Regulation of the nitrate reductase inPisum sativum andtriticum aestivum by irradiation

Nitrate reductase (NR) activity of bothPisum sativum L. cv. Bonneville andTriticum aestivum L. cv. Sonalika seedlings was influenced by the phytochrome system. Short durations of “red” irradiation (R) increased extractable levels of NR whereas subsequent short “far-red” irradiation (FR) partially inhibited the R modulated increases. Qualitatively, a negative correlation existed between thein vitro NR activities andin vivo phytochrome levels inPisum. “Blue” irradiation (B) also increased extractable levels of NR inTriticum. A partial action spectrum study made by exposing excised etiolated leaves ofTriticum and shoot apices ofPisum revealed a maximum increase in extractable NR activities and tissue nitrate level (inTriticum) at 656 nm. A partial action spectrum for the extracted enzyme ofTriticum indicated that at 700 nm the level of activity was increased (as compared to dark controls) more than by R (656 nm), FR (725 nm) or B (425 or 450 nm) irradiation, although all wavelengths used increased NR activity.     

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi)

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.     

Induction of resistance against downy mildew on sunflower by rhizobacteria

Abstract

Induction of resistance to downy mildew caused by Plasmopara halstedii in sunflower was studied after treatment with PGPR (plant growth promoting rhizobacteria) strain INR7 (Bacillus spp). Treatment of sunflower seeds with 1×10⁸cfu/ml of PGPR strain INR7 resulted in decreased disease severity and offered 51 and 54% protection under green house and field conditions, respectively. The induction of resistance to P. halstedii by PGPR strain INR7 was accompanied by the accumulation of various host defense-related enzymes in susceptible sunflower seedlings. Enhanced activation of catalase (CAT), phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and chitinase (CHI) was evident at 6, 9, 12, 12 and 12h post inoculation, respectively, in sunflower seedlings raised from seeds treated with PGPR strain INR7. This enhanced and early activation of defense-related responses in the susceptible cultivar after treatment with PGPR strain INR7 was comparable to that in the resistant cultivar. The results indicate that PGPR strain INR7 induced resistance against P. halstedii in sunflower is mediated through enhanced expression of defense mechanism.     

Electron microscope autoradiography of14C photosynthate distribution at the haustorium-host interface in powdery mildew ofPisum sativum

Summary Haustorial complexes were isolated from leaves ofPisum sativum infected withErysiphe pisi and exposed to¹⁴CO₂ for 2 hours. The constituents of the isolated fraction were quantified and ultrastructurally described and the distribution of¹⁴C studied by electron microscope autoradiography and statistical treatment. Most (86%) of the isotope in the fraction was associated with haustorial complexes. Three classes of haustorial complexes were distinguished by degree of labelling and ultrastructure. Most of the haustorial complexes were termed healthy (i.e., they showed a normal ultrastructure) and were heavily labelled; necrotic complexes were unlabelled; and a class with intermediate labelling, modified extrahaustorial membrane and usually a normal haustorial cytoplasm was termed pre-necrotic. In healthy haustorial complexes the haustorial lobes and extrahaustorial membrane showed the highest grain densities and the body and extrahaustorial matrix were also significantly labelled. Comparison of the results suggest that ultrastructural modifications leading to necrosis were caused by dehydration which in turn determined reduction in photosynthate transfer. Other factors influencing transport into haustoria and the status of the extrahaustorial matrix are also discussed.U.V. fluorescence microscopy with acetamido-isothiocyanatostilbene salt was used to distinguish healthy and pre-necrotic haustorial complexes. It is recommended as a simple technique to monitor the functional quality of isolated haustorial complexes.     

Control of pearl millet downy mildew by seed treatment with metalaxyl*

Three formulations of the systemic fungicide metalaxyl were tested in various seed treatments for the control of pearl millet downy mildew in three field experiments with downy mildew-susceptible pearl millet hybrid NHB-3. Uniform, high levels of sporangial inoculum of the causal fungus, Sclerospora graminicola, were provided throughout the growth of the test crops from inoculated infector rows of NHB-3, planted earlier between the test plots. Significant reductions in downy mildew were obtained with all fungicide treatments. Best control was obtained when seed was soaked in a 0.5% aqueous solution of a liquid formulation (mean infection index of 9.8% compared with 94.8% in the untreated check). The degree of control with the wettable powder formulations was directly related to fungicide dosage, and there were no significant effects of application method. Simple dusting of seed at 2 g a.i./kg, a rapid and simple operation requiring small quantities of fungicide and no special application equipment, gave a high level of control (infection index of 12.6% compared with 78.9% in the untreated check). In two experiments grain yields from all the treated plots were significantly greater than from the untreated plots (means of 1234 and 1534 kg/ha for treated plots compared with 485 and 743 kg/ha, respectively), and in the third, the treatment with the least downy mildew gave significantly more grain than the untreated check (1228 compared with 727 kg/ha).     

