A greenhouse assay to screen sunflower for resistance to Alternaria helianthi

A greenhouse assay to screen sunflower for resistance to Alternaria helianthi is described. A comparison of conditions led to the following standard conditions being recommended. The first or second pair of leaves of seedling plants at the V8 growth stage are inoculated using inoculum grown on sunflower leaf extract agar for 5–10 days at an inoculum density of 1–2 spores cm² of leaf tissue. A 48 h dew period should be applied to plants covered by a plastic tent. A dew period temperature of 26/26°C night/day and a post-dew period temperature relative to that experienced under local growing conditions should be applied. Lesions are measured 7 days after inoculation, and mean lesion size per plant is calculated. Mean lesion size of lines being tested is expressed as a proportion of the mean lesion size of a susceptible standard included in each screening experiment.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Influence of pollen selection for Alternaria helianthi resistance on the progeny performance against leaf blight in sunflower (Helianthus annuus L.)

Seven genotypes of sunflower, including populations and hybrids, showing differential susceptibility to Alternaria leaf and stem blight were crossed to CMS 234A with pollen selection and without pollen selection. The pathogen culture filtrate was used as selective pressure at stylar tissue by applying it 1 h before pollination. Distilled water applied to stigmas and styles served as control. Two sets of seven hybrids, one set from selective and the other from non-selective fertilization, were evaluated for reaction to Alternaria leaf and stem blight during the rainy season under natural epiphytotic conditions. Selection for resistant pollen on the stigmatic surface resulted in a corresponding increase in progeny resistance. The study demonstrates the transmission of the selected trait from the pollen generation to progeny. Further, it was observed that the effect of pollen selection was high in the progenies of moderately resistant parents compared to progenies of highly susceptible parents. The effect of successive pollen selection was studied by backcrossing the progeny derived through selective fertilization to the fertile parent using selective fertilization. Successive pollen selection further improved disease resistance of progeny. However, the improvement was not very great. Hence, repeated cycles of selection are required to achieve a useful level of resistance in the case of sunflower, since resistance is polygenetically controlled. Received: 17 May 1999 / Revision accepted: 13 September 1999     

Marker-assisted selection for two rust resistance genes in sunflower

In this study we report on the identification of molecular markers, OX20₆₀₀ and OO04₉₅₀, linked to the geneR _Adv in the proprietary inbred line P2. This gene confers resistance to most of the pathotypes of Puccinia helianthi identified in Australia. Analysis indicates these RAPD markers are linked to the resistance locus at 0.0 cM and 11 cM respectively. SCAR markers SCX20₆₀₀ and SCO04₉₅₀ derived from these two RAPD markers, and SCT06₉₅₀ derived from a previously reported RAPD marker linked at 4.5 cM from the R ₁ rust resistance gene were developed. SCX20₆₀₀ and SCO04₉₅₀ were linked at similar distances from their resistance locus as the RAPD markers. SCTO6₉₅₀ co-segregated completely with rust resistance. The robustness of the R ₁ SCAR marker was demonstrated through the amplification of the marker in a diverse range of sunflower germplasm considered to possess the R ₁ gene. The SCAR markers forR _Adv were not amplified in the sunflower rust differential set thereby supporting the contention that this is a novel resistance gene. They did amplify in a number of proprietary lines closely related to the line P2. This locus is under further investigation as it will be useful in our attempts to use molecular-assisted breeding to produce durable resistance in sunflower to P. helianthi.     

Benzothiadiazole Induces Resistance to Plasmopara helianthi in Sunflower Plants

A novel synthetic chemical CGA 245704 [benzo (1,2,3) thiadiazole-7-carbothioic acid S methyl ester (acibenzolar-S-methyl) (BTH)] applied as a soil drench and foliar spray was shown to provide good protection against Plasmopara helianthi in sunflower plants. Soil drenches at the rates of 150 and 200 mg/kg soil, were applied 3 days before the inoculation and induced high levels of protection (80–82%) against downy mildew. At the highest concentrations (up to 300 mg/kg soil) about 90% protection was observed but at these rates phytotoxic symptoms were noted. This compound also provided good protection against P. helianthi when applied 1 day before the inoculation as well as when applied 1 day post-inoculation. The chemical had no antifungal activity in vitro against P. helianthi . In addition the combination of CGA 245704 and metalaxyl, as a seed dressing at low rates, provided a high level of control against the disease; however, the activity of this combination was not significantly different from metalaxyl alone. CGA 245704 applied as a soil drench and foliar spray also protected sunflower plants from P. helianthi foliar infection.     

