Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection

We report a comprehensive primary metabolite profiling of sunflower (Helianthus annuus) genotypes displaying contrasting behavior to Sclerotinia sclerotiorum infection. Applying a GC-MS-based metabolite profiling approach, we were able to identify differential patterns involving a total of 63 metabolites including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids and few soluble secondary metabolites in the sunflower capitulum, the main target organ of pathogen attack. Metabolic changes and disease incidence of the two contrasting genotypes were determined throughout the main infection period (R5.2-R6). Both point-by-point and non-parametric statistical analyses showed metabolic differences between genotypes as well as interaction effects between genotype and time after inoculation. Network correlation analyses suggested that these metabolic changes were synchronized in a time-dependent manner in response to the pathogen. Concerted differential metabolic changes were detected to a higher extent in the susceptible, rather than the resistant genotype, thereby allowing differentiation of modules composed by intermediates of the same pathway which are highly interconnected in the susceptible line but not in the resistant one. Evaluation of these data also demonstrated a genotype specific regulation of distinct metabolic pathways, suggesting the importance of detection of metabolic patterns rather than specific metabolite changes when looking for metabolic markers differentially responding to pathogen infection. In summary, the GC-MS strategy developed in this study was suitable for detection of differences in carbon primary metabolism in sunflower capitulum, a tissue which is the main entry point for this and other pathogens which cause great detrimental impact on crop yield.     

Changes in antioxidant systems in sunflower partial resistant and susceptible lines as affected by Sclerotinia sclerotiorum

Sunflower, Helianthus annuus L., is a major oil-seed crop widely cultivated throughout the globe. White mold, caused by necrotrophic pathogen Sclerotinia sclerotiorum (Lib.) de Bary, is a common and widespread disease of sunflower. Changes in various physiological activities such as production of malondialdehyde (MDA) as the main end product of lipid peroxidation, enzymatic and non-enzymatic activities and proline content, were investigated in leaves and stems of sunflower lines either resistant or susceptible to S. sclerotiorum. The results showed accumulation of high amount of free proline in the leaves of the resistant line. S. sclerotiorum invasion resulted in increase in the MDA content in both lines compared to the controls. Superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase activities significantly increased in the stem and leaves of inoculated lines. In the resistant line, Sclerotinia infection significantly induced catalase (CAT) activity both in the stem and leaves of contaminated plants. The activity of CAT was significantly decreased in susceptible line. Based on our results the activity of antioxidant enzymes was much pronounced in sunflower resistant line than in susceptible one. Studied lines showed distinctive activity considering different biochemical parameters, which may point to further directions in exploring host-pathogen interaction and lead to selection and production of new lines to achieve an improvement of plant adaptation to pathogen.     

Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species

The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3–4 months and set seeds, of which 70–80% germinated. Received: 18 February 1997 / Revision received: 25 March 1997 / Accepted: 15 May 1997     

Fungal populations on sunflower ( Helianthus annuus ) anthosphere and their relation to susceptibility or tolerance to Sclerotinia sclerotiorum attack

Analysis of the fungal flora from different floret parts of various sunflower (Helianthus annuus) varieties showed that there are differences in both fungal species and frequency, depending on whether the sunflower variety is susceptible (SV) or tolerant (TV) to attack of the flower heads by the ascomycete pathogen Sclerotinia sclerotiorum. The sunflower varieties analyzed were SV: HA 300 and Z 20028, and TV: HA 302, Z AV 8410 and Z 30629. The isolates showed different “in vitro” behavior as biocontrol agents. The most common types of interaction with Sclerotinia sclerotiorum were D2 and D2+ (hyphal contact) for isolates from SV and TV, while some of the isolates from TV displayed antibiosis. The microorganisms that colonize TV florets play a part in an indirect mechanism that protects flowers from ascospore germination and pathogen growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

QTL mapping of <Emphasis Type="Italic">Sclerotinia</Emphasis> midstalk-rot resistance in sunflower

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F₃ families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F₂ genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F₃ families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h²=0.55–0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.     

