Effect of some antibiotics on Sclerotium cepivorum Berk., the cause of white rot of onion

The effect of some antibiotics onSclerotium cepivorum, the cause of white rot of onion was studied in agar culture and soil. The growth ofS. cepivorum was inhibited in Czapek Dox yeast agar containing 50µg of gliotoxin, viridin, actidione and 100µg of patulin per ml of the medium. Lower concentrations of the antibiotics retarded the growth of the fungus. In soil, patulin had no effect in the control ofS. cepivorum infection of onion seedlings. Concentration of actidione of 5µg/g of soil completely controlled white rot infection but severely stunted the growth of onion seedlings; 40µg/g of actidione killed the seedlings. Despite the importance of actidione as a fungistatic agent its use on onion is limited by its phytotoxicity.     

Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

Antagonism against fomes annosus. Comparison between different test methods in vitro and in vivo

The antagonism of Trichoderma harzianum, Scytalidium album and an unidentified fungus (isolated from Monotropa hypopitys) against the root rot fungus Fomes annosus was studied in vitro and in vivo. Antibiosis was demonstrated on malt extract agar and on powdered wood. Competition between F. annosus and its antagonists was studied in stem discs and in living stems of Norway spruce. Effective antibiotics on agar and sawdust did not always bring about effective antagonism against F. annosus in stem discs or living trees. Factors such as growth rate of the antagonists in wood and their ability to invade the substrate during competition may influence the effectiveness of F. annosus suppression. The results from studies on antagonism in stem discs agreed with the results from tests in living trees. The frequency of penetrations through the wood cell walls was drastically lowered by the presence of the antagonists but was not closely correlated with the abundance of F. annosus.     

Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method

AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.     

Germination and Infectious Potential of Microconidia of Sclerotium cepivorum

Observation with the scanning electron microscope showed that microconidia of Sclerotium cepivorum are able to germinate on onion plants. A young hypha penetrated the plant cuticle. Cytochemical techniques gave evidence of phenoloxydase and esterase activities in microconidia and thus confirmed their infectious potential. Massive production of microconidia was induced by immersion of the fungus in water. These elements suggest that in high moisture conditions microconidia of S. cepivorum might play a crucial role in survival and dissemination of the fungus.     

Medium- and Light-dependence of Growth and Conidiomata Production in Phaeocytosporella zeae Pathogenic to Maize

Culture appearance, mycelium growth and pycnidial conidiomata formation in Phaeocytosporella zeae cultivated on potato dextrose agar (PDA), Leonian agar and carnation leaf agar (CLA) during permanent dark (25 C) or a 12-h photoperiod (24/18°C) are described. Different media and light conditions significantly affected fungus growth and the occurrence of conidiomata.
Dark conditions favoured more mycelium growth of the fungus than alternating light and dark. Moreover, fungus growth was more rapid on PDA and CLA media than on Leonian agar. Carnation leaf agar and a 12-h photoperiod provided excellent conditions for the promotion of rapid conidiomata formation in a number of P. zeae isolates.     

Biological control of white rot of onion

Summary Interactions of 123 isolates of fungi, 17 of bacteria and 22 of actinomycetes, respectively, withSclerotium cepivorum were studied in agar culture. They were grouped into 4 different types of reactions. Amongst themTrichoderma viride, Fusarium graminearum, Coniothyrium minitans andGliocladium roseum inhibited the growth ofS. cepivorum and later grew over its colony.T. viride showed a characteristic coiling around the hyphae ofS. cepivorum. T. viride andF. graminearum prevented the development of sclerotia.C. minitans was found to parasitize the sclerotia ofS. cepivorum and produced its pycnidia within them.Aleurisma carnis, Cladosporium elatum, Penicillium expansum, P. nigricans, P. notatum, P. piscarium, P. puberulum, P. rolfsii, P. urticae, P. variabile, Tilachlidium humicola andHelminthosporium sp. inhibited the growth ofS. cepivorum at a distance. Eleven isolates of bacteria and 3 ofStreptomyces sp. showed pronounced antagonistic properties againstS. cepivorum.Experiments were carried out to study the effects on white rot development in soil of organisms selected from agar plate tests. None of the antagonistic micro organisms had any deleterious effects on onion growth. Of the organisms testedP. nigricans gave the best results in controlling white rot infection.This work was carried out at the Department of Botany, The University, Birmingham, England. I wish to thank Prof.C. J. Hickman for his invaluable advice and encouragement.     