Determination of Physiological Races and Evaluation of Sunflower for Resistance to Puccinia helianthi Schw

Sunflower rust, caused by Puccinia helianthi Schw., is a widespread disease of sunflower (Helianthus annuus L.) in China. To study physiological races, sunflower field surveys were undertaken in major sunflower growing areas of China in 2010. Forty‐four rust‐infected sunflower leaf samples were collected from 25 geographical locations. Freshly produced spores were used to study physiological race differentiation on a set of nine differentials. Race 300 was the most prevalent race observed over all locations with a 59% frequency followed by races 735, 310, 500, 724 and 737. To evaluate hybrids and varieties for resistance screening, spores of race 300 were used to inoculate 65 hybrids, and five open‐pollinated varieties selected from breeding programmes and from the seed market. None of the confection hybrids and open‐pollinated varieties was immune to race 300. Conversely, among oilseed hybrids, 3% of them showed immunity, 12% highly resistant, 59% resistant and 26% showed susceptible reactions. Open‐pollinated varieties were the most susceptible to race 300 followed by confection and oilseed sunflower hybrids. Results from this study are projected to assist breeders in selection of hybrids and varieties against prevalent race as our results showed a diversity of resistance levels to race 300.     

The infection process, sporulation and survival of Alternaria helianthi on sunflower

Electron microscopy was used to study the infection of sunflower leaves by Alternaria helianthi. Conidia germinated by producing one to many germ tubes which grew across the leaf surface before forming appressoria. The fungus directly penetrated its host through the cuticle and epidermis. Entry into the host through wounds and stomates was also observed. Extracellular sheaths were found to be associated with germ tubes and intercellular hyphae of A. helianthi. Conidiophores developed through collapsed stomates, from leaf veins, trichomes and also from mycelium growing across the host leaf surface. Microcylic conidia were produced directly from parent conidia under certain conditions. Studies using a volumetric spore trap showed that the airborne spore concentration followed a distinct periodicity with peaks occurring between 0900 and 1100 h each day. Laboratory studies showed that safflower, noogoora burr and bathurst burr could serve as alternative hosts for A. helianthi. The pathogen was readily isolated from sunflower crop debris from a diseased crop that had been harvested 1 yr earlier.     

Effect of carbendazim and mancozeb combinationon Alternaria leaf blight and seed yield in sunflower (Helianthus annus L.)

Abstract

Leaf blight caused by Alternaria helianthi (Hansf.) Tubaki & Nishihara, is the major disease of sunflower affecting the successful cultivation across India. Five individual fungicides and two combination fungicides were evaluated against this pathogen in laboratory and in field experiments. Among them, the combination of carbendazim + mancozeb completely (100%) inhibited the mycelial growth of A. helianthi, irrespective of the concentrations tested followed by carbendazim alone and metalaxyl + mancozeb under in vitro condition. In field conditions, the combination of carbendazim + mancozeb was found to be highly effective in reducing the leaf blight disease of sunflower in all the three experiments as compared to other fungicides and unsprayed control. The reduction of Alternaria leaf blight was also directly associated with an increase in seed yield. The economics of the fungicides spray has been worked out and the benefit cost ratio for the combination of carbendazim + mancozeb at 2.0 g/l was 7.1 as compared to unsprayed control. The overall analysis of the results revealed that the combination of carbendazim + mancozeb at 2.0 g/l can be used for the management of foliar diseases such as Alternaria leaf spot/blight in agricultural crops.     

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers—ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10?pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.     