Thermal imaging of cucumber leaves affected by downy mildew and environmental conditions

Pathogenesis of Pseudoperonospora cubensis causing downy mildew of cucumber resulted in changes in the metabolic processes within cucumber leaves including the transpiration rate. Due to the negative correlation between transpiration rate and leaf temperature, digital infrared thermography permitted a non-invasive monitoring and an indirect visualization of downy mildew development. Depending on the stage of pathogenesis and the topology of chloroses and necroses, infection resulted in a typical temperature pattern. Spatial heterogeneity of the leaf temperature could be quantified by the maximum temperature difference (MTD) within a leaf. The MTD increased during pathogenesis with the formation of necrotic tissue and was related to disease severity as described by linear and quadratic regression curves. Under controlled conditions, changes in temperature of infected leaves allowed the discrimination between healthy and infected areas in thermograms, even before visible symptoms of downy mildew appeared. Environmental conditions during thermographic measurement, in particular air temperature and humidity, as well as water content and age of the leaf influenced the temperature of its surface. Conditions enhancing the transpiration rate facilitated the detection of changes in leaf temperature of infected leaves at early stages of infection. As modified by environmental conditions, MTD alone is not suitable for the quantification of downy mildew severity in the field.     

A region of maize chromosome 2 affects response to downy mildew pathogens

Quantitative trait loci (QTLs) for downy mildew resistance in maize were identified based on co-segregation with linked restriction fragment length polymorphisms or simple sequence repeats in 220 F2 progeny from a cross between susceptible and resistant parents. Disease response was assessed on F3 families in nurseries in Egypt, Thailand, and South Texas and after inoculation in a controlled greenhouse test. Heritability of the disease reaction was high (around 93% in Thailand). One hundred and thirty polymorphic markers were assigned to the ten chromosomes of maize with LOD scores exceeding 4.9 and covering about 1,265 cM with an average interval length between markers of 9.5 cM. About 90% of the genome is located within 10 cM of the nearest marker. Three putative QTLs were detected in association with resistance to downy mildew in different environments using composite interval mapping. Despite environmental and symptom differences, one locus on chromosome 2 had a major effect and explained up to 70% of the phenotypic variation in Thailand where disease pressure was the highest. The other two QTLs on chromosome 3 and chromosome 9 had minor effects; each explained no more than 4% of the phenotypic variation. The three QTLs appeared to have additive effects on resistance, identifying one major gene and two minor genes that contribute to downy mildew resistance.     

Purification and characterization of proline/hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components, which are also involved in response to pathogen attack. In pearl millet, deposition and cross-linking of HRGPs in plant cell walls was shown to contribute to the formation of resistance barriers against the phytopathogenic oomycete Sclerospora graminicola. In the present study, the purification and characterization of HRGPs that accumulated in coleoptiles of pearl millet seedlings in response to S. graminicola inoculation has been carried out. Periodic acid Schiff's staining revealed that the purified protein was a glycoprotein. The protein to carbohydrate ratio was determined to be 95.5%:4.5% (w/w). Proline amounted for 20 mol% of the total amino acids as indicated by amino acid composition analysis. The isolated protein had a pI of 9.8 and was shown to be composed of subunits of 27, 17, and 14 kDa. Cross reactivity with the monoclonal antibody MAC 265 and the presence of the signature amino acid sequence, PVYK, strongly suggested to classify the purified glycoprotein as a member of the P/HRGPs class. In the presence of horseradish peroxidase and H2O2 the purified glycoprotein served as a substrate for oxidative cross-linking processes.     

The cytology of cotyledon cells and the induction of giant polytene chromosomes inPisum sativum

Summary The cotyledon cells ofPisum sativum have high DNA contents. By appropriate culture techniques, some of these cells can be triggered into division. Two types of dividing nuclei were seen. Firstly those that were polyploid with metaphases containing chromosome numbers ranging in value from 4 x to 32 x. Included among these were unexpected numbers equivalent to 12 x and 14 x. Secondly there were cells containing giant polytene chromosomes and these progressed from prophase to a metaphase where the polytene chromosomes separated into constituent single chromosomes.     