Stimulation of sclerotial germination ofSclerotium cepivorum by host-plant extract

Summary Extract from onion bulbs and diffusate from roots of onion seedlings were fractionated by column chromatography. The stimulatory effects of the different fractions of onion extract on sclerotial germination ofSclerotium cepivorum were studied. The sugar fraction was the most stimulatory, whereas, the amino acid fraction was not effective. Paper chromatographic analysis revealed the presence of glucose, fructose and no amino acids in the root diffusate. These two sugars and 13 amino acids were identified in the onion extract. When various sugars and amino acids were supplied individually to autoclaved soil, only glucose, fructose, mannose and maltose effectively induced sclerotial germination. Partial stimulation occured in nonsterile soil amended with high glucose concentrations. Studies on the antibiotic effect of the different fractions against some soil fungi by the spore germination method showed that, the sugar fraction inhibits completely the spore germination of all the fungi, tested, whereas, the amino acid fraction was non-inhibitory. Both fractions did not show antibiotic activity when tested by the filter paper disc method. Attempts to extract inhibitory substances from soil which inhibit sclerotial germination were unsuccessful. It was suggested that onion extract plays a twofold role in stimulating sclerotial germination in natural soil: (a) a direct nutritional influence; (b) an antibiotic effect on soil mycoflora which reduces competition for nutrients.     

Comparison of gentamicin with other antibacterials and variation of incubation temperature on growth of Trichophyton mentagrophytes from skin scales

Neutral (Emmons modification) Sabouraud medium, Dermatophyte Test Medium and Littman oxgall agar, with substitutions in type of antibacterial antibiotic, were quantitatively evaluated by colony counts for ability to support growth when suspensions of trypsintreated guinea pig skin scales, infected withTrichophyton mentagrophytes, were used as inocula. Both liquid and powder form of gentamicin sulfate were used separately in place of other routinely used antibacterial antibiotics. Also evaluated were three isolation temperatures. No differences in ability to support growth were noted among media. No difference was noted between the two forms of gentamicin. No data were available on the antibacterial activities of the antibiotics because no bacterial growth was produced on any of the media. Room temperature (24 °–26 °C), 30 °C and 37° C were found to support growth similarly as primary isolation temperatures for this fungus.     

Studies on the nature of resistance in tomato plants to Verticillium albo-atrum

Extracts of healthy resistant and of healthy susceptible plants of tomato had the same effect on growth of Verticillium albo-atrum in vitro. Tracheal saps from resistant and from susceptible plants showed no difference in their effect on spore germination and mycelial growth of V. albo-atrum. Cuttings from resistant plants, inoculated with V. albo-atrum and fed with low concentrations of casamino acids, or glucose, at first wilted more than controls but soon recovered. Continuous treatment with dilute ethanol solutions for 2 weeks induced marked wilting in inoculated cuttings of resistant plants: treatment for shorter periods caused less severe symptoms, from which cuttings recovered slowly. Metabolic inhibitors did not break resistance of cuttings, but the pathogen survived longer in cuttings treated with sodium diethyldithiocarbamate, salicylaldoxime or 8-hydroxyquinoline than in controls. When one end of segments of stems of resistant plants was inoculated with the pathogen, and 48 h later the uninoculated end was placed near a colony of V. albo-atrum on agar, growth of the fungus colony towards the stem segment was sometimes inhibited. There was no such inhibition when segments from susceptible plants were used. Both tracheal sap and diffusates from segments of inoculated resistant plants supported less growth of germ tubes of V. albo-atrum than sap and diffusates from uninoculated plants. These differences were not obtained with the susceptible variety and production of fungitoxic substances in resistant plants after infection is inferred.     

