Production and use of calli from yellow birch buds for in vitro assessment of virulence of Nectria galligena isolates

Field studies of perennial Nectria canker of northern hardwoods, caused by the ascomycete fungus Nectria galligena, are time-consuming since the disease develops slowly on the stem of trees. In this report, an in vitro bioassay is described for determining and comparing the virulence of different isolates of Nectria galligena by using calli produced from yellow birch buds. The technique facilitates the distinction between highly virulent and less virulent isolates of the pathogen within one week following the inoculation.     

Lytic enzymes in the autolysis of filamentous fungi

The degrees of autolysis attained by five different genera of filamentous fungi during an incubation period of 60 days, under the same culture conditions were: 87.3% for Penicillium oxalicum; 65.9% for Neurospora crassa; 62.7% for Polystictus versicolor; 51.7% for Aspergillus niger and 23.5% for Nectria galligena. N. crassa, A. niger and P. versicolor reached the end of the autolysis during this incubation period (60 days), whereas P. oxalicum and N. galligena did not.The excretion of the lytic enzymes -N-acetyl-glucosaminidase, -1–3 glucanase, chitinase, invertase and acid phosphatase into the culture medium during growth and autolysis was investigated. The excretion of these enzymes was consistent with the degree of autolysis reached, the maximum excretion belonging to P. oxalicum and the minimum to N. galligena. The N. crassa invertase was excreted into the culture liquid at levels very much higher than the other enzymes studied, and at levels very much higher than the invertases excreted by the other fungi.     

Pith Autolysis in Herbaceous Dicotyledonous Plants. A Physiological Ecological Study of Pith Autolysis under Native Conditions with Special Attention to the Wild Plant Impatiens capensis Meerb.

Pith autolysis, in the past studied only in herbaceous cultivateddicotyledonous plants, commonly occurs in wild plants. A surveyof pith autolysis in wild plants was conducted in several differentbiomes in both North Carolina, USA and in Belgium. An objectivemethod and a subjective method were developed and used to estimatethe amount of pith autolysis in the internodes of bean (Phaseolusvulgaris L.) and the wild plant jewelweed (Impatiens capensisMeerb.) under experimental conditions. A ranking system alsowas used to quantify pith autolysis in other wild plants. Inthe field, a positive correlation was found between the rateof internode elongation and pith autolysis. Injections of sucrose solution were found to reduce the amountof pith autolysis developed in the upper internodes of jewelweedwhen compared to injections of control solution. Extracts fromthe nodal tissues of jewelweed increased the amount of pithautolysis developed by both jewelweed in the field and beanin the greenhouse. This increase in pith autolysis may be dueto a principle present in the jewelweed nodes that signals orcauses the supertending internodes to become hollow.Copyright1995, 1999 Academic Press Pith, autolysis, Phaseolus vulgaris L., Impatiens capensis Meerb., sucrose, internode, hollow stem     

The infection of apples, cv. Bramley's Seedling, by Nectria galligena Bres

In laborator experiments germinating conidia penetrated leticels and wounds but not the intact surfaces of apples. Date of harvest had no significant effect on the numbers of apples infected with Nectria galligena but the earliest picks rotted first in barn store. Inoculations of unpicked apples resulted in small arrested lesions which only developed into progressive rots after a considerable period in store. Rots developed mosy quickly from inoculations made between mid-August and mid-September. The size of rot increased with spore number and many inoculations with 10–100 conidia remained as arrested lesions. Arrested lesions developed 10–15 days after unripe apples were inoculated and consisted of a zone of fungal colonization surrounded by suberized, necrotic cells in which compounds toxic to both N. galligena and Penicillium expansum were detected. No antifungal compounds were found in progressive rots to mature apples or in healthy apples of any age. Antifungal activity, measured by inhibition of P. expansum, was greatest 15–20 days from inoculation of unripe apples with N. galligena but decreased after a total of 35 days incubation at 20 d? C. Much less antifungal activity was produced in ripe or desert apples.     

Stability of the Cell Walls of Neurospora crassa during Autolysis

The influence of autolysis upon the cell walls of Neurosporacrassa has been studied. This fungus was grown at 24 °Cin agitated and aerated cultures in a synthetic medium during60 days. At convenient intervals samples of culture were taken,mycelium separated, and dried to constant weight. From aliquotsof these mycelia cell walls were prepared, dried, weighed, andanalysed for total nitrogen, phosphorus, amino acids, lipids,and protein. No changes in the chemical composition of the wallscould be detected. The percentage of walls continuously increasedduring autolysis. These results strongly suggest that cell wallsof N. crassa are unaffected by autolysis. Examination of thefine structure of the whole mycelium at different ages duringautolysis seemed to confirm these findings.     