Linkage analysis of er-1, a recessive Pisum sativum gene for resistance to powdery mildew fungus (Erysiphe pisi D.C.)

Linkage analysis was used to determine the genetic map location of er-1, a recessive gene conditioning resistance to powdery mildew, on the Pisum sativum genome. Genetic linkage was demonstrated between er-1 and linkage group 6 markers after analyzing the progeny of two crosses, an F₂ population and a set of recombinant inbred lines. The classes of genetic markers surrounding er-1 include RFLP, RAPD and allozyme markers as well as the morphological marker Gty. A RAPD marker tightly linked to er-1 was identified by bulked segregant analysis. After DNA sequence characterization, specific PCR primers were designed to convert this RAPD marker into a sequence characterized amplified region (SCAR).     

Some observations on the economic importance of sugar-beet downy mildew in England

Sugar-beet downy mildew is most prevalent in England in the sugar-beet and mangold seed-growing area of South Lincolnshire and West Norfolk. The most widespread and severe recent outbreaks were in 1957, and in 1965 when 6412 acres were reported with more than 10% infected plants. The fungus usually overwinters in sugar-beet and mangold seed crops, and in England other ways of overwintering are seldom important. Steckling beds are infected in the autumn, and the disease may increase rapidly in the seed crop in early spring. Summer-sown stecklings get more downy mildew than stecklings sown in spring under a cereal cover crop, and direct-drilled seed crops get more downy mildew than transplanted crops.     

Zoosporogenesis in Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

During sporulation of Pseudoperonospora cubensis on cucumber leaves ( Cucumis saliva ) zoosporangia are formed on the dichotomously branched sporangiophore. The mature zoosporangium has a preformed discharge papilla and the cytoplasm is uncleaved. The zoosporangium wall is decorated and the outer layer of the wall is electron opaque in ultrathin sections. As the zoosporangium is able to survive freezing (- 18°C) for prolonged periods of time (3–4 months) the zoosporangium may serve as the resting structure which survives overwintering in Northern latitudes in the absence of oospore formation.
Zoospore cleavage can be synchronized by placing freshly harvested zoosporangia in distilled water. Cleavage of the zoosporangial cytoplasm is by means of the fusion of small vesicles apparently derived from dictyosomes which become highly active after zoosporogenesis is induced.
Vesicles with an osmiophilic electron opaque content are the dominant type of vesicle found in the zoosporangia. The content of these vesicles undergoes dynamic changes during zoosporogenesis and during the late stages of sporogenesis the content becomes finely striated as is typical of these vesicles when observed in the zoospore. On the basis of the results presented here it is suggested that zoosporangium formation and zoosporogenesis in P. cubensis could serve as a model system for assays with obligate oomycetous plant pathogens, also in relation to fungicide mode of action studies.     

Internal mycelium of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

The internal mycelium of Pseudoperonospora cubensis has been observed in transmission and scanning electron microscopic preparations. The internal mycelium may be inter- or intracellular. Haustoria of short swollen bundles of hyphae have been observed. Actively growing hyphae contain numerous mitochondria, nuclei, active dictyosomes, low amounts of storage materials (lipid) and microbody-like structures with a laminate inclusion. Thick walled hyphae with a diameter which is smaller than the actively growing hyphae have been observed. These thick wallcd hyphae contain large amounts of reserve material (lipid) and it is suggested that they may function as resting propagules.     

The zoospore of Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew

The zoospore of Pseudosporonospora cubensis is typical of the secondary zoospore of the Peronosporales. The reniform zoospore contains a central nucleus with a prominent beak-like extension to the kinetosomes on the lateral side of the spore in the groove region. "Fuzzy" vesicles derived from dictyosomes surround and fuse with the contractile vacuole. Mitochondria and microbodies are located in the peripheral cytoplasm of the zoospore but the latter are confined to the groove region of the spore. The microbodies usually contain a laminate inclusion and the microbodies are not in a fixed position in relation to the peripheral cisternae. Neither a microbody-lipid body complex nor a "U-body" were observed.
The kinetosomes of the spore are almost perpendicular to each other at the distal end of the beak-like extension of the nucleus. A complex system of cytoplasmic microtu-bules flare out from the kinetosomes to surround the nucleus and bundles of cytoplasmic microtubules extend under the plasmalemma of the spore. The zoospore contain numerous vesicles with osmiophilic inclusions which are finely striated; these are the so-called finger-print vesicles.     