Regeneration of interspecific somatic hybrids between Helianthus annuus L. and Helianthus maximiliani (Schrader) via protoplast electrofusion

Helianthus maximiliani is one of the wild Helianthus species with the genes for resistance to many pathogens including Sclerotinia sclerotiorum. Unfortunately, a transfer of disease resistance genes from this species into the cultivated sunflower is limited by its poor crossability with the cultivated sunflower and sterility of interspecific hybrids. To overcome this problem, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clone of H. maximiliani were electrically fused with etiolated hypocotyl protoplasts of the cultivated sunflower inbred line PH-BC₁-91A. Fusion products were embedded in agarose droplets and subjected to different regeneration protocols. Developed microcalluses were released from the agarose and transferred into solid media. Shoot regeneration was achieved by culture of calluses on regeneration medium containing 2.2 mg l⁻¹ BAP and 0.01 mg l⁻¹ NAA after the treatment with a high concentration of 2,4 D for a limited period of time. A morphological and RAPD analysis confirmed a hybrid nature of the regenerated plants.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commericial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. Withthe discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race, 4 or 5.     

New Races of Sunflower Downy Mildew (Plasmopara Halstedii) in Germany

Plasmopara halstedii, the causal agent of downy mildew of cultivated sunflower (Helianthus annuus), was documented in Germany for the first time in commercial fields. The pathogen was first observed in the Württemberg area, where races 1 and 4 were identified using a set of differential lines. Later, commercial fields near Baden were found to be infected by race 5, which is the first occurrence of that race outside of North America. With the discovery of race 5, there are now eight races of the sunflower downy mildew fungus that have been found in Europe. The sunflower cultivars most frequently grown in Germany were investigated for resistance to race 1, 4 and 5; while all were resistant to race 1, none were resistant to either race 4 or 5.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

Metabolic processes and carbon nutrient exchanges between host and pathogen sustain the disease development during sunflower infection by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Interactions between the necrotrophic fungus Sclerotinia sclerotiorum and one of its hosts, Helianthus annuus L., were analyzed during fungal colonization of plant tissues. Metabolomic analysis, based on ¹³C- and ³¹P-NMR spectroscopy, was used to draw up the profiles of soluble metabolites of the two partners before interaction, and to trace the fate of metabolites specific of each partner during colonization. In sunflower cotyledons, the main soluble carbohydrates were glucose, fructose, sucrose and glutamate. In S. sclerotiorum extracts, glucose, trehalose and mannitol were the predominant soluble carbon stores. During infection, a decline in sugars and amino acids was observed in the plant and fungus total content. Sucrose and fructose, initially present almost exclusively in plant, were reduced by 85%. We used a biochemical approach to correlate the disappearance of sucrose with the expression and the activity of fungal invertase. The expression of two hexose transporters, Sshxt1 and Sshxt2, was enhanced during infection. A database search for hexose transporters homologues in the S. sclerotiorum genome revealed a multigenic sugar transport system. Furthermore, the composition of the pool of reserve sugars and polyols during infection was investigated. Whereas mannitol was produced in vitro and accumulated in planta, glycerol was exclusively produced in infected tissues and increased during colonization. The hypothesis that the induction of glycerol synthesis in S. sclerotiorum exerts a positive effect on osmotic protection of fungal cells and favors fungal growth in plant tissues is discussed. Taken together, our data revealed the importance of carbon–nutrient exchanges during the necrotrophic pathogenesis of S. sclerotiorum.     

Reaction and survival of four types of sunflowers against Sclerotinia sclerotiorum under controlled conditions

Sclerotinia wilt of sunflower caused by Sclerotinia sclerotiorum (Lib.) De Bary is the major disease of sunflower (Helianthus annuus L.) in Iran. North-western areas of Iran demonstrate the most yield losses through the disease. Development of cultivars with adequate genetic resistance is necessary to avoid these losses. Evaluation of sunflower genotypes varies based on researchers and environmental conditions. Meanwhile, greenhouse tests of the sunflower genotypes are more reliable because of controlled conditions during the assessment activities. To study the reaction of the host plants under controlled conditions, three stem inoculation techniques, including mycelium plug (MP), oxalic acid solution (OAS) and wheat seeds infested (IWS) with Sclerotinia mycelium, were employed. Wounded and non-wounded treatments were used in the experiment to find their effect on the disease progress. Four genotypes, including Ghalami (local variety in market), Confeta, Alstar and Master, were inoculated in this study. The factor was the lesion length to evaluate the effectiveness of different inoculation procedures. The lesion length was measured after 3, 7, 10 and 14 days post inoculation. The analysis of variance demonstrated significant differences between IWS and two other methods (MP and OAS), where the IWS produced the longest lesion lengths. In contrast, the non-significant differences between MP and OAS methods might help researchers to employ the pathogenicity factor (oxalic acid) as an alternative inoculum for their studies. Master variety was the most tolerant genotype among the treatments and its viability was 100% even at 14?days after inoculation and incubation. Interestingly, there was no mortality in all cultivars before 7-day incubations, regardless of resistant or susceptible reactions.