Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Isolation of Solanapyrones A, B and C from Culture Filture and Spore Germination Fluids of Ascochyta rabiei and Aspects of Phytotoxin Action

Nine isolates of the fungus Ascochyta rabiei have been assayed for their ability to produce solanapyrone toxins. All isolates formed solanapyrone A, B and C which were secreted into the culture medium. Pronounced production of the toxins only occurred after onset of sporulation. The identification of the fungal products was achieved by cochromatography (TLC, HPLC), ¹H-NMR (solanapyrone A and B) and mass spectrometry (solanapyrone B). Work with A. rabiei isolate X showed that cultivation in chickpea seed extract medium in a surface culture provided best conditions for maximal toxin production. The accumulation of solanapyrones over the growth cycle was monitored. Germinating spores produced solanapyrones C and B whereas solanapyrone A was formed from the 6th day of the culture period on. Application of a mixture of solanapyrones A, B and C to leaflets of intact plants from an A. rabiei resistant cultivar (ILC 3279) and a susceptible cultivar (ILC 1929) led to characteristic changes in leaf morphology which had earlier been obsevad in susceptible plants following infection with spores of A. rabiei. Attempts to demonstrate the occurrence of toxins in the infected leaf were unsuccessful. Application of solanapyrones to solanapyrones to chickpea cell suspension cultures (derived from both cultivars) led to pronounced losses in viability and to plasmolysis of cells.     

Infection Studies of Cicer arietinum (L.) with GUS-(E. coli β-glucuronidase) Transformed Ascochyta rabiei Strains

Ascochyta rubiei (Pass) Labr. was transformed with the GUS-(β-glucuronidase) reporter gene of E. coli. Integration of vector-DNA into the A. rabiei genome was documented by Southern analysis. To verify that the transformation process had not adversely affected biochemical traits potentially important for fungal pathogenicity. i.e. hydrolytic exoenzymes. the extent of solanapyrone-toxin production and phytoalexin degradation were measured, Significant differences between the transformants and the wildtype were not observed. Transformants also showed the same degree of virulence as the wildtype A. rabiei strain. Histological studies with the GUS-transformed genotypes were performed and the time course of infection was monitored by light microscopy. A. rabiei penetrates its host directly through the cuticle and penetration through hydathodes has also been found. After penetration. A. rabiei spreads mainly in the apoplast. When growing through leaflets towards petioles, the fungus could mainly be found in the apoplast and in the cells of phloem, but rarely in xylem. The infection process leads to a total collapse of plant tissue with extensive formation of pycnidia near the vascular tissue.     

The Influence of Human Growth Hormone (GH) and Thyroxine (T4) on the Differentiation of Adipose Tissue in the Fetus

Late term fetuses from genetically obese dams have slightly larger fat cells, greater adipose tissue lipoprotein lipase (LPL) activities, elevated levels of thyroid hormones, and depressed growth hormone (GH) levels when compared to fetuses from lean dams. We have investigated the influence of thyroid hormone and GH status per se on these and other adipose tissue traits by chronically treating hypophysectomized (hypox) fetuses (day 70) between day 90 and 105 of gestation with either thyroxine (T4) or human GH. Treatment with T4 decreased body weights (P<.05), increased serum T₄ levels (P<.05), and enhanced skin and hair development (P<.05). Quantitative analysis of sections of perirenal and subcutaneous adipose tissue indicated that T₄ increased LPL activity (P<.05), slightly increased fat cell size, and more than doubled (P<.05) lipid accretion. A hypox induced deficit in fat cell cluster number in the outer layer of subcutaneous tissue was normalized by T₄ (P<.05). Conversely, human GH (hGH) treatment had no influence on body weight, increased serum hGH levels, decreased fat cell size (P<.05) and LPL activity (P<.05) but had no influence on lipid accretion. Quantitative analysis of adipose tissue sections provided direct and indirect evidence of a “critical” or “sensitive” period between 90 and 105 days, since fetal hypox at day 70 severely impeded preadipocyte recruitmentheplication during this period. Furthermore, T₄ but not GH effectively normalized this hypox-induced deficiency in preadipocyte development. Therefore, T₄ may have a major role in preadipocyte recruitmentheplication during late fetal life.     

