached leaf technique was employed, whereas in the glasshouse and in the field, 6—14 weeks old seedlings and adult plants, respectively, were artificially inoculated with the pathogen grain inoculum. The results of the studies showed that great genotypic variability occurs within diploid (2×)and tetraploid (4×)red clover in response to that disease. Generally, tetraploid red clover, in comparison to diploid, is more resistant to SCR. The laboratory, glasshouse and field experiments showed that the effect of induced autotetraploidy on SCR resistance is positive. The hardening of plant with light and temperature enlarged the differences in reponse to SCR between ploidy levels, family pairs and individual strains with every testing method. Other factors, like plant age and flowering time, influencing red clover response to SCR are also discussed.     

甘蓝型油菜菌核病抗性的遗传分析

利用牙签接种和花瓣接种鉴定了甘蓝型油菜DH821（抗）×DHBao604（感）组合P₁、P₂、F₁、BC₁、BC₂、F₂ 6个世代群体菌核病的抗性，通过主基因-多基因混合遗传模型分析表现，牙签接种3 d的抗性受2对加性主基因+加性-显性多基因控制，BC₁、BC₂、F₂ 群体主基因遗传率为12.49%~28.48%，多基因遗传率为21.08%~27.87%；5 d的抗性由2对加性-显性-上位性主基因控制，显性及显性互作效应明显，无多基因修饰，BC₁、BC₂、F₂群体主基因遗传率为25.19%~38.69%；3~5 d的菌丝扩展抗性由2对加性-显性-上位性主基因+加性-显性多基因控制，主基因效应明显，多基因效应不明显，BC₁、BC₂、F₂ 群体主基因遗传率为0.72%~54.06%，多基因遗传率为7.34%~46.79%。花瓣接种15 d的抗性由2对加性-显性-上位性主基因+加性-显性-上位性多基因控制，2对主基因的加性效应和显性效应为－3.6051~ －3.5225，互作效应为3.5190~3.6089，主基因遗传率为0.91%~41.79%，多基因遗传率为19.32%~73.69%。     

Indicators of resistance of sunflower plant to basal stalk rot (Sclerotinia sclerotiorum): Symptomatological,biochemical, anatomical,and morphological characters of the host

Summary The object of this work was a study of the relationship between the field reactions of different sunflower genotypes to basal stalk rot (in terms of severe (dead plants) and incipient wilting, and lesion length) and some biochemical (phenol concentration), morphological (plant height, and stem and flower-bud diameters) and anatomical (xylem and cortical indexes) characters of the host. Plants from 8 inbred lines at closed flower-bud stage were artificially inoculated with mycelium at the base of the stem. The percentage of dead plants for each inbred line and the lesion length and wilting range for individual plants after 7 days were recorded.A positive and highly significant correlation coefficient between the percentage of dead plants and lesion length was found for the three years of the study (r=0.83; P<0.01). A highly significant association between lesion length and wilting range for individual plants was always found (P=0.00). Postinfectional phenol content exhibited a strong negative correlation with lesion length and the percentage of dead plants in all the experiments (P=0.05). Association between postinfectional phenol content and wilting range for individual plants was significant for all the years studied (P<0.05). No correlation between phenol levels in healthy plants of the different sunflower genotypes and their susceptibility was found. Morphological characters positively correlated with lesion length but only plant height exhibited significant values for the three years. Associations between wilting range and morphological characters for individual plants were significant for one of the two years analyzed (P<0.05). Xylem index showed a negative correlation with lesion length which was significant one of the two years studied.The lesion length measure seems to be a simple and direct method for resistance screening before the flowering period. Although strong relations with postinfectional phenol levels were found, their determinations would be too much time consuming and not completely reliable. The relationships between other characters measured and disease resistance would indicate that physiological mechanisms could be related to resistance.