The effect of seed treatment with benzimidazole-based fungicides on infection of the foliage of overwintered salad onions by Botrytis cinerea

Seed treatments of carbendazim (Bavistin 50% W.p.) and thiophanate-methyl (Mildothane 50% W.p.) applied to overwintered salad onions at 250 g a.i./kg seed protected the foliage of plants from infection by Botrytis cinerea during the seasons 1973–1976. Crop establishment and yield were also improved. Seed treatment with calomel was not effective. Chemical analysis of treated non-viable seeds, retrieved from the soil, indicated that 73% carbendazim and 46% thiophanate-methyl remained attached to the seeds after 9 months in the soil. Analyses of onion leaves revealed that each fungicide was represented by similar quantities of carbendazim, 5 μg/g fresh weight in October 1975 reducing to 1 μg/g fresh weight in May 1976. Bioassay tests showed that the fungicide was acropetally distributed and was present in all leaves early in the season (October) but was absent from some new leaves formed in the following spring. Carbendazim-insensitive isolates of B. cinerea occurred after three seasons' use of this chemical. Sensitive isolates failed to grow on agar containing 1 μg/ml benomyl but all insensitive isolates (31 total) grew normally at this concentration and some were capable of growth on agar containing 1000 μg/ml benomyl. The emergence of foliar isolates of the fungus insensitive to the benzimidazole-based compounds used in the treatment of seeds indicated that these fungicides did not provide a permanent solution to the disease problem.     

STUDIES OF THE BIOLOGY OF SCLEROTIUM CEPIVORUM BERK.

Sclerotium cepivorum did not grow from agar into unsterile soil, but growth of restricted duration and extent was observed from infected onion tissue. Growth depended on the food reserves and its extent was determined by the size and age of the infected tissue. In sterile soil the mycelium grew only very slowly even in the presence of organic supplements. In addition to being inadequate nutritionally, unsterile soil has a microflora that interferes with the growth of S. cepivorum. It is concluded that S. cepivorum does not persist as growing mycelium in natural soils.     

The Role of White Mold-Infected White Bean (Phaseolus vulgaris L.) Seeds in the Dissemination of Sclerotinia sclerotiorum (Lib.) de Bary

Sclerotinia sclerotiorum survived in infected seeds of white beans as dormant mycelium in testa and cotyledons. The rate of survival averaged 85 to 89% and did not change appreciably over a 3-year period. When the infected bean seeds were sown in soil or sand, 88 to 100% failed to germinate. The seeds that failed to germinate, depending on the severity of seed infection, were rotted by S. sclerotiorum. In place of each seed, 3 to 6 sclerotia were formed. A low percentage of these sclerotia germinated carpogenically with or without preconditioning, (2.5 and 11.5% respectively). Myceliogenic germination of sclerotia with and without preconditioning was 35.5% and 70.5% on water agar and 81.0% and 93.0% on glucose agar, respectively. Both, preconditioning and nonpreconditioned sclerotia which were scattered on soil surface could germinate myceliogenically and infect bean leaves by contact. It is therefore, concluded that dormant mycelia in the infected seeds play an important role notonly in dissemination of the fungus but also in epidemiology of the disease.     

Development of a method for the quantitative assessment of microorganism sensitivity to antibiotics using discs. The study of different nutrient media for determining microorganism sensitivity to antibiotics

Five nutrient media used for determination of microbial sensitivity to antibiotics, i.e. beaf-peptone agar, Hottinger pancreatic beaf infusion agar, sprat hydrolysate nutrient agar of the Dagestan Research Institute of Nutrient Media, Muller-Chintone agar from Bulgaria and "Oxoid" agar for determination of microbial sensitivity were studied comparatively. The media were compared with respect to the growth density with the use of different test-cultures and the clearance of the inhibition growth zones around the discs containing different antibiotics. The best results were obtained with the use of sprat hydrolysate nutrient agar. Further studies on the medium standardization are necessary.     