A novel technique for greenhouse evaluation of plant extracts for management of downy mildew of sorghum

Antipathogenic potential of 16 plants was evaluated in the form of aqueous extracts against Peronosclerospora sorghi, causing downy mildew of sorghum. A novel method was developed in which conidial suspension and plant extracts were mixed individually and allowed to stand for 5?min and then used to inoculate the host by sprout-dip method. The sprouts thus inoculated were grown in pots, and disease incidence was observed. Three extracts (Parthenium hysterophorus, Duranta repens and Oxalis latifolia) suppressed the disease at par with chemical fungicide (Mancozeb 75%). In form of spray, D. repens extract with tween-20 adjuvant (1.9% disease) performed closest behind mancozeb (0% disease), while negative control showed 21% disease incidence. Organic management of airborne inoculum of downy mildew of sorghum is feasible and preferable as compared to the use of chemical fungicides, considering human and environmental health concerns. Use of water extract keeps the technology simple so that it can be directly prepared and used by farmers. Short listing of three most effective water extracts would help in self-reliance of farmers, reducing their dependence on commercial products.     

Effect of climate change on infection of grapevine by downy and powdery mildew under controlled environment

Plant responses to elevated CO2 and temperature have been much studied in recent years, but effects of climate change on pathological responses are largerly unknown. The pathosystems grapevine (Vitis vinifera) - downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necatrix) were chosen as models to assess the potential impact of increased CO2 and temperature on disease incidence and severity under controlled environment. Grapevine potted plants were grown in phytotrons under 4 different simulated climatic conditions: (1) standard temperature (ranging from 18 degrees to 22 degrees C) and standard CO2 concentration (450 ppm); (2) standard temperature and elevated CO2 concentration (800 ppm); (3) elevated temperature (ranging from 22 degrees to 26 degrees C, 4 degrees C higher than standard) and standard CO2 concentration; (4) elevated temperature and CO2 concentration. Each plant was inoculated with a spore suspension containing 5x10(5) cfu/ml. Disease index and physiological parameters (chlorophyll content, fluorescence, assimilation rate) were assessed. Results showed an increase of the chlorophyll content with higher temperatures and CO2 concentration, to which consequently corresponded an higher fluorescence index. Disease incidence of downy mildew increased when both CO2 and temperatures were higher, while an increase in CO2 did not influenced powdery mildew incidence, probably due to the increased photosynthetic activity of plants under such conditions. Considering that the rising concentrations of CO2 and other greenhouse gases will lead to an increase in global temperature and longer seasons, we can assume that this will allow more time for pathogens evolution and could increase pathogen survival, indirectly affecting downy and powdery mildews of grapevine.     

Emerging virulence arising from hybridisation facilitated by multiple introductions of the sunflower downy mildew pathogen Plasmopara halstedii

The sunflower downy mildew pathogen Plasmopara halstedii is an invasive plant pathogen in Europe of American origin. Despite efforts to produce resistant host varieties, nationwide monitoring in France has revealed the rapid emergence of new virulent races increasing the number from one founder identified in 1966 to as many as 14 today. We have genotyped 146 samples (including all 14 races) using 13 nuclear and one mtDNA marker. Samples of the same race were found to share alleles/mtDNA haplotype and the two most common races had individuals with multiple matching genotypes. Cluster analyses confirmed that the samples form three groups to which races strongly adhere. Clusters were highly differentiated (F(ST) 0.65) and characterised by high inbreeding coefficients. Despite this, samples of recently emergent races, including six that are unique to France had mixed ancestry between the groups suggesting they have arisen in situ due to hybridisation. Five such samples also had conflicting mtDNA and nuclear DNA profiles. This demonstrates that multiple introductions have aided the establishment of this pathogen in France, and suggests recombination facilitated by these introductions is driving the emergence of new and endemic races in response to host resistance.     

A cytochemical kinetic investigation of the feulgen hydrolysis of fixed chromatin

Summary Measurements of the initial slope of the Feulgen hydrolysis curve under various conditions of preparation, temperature, and acid concentration demonstrate that this slope has the kinetic properties of the reaction rate of a simple hydrolytic reaction. This result allows a considerable refinement in both the kinetic analysis of chromatin fractions and the cytophotometric measurement of DNA amounts.