RAPD Analysis of Pathogenic Variability in Ascochyta rabiei

Thirty Italian isolates of the phytopathogenic fungus Ascochyta rabiei (Pass.) Labr., the causal organism of Ascochyta blight on chickpea (Cicer arietinum L.), were analysed by a random oligonucleotide primer dependent polymerase chain, reaction (PCR) technique called random amplified polymorphic DNA analysis (RAPD) using three decamer primers. In previous investigations these isolates had been differentiated in six pathogenic groups. RAPD results were summarized in an analysis using the program PAUP. With each of the primers several amplification products were observed which were common to all isolates. The results of the RAPD analyses also showed that all isolates could be identified by a unique RAPD pattern. No correlation between RAPD patterns and the division of the isolates in pathogenic groups could be established. The application of the RAPD technique for cataloguing isolates and to obtain specific genetic markers for all isolates of the species Ascochyta rabiei is discussed.     

Degradation of the pterocarpan phytoalexin medicarpin by Ascochyta rabiei

Four strains of Ascochyta rabiei pathogenic to chickpea (Cicer arietinum L.) were shown to efficiently degrade medicarpin (3-hydroxy-9-methoxypterocarpan), the main phytoalexin of this plant. Degradative studies were performed with mycelium preparations or with crude protein extracts of the fungus. Isolation and structural elucidation of 10 catabolites by chromatographic and spectroscopic techniques revealed that medicarpin degradation involves 1. reductive conversion to a 2-hydroxyisoflavan, 2. O-demethylation, 3. aromatic hydroxylation in ring A and 4. formation of a 1a-hydroxy-pterocarp-1,4-diene-3-one. As terminal aromatic catabolite 2,4-dihydroxybenzoic acid was found. A catabolic sequence for medicarpin is postulated and the results are discussed with regard to pterocarpan dissimilation by other phytopathogenic fungi.     

Characterization and Identification of an Epidemic Strain of Ascochyta rabiei on Chickpeas in Northwest China

The chickpea (Cicer arietinum L.) is the primary cultivated rain fed crop in the Xinjiang region of Northwest China. In 2007, chickpeas suffered an epidemic disease that caused significant economic losses. To identify the causative agent, debris and seeds of infected chickpea plants were collected from cultivated fields. We isolated and purified the responsible pathogen and tested its pathogenicity both in greenhouse and in the field. Morphological analysis using tissue culture as well as molecular techniques confirmed that the pathogen was the blight fungus Ascochyta rabiei.     

UP-PCR Genotyping and rDNA Analysis of Ascochyta pisi Lib

Isolates of Ascochyta pisi, previously identified as separate pathotypes, were genotyped by rDNA-RFLP and UP-PCR using 8 UP-primers and 2 arbitrary primers individually or in pair-wise combinations. The appearance of polymorphic UP-PCR products for all studied isolates facilitated their differentiation. The markers were suitable in identifying isolates, and may be useful for developing isolate- or pathotype-specific PCR-based diagnostic assays. In studying genetic relatedness by UP-PCR among A. pisi isolates, two distinct clusters were revealed. rDNA analysis of the isolates based on endo-ntjclease digestion of amplified ITSl and 1TS2-25S nuclear rDNA regions did not separate the isolates, whereas the variabihty of the isolates using UP-PCR was more than 40%.     

Virulence diversity of Ascochyta rabiei the causal agent of Ascochyta blight of chickpea in the western provinces of Iran

Chickpea is the third most important food legume in the world. The most important limiting factor for the chickpea production in the world, including Iran, has been the Ascochyta blight. The pathogenic variation of 40 Ascochyta rabiei isolates from the western provinces of Iran was assessed on eight chickpea differential lines. The results revealed that A. rabiei population is diverse in the western provinces of Iran and the virulence rating of isolates across differential lines showed a large but continuous pathogenic variability. Based on the statistical analysis and the continuous response in differential lines, it was not possible to categorise A. rabiei isolates in the present study into pathotypes or races. Information obtained from the current study can be valuable in developing quarantine methods aimed to prevent dissemination of highly virulent isolates and in the development of durable resistant cultivars against the Ascochyta blight of chickpea.     