Gel formulations for the treatment of pruning wounds.

Gels incorporating carbendazim and triadimefon were prepared from sodium alginate, a xanthan gum or an esterified alginic acid and evaluated as wound treatments on apple and plum trees. Fungicide movement and persistence were similar with sodium alginate and xanthan gels but movement was reduced and persistence increased with the esterified material. Callus tissue formation was not inhibited by any formulation. Adding acid to fungicides to form salts had little effect on fungicide movement or persistence and caused unacceptable phytotoxicity. Thiophanate-methyl or thiabendazole were not superior to carbendazim, nor imazalil to triadimefon, for protection against Nectria galligena and Chondroster-eum purpureum respectively. Sealants containing octhilinone, carbendazim plus captan, or mercuric oxide quickly established sufficient fungicide in the wood to eradicate N. galligena but possibly insufficient to protect adequately against C. purpureum. Gel formulations are valid alternatives to sealants as fresh wound pathogen treatments but fungicide persistence is insufficient for them to be recommended for treatment against mature wound pathogens.     

Sur la dégradation de l'acide indolyl-3-acétique par Nectria galligena Bres. var. major Wr.

Summary Nectria galligena is grown in synthetic medium. Experiments are carried out with suspensions of washed mycelium, incubated and stirred with indole-3-acetic acid (IAA) or IAA 2-¹⁴C. Auxin degradation is quicker with either acid pH or young mycelium. Two indolic compounds are identified in the course of this metabolism: indole-3-aldehyde (IAld) and indole-3-carboxylic acid (ICA).A correlation is supposed to exist between the increase of auxin contained in cultures of Nectria and IAA catabolism as it lessens with age and alcalin pH.

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Dans le texte les abréviations suivantes seront employées IAA Acide Indolyl-3-acétique - IAld Indolyl-3-aldéhyde - ICA Acide Indolyl-3-carboxylique - DPH 2,4-dinitrophénylhydrazine - DMCA Paradiméthylaminocinnamaldéhyde     

The use of fungicidal paints and gels to treat existing apple cankers

Four paints containing mercuric oxide, octhilinone, thiophanate-methyl or triad-imefon and five gel formulations (carbendazim/triadimefon in sodium alginate or xanthan gum, carbendazim/triadimefon/hypophosphorous acid and thiophanate-methyl/oxycarboxin in xanthan or PP969/alginate) were tested on natural cankers caused by Nectria galligena on apple trees. Scraping cankers decreased their size 7–9 months later due to stimulation of callus formation, even where no fungicide was applied, and decreased perithecial, but not conidial production. Only the paints were applied to scraped cankers and that containing mercuric oxide was the most effective in decreasing canker size and restricting new canker growth. All formulations except the octhilinone paint were applied to unscraped cankers. Only mercuric oxide paint and acidic carbendazim/triadimefon/xanthan gel stimulated callus formation and only the former caused a decrease in canker size. Every treatment, except triadimefon paint and PP969/alginate, reduced conidial production. Bioassay with TV. galligena showed that the carbendazim and thiophanate-methyl gels achieved some movement of fungitoxicant into plant tissue.     

Control of apple canker using fungicidal paints and gels

Paints containing mercuric oxide or thiophanate-methyl were applied to either scraped or unscraped cankers caused by Nectria galligena on apple trees at two sites. Three gel formulations (carbendazim/triadimefon in alginate or xanthan and carbendazim/imazalil in xanthan) and a solvent-based PP969 formulation were applied to unscraped cankers only. Assessments were made 9 and 21 months after treatment. Mercuric oxide was more effective than thiophanate-methyl after 21 months on both scraped and unscraped cankers. All gel formulations reduced spore production and fungitoxicant could still be detected in bark and wood after 21 months. The solvent-based PP969 formulation did not perform as well as the gels.     

The seasonal release of spores of Nectria galligena from apple cankers in Northern Ireland

Trapping of ascospores and conidia of Nectria galligena Bres. released from cankers on Bramley's Seedling apple trees over a period of three years showed that ascospores were most prevalent in the spring and early summer, and conidia from early summer to late autumn. Few ascospores were released in late summer or mid-winter but a minor period of discharge occurred in autumn. The distribution of cankers within trees in various orchards was also examined, and most were found to occur at the junctions of different seasons' growth. Possible reasons for this are discussed.     

