Biochemical Changes in Cultures of Nectria galligena during the Autolytic Phase of Growth

Some biochemical changes occurring in cultures of Nectria galligenaduring its autolytic phase of growth have been investigated.In nitrate-grown and autolysed cultures of this fungus the degreeof autolysis amounted to 57 per cent. The amount of myceliallipids decreased continuously with the age of the culture. Totalmycelial nitrogen did not substantially change within the first50 days of autolysis. The constancy in the amount of bound aminoacids released from mycelium of Nectria galligena strongly suggeststhat mycelial protein are not affected by autolysis.     

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.     

Location of pathogenicity genes on dispensable chromosomes inNectria haematococca MPVI

Nectria haematococca MPVI can be found in many different biological habitats but has been most studied as a pathogen of pea (Pisum sativum). Genetic analyses of isolates obtained from a variety of biological sources has indicated that a number of genes control pathogenicity on pea but that one important PEa Pathogenicity (PEP) gene isPDA, which confers the ability to detoxify the pea phytoalexin pisatin. In these studies, all naturally occurring isolates that lackedPDA (i.e. Pda^– isolates) and all Pda^– progeny were essentially non-pathogenic on pea. However, we have demonstrated recently that Pda^– mutants created by transformation-mediated gene disruptions, while having a modest reduction in virulence, and more virulent than any naturally occurring Pda^– isolates. In addition we know thatPDA genes are on dispensable (DS) chromosomes in this fungus. We believed that the gene disruption mutants have allowed the detection of otherPEP genes that are present on the DS chomosomes along withPDA and that naturally occuring Pda^– isolates usually lack this DS chromosome. This would explain why naturally occurring Pda^– isolates are always low in virulence. We propose that the DS chromosomes in fungi are analogous to bacterial plasmids which allow those microorganisms to colonise different habitats, i.e. the DS chromosomes ofNectria haematococca contain genes that allow individual isolates of this broad host range pathogen to occupy different biological niches.     

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.     

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.     

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.     

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.     

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.     

Control of Mn status and infection rate by genotype of both host and pathogen in the wheat take-all interaction

Take-all is a world-wide root-rotting disease of cereals. The causal organism of take-all of wheat is the soil-borne fungus Gaeumannomyces graminis var tritici (Ggt). No resistance to take-all, worthy of inclusion in a plant breeding programme, has been discovered in wheat but the severity of take-all is increased in host plants whose tissues are deficient for manganese (Mn). Take-all of wheat will be decreased by all techniques which lift Mn concentrations in shoots and roots of Mn-deficient hosts to adequate levels. Wheat seedlings were grown in a Mn-deficient calcareous sand in small pots and inoculated with four field isolates of Ggt. Infection by three virulent isolates was increased under conditions which were Mn deficient for the wheat host but infection by a weakly virulent isolate, already low, was further decreased. Only the three virulent isolates caused visible oxidation of Mn in vitro. The sensitivity of Ggt isolates to manganous ions in vitro did not explain the extent of infection they caused on wheat hosts. In a similar experiment four Australian wheat genotypes were grown in the same Mn-deficient calcareous sand and inoculated with one virulent isolate of Ggt. Two genotypes were inefficient at taking up manganese and were very susceptible to take-all, one was very efficient at taking up manganese and was resistant to take-all, and the fourth genotype was intermediate for both characters. All genotypes were equally resistant under Mn-adequate conditions.     

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.     

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.     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.     

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.     

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     

Columnaris infection among cultured Nile tilapiaOreochromis niloticus

Flexibacter columnaris was isolated from 13 culturedOreochromis niloticus showing respiratory disorders. The isolates developed typical swarming rhizoid colonies on Cytophaga agar medium. Antibiotic sensitivity test revealed the susceptibility ofF. columnaris isolated to oxytetracycline, chloramphenicol and erythromycin. A marked difference in the pathogenicity of seven tested isolates was observed: two were highly virulent, one was moderately virulent and four were avirulent. No experimental infection could be induced with the highly virulent isolates except after injuring one of the natural barriers of the fish body. The severity of the disease and the increased median death time shortened by keeping infected fishes with injured gills in water containing ammonia.In naturally infectedO. niloticus, the disease became chronic as indicated by the presence of excessive proliferative and necrotic changes. On the other hand, severe dilatation of branchial blood vessel, oedema and round cell infiltration proved that, the disease among experimentally infected tilapias was acute.     

A new species ofNectria onMauritia flexuosa (Arecaceae) in Ecuador and a key toNectria and allied genera on palms

A new species ofNectria, N. palmicola on rachides ofMauritia flexuosa, is described from Ecuador. It is compared with the similar species.N. calami andN. pseudopezizia. Nectria palmicola is illustrated with light and SEM micrographs and line diagrams. A key and host index to species ofNectria and allied genera on palms is given.     

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.     

Fungi of the Windmill Islands, continental Antarctica. Effect of temperature, pH and culture media on the growth of selected microfungi

Studies were conducted on microfungi isolated from soils in the Windmill Islands, continental Antarctica. Growth responses of Alternaria alternata, Chrysosporium pannorum, Nectria peziza, Thelebolus microsporus, Mycelia sterile and Phoma cf. herbarum to temperature, pH and culture media were investigated. Maximum growth occurred after 16 days, except in Nectria peziza and Thelebolus microsporus, where maximum growth occurred 12 days after inoculation. All isolates showed poor growth at 0°C. Maximum growth was obtained with temperatures ranging from 15 to 25°C. The optimum temperature for all fungi was 20°C. An acid medium favoured growth. Chrysosporium pannorum, Phoma cf. herbarum and Nectria peziza grew best at pH 3–4, whereas Mycelia sterile, Alternaria alternata and Thelebolus microsporus grew best at pH 5–6. The culture medium had little effect on growth, except for nutrient agar, which showed poor growth against all isolates with the exception of Thelebolus microsporus. Received: 11 September 1996 / Accepted: 13 January 1997     

Isozyme polymorphism and virulence of Indian isolates of the rice sheath blight fungus

Inadequate information about the genetic structure of the polyphagous Rhizoctonia solani has made sheath blight resistance breeding a difficult task. To assess the variability in the Indian populations of sheath blight fungus, 18 isolates were collected from different rice growing regions of India and analyzed for virulence and electrophoretic profiles of 13 isozymes. The virulence spectrum of all 18 isolates was examined on susceptible IR50 and tolerant Swarnadhan varieties, based on which the isolates could be grouped as highly virulent, moderately virulent or a virulent. A total of 11 enzyme systems with 153 electrophoretic phenotypes were applied to characterize the genetic variation among the isolates. Cluster analyses based on isozyme patterns resulted in one major cluster comprising 16 virulent isolates, with two a virulent isolates loosely linked to this at 0.13 similarity. Isozyme systems of esterases (both and ) and 6-phosphogluconic dehydrogenase could be used to fingerprint the individual isolates.This revised version was published online in October 2005 with corrections to the Cover Date.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.