Peroxidase: a marker for Ascochyta blight resistance in chickpea

On the basis of incidence of appearance of Ascochyta blight symptoms after artificial inoculation of 25-day-old chickpea seedlings with 10 different pathotypes of Ascochyta rabiei, GL94011, PBG5 and C214 have been classified as resistant, moderately resistant and susceptible, respectively, to Ascochyta blight. In none of the pathotypes, peroxidase (PO) activity could be detected in culture medium and mycelium. Healthy tissues of GL94011 have almost three times the PO activity in comparison with that of C214. Resistant and moderately resistant genotypes showed 30–60% upregulation of PO activity against infection by A. rabiei whereas it was only 3–6% in susceptible genotype C214. These results indicate the possibility of using PO as a marker of Ascochyta blight resistance.     

Studies on the survival of Mycosphaerella pinodes and Ascochyta pisi

Laboratory and field experiments on two pea-pod and leaf-spot pathogens, Mycosphaerella pinodes and Ascochyta pisi, have indicated that the former species may survive overwinter in soil whether introduced as pycnidiospores, mycelium, chlamydospores or sclerotia. The survival of pycnidiospores appears to be due at least in part to their transformation into chlamydospores in the soil. A. pisi was much less successful in soil; the mycelium inoculum survived, somewhat sporadically, and spore viability was reduced, while the total number of spores declined markedly. Investigations on the competitive saprophytic ability of these pathogens showed that by the agar plate method M. pinodes could be classed as a moderately successful saprophyte, though the Butler and Park methods indicated only intermediate and poor success respectively. A. pisi was adjudged a poor saprophyte by all three methods. Saprophytic growth and survival in soil of both fungi was generally better at 2 °C than at 15 °C.     

Association of Phoma aquilina and Ascochyta pteridis with curl-tip disease of bracken

Many spores of Phoma aquilina and Ascochyta pteridis were rinsed from dry infected litter obtained from a site where curl tip was prevalent. More spores were released from litter which had been incubated under moist conditions for 24 h before rinsing. P. aquilina, known to cause curl-tip symptoms in bracken when inoculated via wounds (McElwee, 1983), did not damage healthy plants when spores were applied to intact tissues. A. pteridis appeared to infect undamaged plants under humid conditions causing the initial ‘fleck’ symptoms associated with the disease. Flecks formed on plants by A. pteridis may permit subsequent infection by P. aquilina. For bracken control, treating plants with fungal inoculum in disease-free areas may result in disease if inoculum supplements can be found which permit initial infection by A. pteridis or damage plants sufficiently to allow colonisation by P. aquilina directly.     

The Expression of Peroxisome Proliferator‐Activated Receptor γ in Pig Fetal Tissue and Primary Stromal‐Vascular Cultures

Objective: This study was designed to determine when peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in developing fetal adipose tissue and stromal‐vascular adipose precursor cells derived from adipose tissue. In addition we examined developing tissue for CCAAT/enhancer‐binding protein β (C/EBPβ) expression to see if it was correlated with PPARγ expression. Pituitary function and hormones involved with differentiation (dexamethasone and retinoic acid) were also tested for their effects on PPARγ expression to determine if hormones known to affect differentiation also effect PPARγ expression in vivo and in cell culture. Research Methods and Procedures: Developing subcutaneous adipose tissues from the dorsal region of the fetal pig were collected at different gestation times and assayed using Western blot analysis to determine levels of PPARγ and C/EBPβ. Hypophysectomy was performed on 75‐day pig fetuses and tissue samples were then taken at 105 days for Western blot analysis. Adipose tissue was also taken from postnatal pigs to isolate stromal‐vascular (S‐V) cells. These adipose precursor cells were grown in culture and samples were taken for Western blot analysis to determine expression levels of PPARγ. Results: Our results indicate that PPARγ is expressed as early as 50 days of fetal development in adipose tissue and continues through 105 days. Expression of PPARγ was found to be significantly enhanced in adipose tissue from hypophysectomized fetuses at 105 days of fetal development (p < 0.05). C/EBPβ was not found in 50‐ or 75‐day fetal tissues and was found only at low levels in 105‐day tissues. C/EBPβ was not found in hypophysectomized (hypoxed) 105‐day tissue where PPARγ was elevated. S‐V cells freshly isolated from adipose tissue of 5‐ to 7‐day postnatal pigs showed the expression of PPARγ1. When S‐V cells were cultured, both PPARγ1 and 2 were expressed after the first day and continued as cells differentiated. High concentrations of retinoic acid decreased PPARγ expression in early S‐V cultures (p < 0.05). Discussion: Our data indicate that PPARγ is expressed in fetal adipose tissue very early before distinct fat cells are observed and can be expressed without the expression of C/EBPβ. The increase in PPARγ expression after hypophysectomy may explain the increase in fat cell size under these conditions. Adipose precursor cells (S‐V cells) from 5‐ to 7‐day postnatal pigs also express PPARγ in the tissue before being induced to differentiate in culture. Thus S‐V cells from newborn pig adipose tissue are probably more advanced in development than the 3T3‐L1 cell model. S‐V cells may be in a state where PPARγ and C/EBPα are expressed but new signals or vascularization are needed before cells are fully committed and lipid filling begins.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Ascochitine Production by Fungi Responsible for Ascochyta Diseases of Pea