An assessment of some new treatments for the control of rotting of stored apples

Benomyl (0.025% a.i.) and thiabendazole (0.05 % a.i.) applied in July, August and September to apple trees cvs. Sunset and Cox's Orange Pippin gave slightly better control of Gloeosporium spp. rots in stored fruit than captan (0.1% a.i.) applied at similar intervals. On Cox's Orange Pippin, treatment with captan at 0.1 % a.i. in July and at 0.2% a.i. in August was as effective as three sprays at 0.1 % a.i. in July, August and September in controlling both Gloeosporium spp. and Nectria galligena rots. Benomyl applied in the spring and early summer to control apple scab on orchard trees reduced the incidence of Gloeosporium spp. and Monilia fructigena in stored fruit. Pre-harvest sprays of benomyl and captan gave good control of rotting caused by N. galligena but thiabendazole was ineffective. Post-harvest dips of thiabendazole controlled rotting by Gloeosporium spp. butnot byN. galligena.     

The effect of store conditions on the rotting of apples, cv. Bramley's Seedling, by Nectria galligena

The log of the time interval between inoculation with Nectria galligena in October and the onset of rotting in apples held in air was proportional to the deficit between the temperature of incubation and 25°C, but temperature did not affect the rate of subsequent rot expansion. Rots expanded equally fast whether apples were held in dry or moist air. The quantity of rotted tissue obtained after incubating inoculated apples in atmospheres containing up to 12.5% CO₂ increased with increasing concentrations of CO₂ greater than 2.5%. The quantity of rotted tissue obtained in apples incubated in 10% CO₂ was three times as great as that obtained after incubation in air. The incidence of natural rots was lower in apples stored at 4% CO₂ than in those stored in air and rotting increased with increasing concentrations of CO₂ higher than 4%. Colonies of N. galligena grew faster on malt agar plates incubated in 5% CO₂ than in air, but growth was slower in 10% CO₂ than in air. The quantity of benzoic acid per mg hyphae accumulated in developing lesions was similarly related to the CO₂ concentrations up to 2.5% but decreased at higher concentrations, and the quantities found in apples stored in CO₂ concentrations >5.0% CO₂ were less than in those stored in air.     

Influence of Temperature on the Autolytic Phase of Growth of Aspergillus niger

The variation in the extent to which autolysis of Aspergillusniger van Tieghem occurs in response to alteration of temperatureand of the length of the period of autolysis has been studied.The degree of autolysis at 10, 15, 20, 25, 30 and 35 °Cwas 15–8, 23–9, 35–2, 40–8, 49–1and 56–2 per cent, respectively. Autolysis increased sharplywith the length of the period of autolysis reaching a plateautowards the second day at the lowest temperatures (0–20°C) and towards the fourth day at the higher temperatures.Theextent of the increase of autolysis plotted against temperatureindicates a linear relationship between these two parameters.The release of protein from autolysing mycelium of A. nigeris greatly affected by temperature, amounting to 1–9 percent at 10 °C, whereas at 30 °C it reached 5–1per cent. On the other hand, gradual increase of temperatureduring autolysis seems not to alter the content of mycelialcarbohydrates of A. niger.     

Magnetic capture hybridisation for improved PCR detection ofNectria galligena from lignified apple extracts

In order to reduce the effects of inhibitors present in DNA extracts from lignified apple tissues, a magnetic capture-hybridisation PCR (MCH-PCR) technique was developed forNectria galligena using the ITS 1 region of the rRNA gene repeats as target. The trapping reagent used to coat the magnetic beads was an 81 bp single-stranded DNA oligonucleotide biotin-labelled on the 5é-terminal and designed to be complementary to part of the rRNA gene ITS 1 region ofN. galligena. For specificity, the probe was located from 14 bp downstream from the 3é-terminal nucleotide of theN. galligena forward primer Ch1 to the last ITS 1 nucleotide immediately upstream of the 5.8S rRNA gene. Following hybridisation in a total DNA extract of woody tissue, magnetic recovery of the bead-oligomer-template conjugate separated target template from other DNA species and inhibitory compounds. Magnetic capture-hybridisation was followed by PCR amplification with the previously designed species-specific primers, Ch1 and Ch2. Application of the MCH-PCR technique resulted in increased levels of sensitivity and reliability when compared to PCR without MCH when used on total DNA extracts from lignified tissues.     