Analysis of Cultural and Genetic Diversity in Alternaria helianthi and Determination of Pathogenic Variability Using Wild Helianthus Species

The study aims at assessment of morphological, molecular and pathogenic variability of Alternaria helianthi, incitant of leaf blight of sunflower. Morphological characteristics determined for 26 isolates of A. helianthi from India revealed variations in shape of culture, pigmentation, conidial measurements, number of septa and colony growth. The conidia of isolate Ah‐7 were long, while conidia of isolate Ah‐15 were short. Based on cultural characters, isolates were classified into four groups. Genetic variability of the isolates was assessed by random amplified polymorphic DNA analysis. Good polymorphism was observed and cluster analysis indicated presence of six genetically distinct groups among the isolates. The isolates Ah‐1, Ah‐7 and Ah‐14 were genetically distinct. Resistant sources are not available in cultivated sunflower, while wild Helianthus species possess resistance to multiple stresses. We evaluated reaction of wild Helianthus species to isolates of A. helianthi. Among wild Helianthus species, H. tuberosus followed by H. occidentalis showed moderately to highly resistant reaction to all the isolates and recorded less disease incidence. The species H. argophyllus followed by H. laevigatus showed more disease incidence. The cultivated sunflower recorded susceptible reaction to most of the isolates and recorded high disease incidence. The isolates differed significantly for pathogenic reaction and were grouped into three pathogenicity groups; low, medium and high. Six isolates induced <20% disease incidence and were included in the low pathogenicity group. Majority of isolates were in the medium pathogenicity group. Six isolates i.e. Ah‐9, Ah‐10, Ah‐18, Ah‐20, Ah‐24 and Ah‐26 induced more than 50% disease incidence and were considered high pathogenicity group. Our results demonstrate the existence of considerable variation in resistance of Helianthus species to A. helianthi and also in morphological and genetic characters of A. helianthi isolates prevalent in India.     

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide. Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P. infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable. Quantitative resistance of foliage to late blight was evaluated in five F₁ hybrid families originating from crosses among seven different diploid potato clones. Tuber resistance was evaluated in four of the families. Two of the families were scored for both foliage maturity and vigour. The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed. QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance. Two of those segments also have major R genes for resistance to late blight. The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers. One QTL was detected for tuber resistance and one for foliage vigour. Two QTLs were mapped for foliage maturity. Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato. Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour.     

Comparative genetic analysis of quantitative traits in sunflower ( Helianthus annuus L.) 1. QTL involved in resistance to Sclerotinia sclerotiorum and Diaporthe helianthi

Sclerotinia sclerotiorum and Diaporthe helianthi are important pathogens of sunflower ( Helianthus annuus L.). Two hundred and twenty F2-F3 families were developed from an intraspecific cross between two inbred sunflower lines XRQ and PSC8. Using this segregating population a genetic map of 19 linkage groups with 290 molecular markers covering 2,318 cM was constructed. Disease resistances were measured in field experiments during 3 years (1998, 1999 and 2000) for phomopsis and 2 years for S. sclerotiorum (1997 and 1999). QTL were detected using the interval mapping method at a LOD threshold of 3. A total of 15 QTL for each pathogen resistance were detected across several linkage groups, confirming the polygenic nature of the resistances. These QTL explained from 7 to 41% of the phenotypic variability. The QTL for phomopsis resistance, in the 3 years of tests, mapped in the same region, and this was also true for some forms of S. sclerotiorum resistance in the 2 years of tests. On linkage group 8, QTL affecting resistance to both S. sclerotiorum and D. helianthi mycelium extension on leaves colocalised, suggesting a common component in the mechanism of resistance for these two pathogens. The colocalisation of QTL and breeding for resistance to S. sclerotiorum and to D. helianthi by pyramiding QTL in sunflower are discussed.     