Ascochitine production by 24 isolates of three fungal species causing the Ascochyta disease complex in pea plants, was examined. All eight isolates of Ascochyta pisi Lib. secreted ascochitine to liquid nutrient medium in amounts of 0.1 to 1.2 mg/l. However, no ascochitine was found in culture filtrates of Mycosphaerella pinodes (Berk. and Blox.) Vestergr. or of Phoma medicaginis var. pinodella (Jon.) Boerema although the aggressiveness of these two species as pathogens was shown to be higher than that of A. pisi. Individual isolates of each of the three fungal species examined did not differ in their pathogenicity.     

Effects of Commercial Fatty Acids on Cutinase Release by Ascochyta pisi

The effect of a range of commercially available C₁₆ and C₁₈ fatty acids, on the release of cutinase by Ascochyta pisi, was studied. When juniperic acid was used as sole carbon source, cutinase activity was released in the culture medium, and was markedly enhanced by post-treatment of A. pisi cultures with acetone used as enzyme extractor. Without acetone, less cutinase was naturally released in culture medium containing juniperic acid at 0.5 %, than at 0.0l % or 0.05 %. Upon post-treatment with acetone, the same level of cutinase was released with all three concentrations, thus suggesting that the enzyme was induced, but not completely released in the presence of 0.5 % juniperic acid. When ricinelaidic or ricinoleic acids were supplemented at 0.5 % to cutin in the culture medium, they strongly inhibited the release of cutinase, even with acetone post-treatment. Comparable inhibition by ricinoleic acid was also observed when juniperic acid was used as cutinase inducer, thus suggesting that not only the release, but also the production of cutinase were inhibited.     

Histological Study of Adventttious bud Formation on Lactuca Sativa Cotyledons Cultured In Vitro

Abstract

Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8^th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies.     

In vitro growth of some species of Ascochyta Lib.

Fungi from the genus Ascochyta are generally facultative saprotrophs, which cause diseases in both monocots and dicots. Over 1 000 species belonging to this genus have been identified, 18 of which infect monocot plants from the family Poaceae. This study analyses the effects of temperature and light on the growth of selected fungi which infect monocots (A. agrostidis, A. avenae, A. brachypodii, A. desmazieri, A. digraphidis, A. ducis-aprutii, A. festucae, A. graminea, A. hordei, A. hordei var. americana, A. hordei var. europea, A. hordei var. hordei, A. melicae, A. phleina, A. skagwayensis, A. sorghi, A. stipae, A. zeicola), grown on three types of media; Potato Dextrose Agar (PDA), Coon??s agar (CN) and oatmeal agar (OMA). The fastest growth among the analyzed fungi at low temperatures was found in Ascochyta melicae, while at high temperatures it was A. zeicola. The fastest in vitro growth (average of all fungi) was observed on CN medium at 20°C (3.4 mm/day), while the lowest on OM medium at 5°C (1.0 mm/day). Radial mycelial growth in dark and the light conditions varied. On average, all isolates grew faster in the dark (3.1 mm/day) than in the light (1.9 mm/day). The greatest effect on the production of pycnidia was found for the isolates. Variation in growth and production of pycnidia depended on temperature, medium and lighting for fungi from the genus Ascochyta infecting monocots. Such variation indicates a potential occurrence of these fungi in different environments.     

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

Homogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three-fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue. Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence. Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.