Pith Autolysis in Herbaceous, Dicotyledonous Plants: Experimental Manipulation of Pith Autolysis in Several Cultivated Species

Pith autolysis, a condition in which dicotyledonous herbaceousplants have a hollow stem, results from the autolysis of a plant'sstorage pith. Our central hypothesis concerning the aetiologyof pith autolysis states that the carbon from the pith is transportedto the growth regions of the plant and used at times when theplant cannot meet its carbon needs by photosynthesis alone.According to this hypothesis, accelerated growth should increasepith autolysis. We here provide supporting evidence for thecentral hypothesis. More pith autolysis was found in fastergrowing tomato varieties than in dwarf varieties. More pithautolysis was found in both beans and tomatoes treated withGA₃ than in controls. More pith autolysis was found in leggybean plants grown in low light than in normal plants grown undernormal light conditions. Pith autolysis decreased in both beansand tomatoes when mechanically perturbed or sprayed with paclobutrazol,both treatments that reduced growth. The stems of buckwheatplants that were flowering showed greater pith autolysis andtherefore were more hollow than plants which were not floweringor which had the incipient flowers pinched off. This indicatedthat carbon from the storage pith may also be used in the formationof reproductive structures which require extra carbon. Alsoin support of the central hypothesis is the prevention of pithautolysis by the addition of extra carbon to the plant, in theform of an increased CO₂ concentration of the surrounding air.Copyright1995, 1999 Academic Press Bean, tomato, buckwheat, pith autolysis, CO₂, GA, thigmomorphogenesis, packobutrazol     

Ultrastructure of Paraquat-treated Pine Cells (Pinus elliottii Engelm.) in Suspension Culture

The ultrastructure of liquid suspension cultures of Pinus elliottiiwas studied, noting characteristics of dividing and senescentcells. The cultures were treated with 0.01, 0.1, 1.0, and 10.0mg l^–1 paraquat, an herbicide which stimulates oleoresinsynthesis and resinosis in the xylem of treated pine trees.The ultrastructural effects of the toxin were studied at eachparaquat concentration over a period of 24 days. Destructiveeffects of paraquat on vacuolar and organelle membranes andcytoplasm are observable in one day in cells supplied with 10.0mg l^–1. At 1.0 mg l^–1 vacuolar membranes and cellorganelles are present at seven days, but the cultures continuallydecline until at 14 days they are dead. At concentrations of0.1 and 0.01 mg l^–1 the effects of the toxin on fine structurecannot be separated from normal cell senescence. At low concentrationsof paraquat the amyloplastids accumulate large amounts of starch.No accumulation of oleoresin was detected. The ultrastructuralobservations are correlated with physiological studies in suspensionculture and in living trees. Pinus elliottii Engelm., slash pine, suspension culture, oleoresin, amyloplastids, membrane permeability, autolysis, vacuoles, senescence, tannin, paraquat     

Relationship of Endopolygalacturonase Inhibitor Activity to the Rate of Fungal Rot Development in Apple Fruits

An inhibitor extracted form the cell walls of apple fruits suppressed the activity of endopopygalacruronases (endo-PGs) produced in vivo and in vitro by Nectria galligena, Phomopsis mali, Fusarium Lateritium and Glomerella cingulata but not the endo-PGs produced by Penicillium expansum or Phytophtobora syringae. Of four apple cultivars tested Granny Smith tissue contained the highest levels of inhibitor and Cox's Orange Pippin contained the least. Linear rot expansion in the four apple cultivars inoculated with N. galligena was inversely related to inhibitor activity in the fruit tissue, rot development being slowest in Granny Smith fruits and most rapid in Cox's Orange Pippin fruits. Rot expansion in fruits inoculated with P. expansum bore no such relationship to inhibitor activity in the tissue Apple tissue maceration by the endo-PGs from N. galligena, P. mali. F. lateritium and G. cingulata was similarly related to inhibitory activity in the fruit. The properties of the partially purified inhibitor were consistent with it being proteinaceous but the relative slowness with which it was hear inactivated and the presence of a small percentage of carbohydrate might indicate that it was a glycoprotein.     

Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture     

Activity of Some Hydrolytic Enzymes in Autolysis of the Embryo Suspensor in Tropaeolum majus L.

Assay of amino-peptidase, acid and alkaline phosphatase, esteraseand ß-glucosidase activity in the haustoria and suspensorof Tropaeolum majus embryos at different stages of developmentis made to evaluate the function of hydrolases in autolysisof the suspensor. Enzyme activities rise to a maximum sometimebetween heart-shaped and cotyledonary phases of embryo development. Tropaeolum majus L. nasturtium, embryology, suspensor, autolysis, hydrolytic enzymes