Cytological Observations of the Infection Process by Phomopsis helianthi (Munt.-Cvet) in Leaves of Sunflower

The infection process of Phomopsis helianthi and the specific degradation of infected tissue were studied in detail using light and transmission electron microscopy. In comparison with other vascular pathogens, the infection and degradation process was in some aspects different. The favourite tissue for the pathogen to grow in was the phloem. Parenchymatic cells in and around vascular bundles were extremely sensitive to infection long before hyphae arrived, probably due to a toxin. In the parenchymatic cells the first changes were visible at the chloroplasts where electron-dense material accumulated in the thylakoid space. The chloroplast stroma changed contrast and later the whole cytoplasm also appeared electron dense. In the vascular bundles, first the phloem was destroyed and then hyphae invaded the adjacent mesophyll, the cambium, and finally the vessel elements. In particular, the compact mesophyll of the midvein was severely affected. Vessel elements were lined with electron-dense material and some were filled with flocculent material. Severe wall destruction indicated the action of a complete set of cell wall-degrading enzymes before hyphae entered the tissue; it always started at the innermost wall layer. Wall degradation in vascular tissue and adjacent parenchyma with intercellular spaces was different. Before the degradation of the protoplasts started, the cell walls were completely metabolized and only the secondary walls of the vessels resisted for longer. There were no host–cell reactions visible that could be interpreted as a defence reaction.     

Alternaria metabolites in sunflower seeds

The alternariol and alternariol monomethyl ether contamination in sunflower seeds was determined. The levels of alternariol found ranged between 35 to 792 µg/kg and 90 to 630 µg/kg for alternariol monomethyl ether. The fungicides showed different effect on the mycotoxin production depending on the substrate, strains and toxin analysed. Mercury phenyl acetate, Octave, Metiram, Thiram and Orthene inhibited alternariol monomethyl ether production while for alternariol production only Thiram, Metiram and Octave were effective.     

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.     

Detection of quantitative trait loci for resistance to gastrointestinal nematode infections in Creole goats

This study aimed to identify regions of the genome affecting resistance to gastrointestinal nematodes in a Creole goat population naturally exposed to a mixed nematode infection (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum) by grazing on irrigated pasture. A genome‐wide quantitative trait loci (QTL) scan was performed on 383 offspring from 12 half‐sib families. A total of 101 microsatellite markers were genotyped. Traits analysed were faecal egg count (FEC), packed cell volume (PCV), eosinophil count and bodyweight (BW) at 7 and 11 months of age. Levels of activity of immunoglobulin A (IgA) and activity of immunoglobulin E (IgE) anti‐Haemonchus contortus L3 crude extracts and adult excretion/secretion products (ESPs) were also analysed. Using interval mapping, this study identified 13 QTL for parasite resistance. Two QTL linked with FEC were found on chromosomes 22 and 26. Three QTL were detected on chromosomes 7, 8 and 14 for eosinophil counts. Three QTL linked with PCV were identified on chromosomes 5, 9 and 21. A QTL for BW at 7 months of age was found on chromosome 6. Lastly, two QTL detected on chromosomes 3 and 10 were associated with IgE anti‐L3, and IgE anti‐ESP was linked with two QTL on chromosomes 1 and 26. This study is the first to have identified regions of the genome linked with nematode resistance in a goat population using a genome scan. These results provide useful tools for the understanding of parasite resistance in small ruminants.     

Components of Resistance to Early Blight in Four Potato Cultivars: Effect of Leaf Position

Components of early blight resistance were quantified in leaves of different ages in four potato cultivars. The components of resistance: incubation period (IP), lesion number (LN), early blight severity, lesion expansion rate (LER), latent period (LP) and spore production by lesion area (SPLA), were evaluated separately in the lower, middle and upper leaves of four potato cultivars. Plants of cultivar Aracy (resistant), Delta (moderately resistant), Desirée (susceptible) and Bintje (susceptible) were inoculated with an Alternaria solani isolate at the beginning of the flowering stage. Disease severity varied in different plant parts. In all cultivars, regardless of resistance, the smallest values of LN, and severity were recorded on the upper leaves, suggesting that young tissues are less susceptible. In cultivar Aracy, the IP was long, with small values of LN and LER and consequently, low values of early blight severity in all leaf positions were recorded. Although IP was long in cultivar Aracy, no differences between the moderately resistant cultivar Delta and the susceptible cultivars Bintje and Desirée could be detected for this component. The IP was only influenced by leaf position in cultivar Aracy. Clear differences in resistance levels among cultivars could be detected regarding LN, severity and LER. However, neither LP nor SPLA were associated with resistance level of cultivars or with leaf position. Analyses according to plant part suggest that evaluations on leaves of the middle third part are most suitable for screening for early blight resistance in potato.