An evaluation of straw-extract agar media for the growth and sporulation of Madurella mycetomatis

Four new media, namely Wheat straw extract agar, Bajra straw extract agar, Jowar straw extract agar and Paddy straw extract agar, were evaluated for their potential to stimulate the growth and sporulation of Madurella mycetomatis in comparison with the conventional Sabouraud dextrose agar and Soil extract agar. Vegetative growth of M. mycetomatis on the four types of Straw extract agars was superior to that obtained on Sabouraud dextrose agar. Isolates of M. mycetomatis sporulated better and faster on the Straw extract agars than on the Sabouraud dextrose agar and Soil extract agar. Straw extract agar is recommended as a sporulation medium for M. mycetomatis. It may prove useful especially for studies of the conidium ontogeny of the fungus for elucidating its taxonomic status.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

The Ability of Plant Compost Leachates to Control Black Mold (Aspergillus niger) and to Induce the Accumulation of Antifungal Compounds in Onion Following Seed Treatment

Onion seeds treated with leachates of composts prepared from alfalfa and sunflower stalks, at the dosages of 10% and 20% respectively, were inoculated with Aspergillus niger van Tieghem, causal agent of onion black mold disease. The ability of the leachates to induce the production of antifungal compounds and to control black mold were tested at seedling and set stages. Leachates from both composts were able to reduce disease incidence in sets, but not disease severity in onion seedlings. Extracts from treated seedlings and sets were fractionated by thin layer chromatography for their content of antifungal compounds. There were no significant differences between the fractions of alfalfa and sunflower compost leachates in the inhibition of the mycelium growth of A. niger, with the exception of one fraction. The presence of fluorescent pseudomonads and Pantoae agglomerans [synonym: Erwinia herbicola (Löhnis)] bacteria was determined in both leachates. The population of P. agglomerans was higher in the sunflower compost leachate compared to the alfalfa leachate. The tested strains of both bacteria were able to inhibit mycelium growth of the fungal pathogen in agar tests. This study suggests the possible role of beneficial bacteria in the induction of antifungal compounds in onion against A. niger during seedling and set stages.     

Local and systemic protection of poinsettia (Euphorbia pulcherrima Willd.) against Botrytis cinerea Pers. induced by benzothiadiazole

Benzothiadiazole (BTH) was found to be highly effective in increasing resistance of two poinsettia cultivars — ‘Coco White’ and ‘Malibu Red’, moderately susceptible to the fungus Botrytis cinerea. BTH applied at a concentration of 0.3 mM on the discs cut out from the leaves of these poinsettia cultivars reduced disease symptoms by more than 60 % in comparison to the control discs treated with water and exposed to infection. It was also observed that the applied inducer at a concentration of 0.03 and 0.3 mM had a favourable influence on the increase of poinsettia systemic resistance of SAR type (systemic acquired resistance). The effectiveness of BTH was much less when disease development was examined on detached leaves (a 20 % reduction of lesion area) in comparison with a pronounced inhibition of grey mould development on intact leaves of previously induced plants (a 80 % protection of intact plants). Benzothiadiazole in the concentration range from 0.03 to 1.4 mM added to in vitro agar medium was not found to have an inhibitory influence on Botrytis cinerea mycelium growth and sporulation.     

The toxicity of legume seed diffusates toward rhizobia and other bacteria

Summary Seed coat diffusates from the legumesCentrosema pubescens and subterranean clover were found to contain a water-soluble, thermostable, antibiotic inhibiting a wide range of rhizobia and other gram-negative organisms as well as gram-positive organisms. Lucerne seed diffusate showed little activity. The extent of inhibition varied with the micro-organism. Diffusate from subterranean clover was generally more active than fromC. pubescens.It was shown that seed diffusates can have a depressive or a stimulative effect on multiplication of organisms around seeds germinating in sand and soil.The significance of the results, particularly with respect to inoculation with Rhizobium is discussed.     

Antimicrobial solid media for screening non-sterile Arabidopsis thaliana seeds

Stable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step. Adding a combination of terbinafine (1 μM) and timentin (200 mg l⁻¹) to Murashige and Skoog agar delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from 10 different wild-type and mutant Arabidopsis thaliana accessions. We named this antimicrobial solid medium “MSTT agar”. Seedlings sown in non-sterile conditions could be maintained on MSTT agar for up to a week without observable contamination. This medium was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as 4 days after stratification, and transferred to soil before the onset of visible microbial contamination. By using MSTT agar we were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.