Soil Extract Agar Research Articles

Selective effects of fumigation with carbon disulphide on the soil fungus flora

Kettering soil (pH 5–6) was fumigated with carbon disulphide in Evans soil recolonization tubes (vol. c . 220 ml.) at dosage rates of 0·05–1·0 ml. per tube, over a period of 96 hr. at 25°C. Soil-dilution plates of glucose-nitrate soil-extract agar+rose bengal (1:15,000) were made from soil samples collected from the side-arms of the recolonization tubes at 1 and again at 36 days after the end of the fumigation period. The twenty-four species of soil fungi most commonly occurring on the dilution plates have been listed in approximate order of their tolerance of carbon disulphide. Trichoderma viride showed only a moderate degree of tolerance (10th in the list of 24 species), but this was combined with a high recolonizing ability after the optimum dose of carbon disulphide (0·1 ml. CS 2 /tube). Recolonizing ability was expressed by the ratio of the plate count at 36 days after fumigation to that at 1 day, and was 9·2 for T. viride ; the ratio for this fungus was higher than that for any of the other twenty-three species. For T. viride and other species possessing a medium to high degree of fumigant tolerance, the dose of carbon disulphide optimum for soil recolonization was generally lower than the maximum dose permitting survival. This suggests that there is a limiting value of inoculum potential necessary for soil recolonization, below which a species is unable to recover from the effects of fumigation.

SOME STUDIES OF THE COLONY COUNT TECHNIQUE FOR SOIL BACTERIA

SUMMARY: A series of co‐operative experiments was conducted to compare the bacterial colony counts of soil obtained by workers in different laboratories, using soil extract agar and other media for the determinations.In the earlier experiments it was not possible to obtain a reasonable degree of reproducibility of results between laboratories even when the plating technique was carefully prescribed. By modification and more rigid standardization of the technique closer agreement was obtained in subsequent experiments. It is suggested that when co‐operative investigations are contemplated the participating laboratories should check their technique by the examination of ‘control’ soils.No evidence could be obtained to support the suggestion that higher colony counts are obtained by the use of soil extract media containing extract prepared from the same soil as the sample tested. The source appears to be immaterial so long as the soil for extract preparation is not of extreme type and has been well manured and cultivated.

Plate counts of bacteria and fungi in a saline soil.

Numbers of bacteria and of fungi in a saline soil were about one-fifth of numbers in a Red River clay soil. Bacterial counts on two soil-extract agar media, one prepared from the saline soil and the other from Red River soil, were the same, and the populations on the two were the same as shown by the replica plating technique. They were larger than counts on sodium albuminate agar or on asparagin–mannitol agar. Likewise, fungal counts on either soil-extract agar, with 0.02% dipotassmm phosphate, 0.1% glucose, and 0.1% peptone, were higher than counts on Waksman's acidified medium, or on Martin's medium, or on Smith and Dawson's medium. Interestingly, fungal counts on a medium with the same three chemicals as the soil-extract media but with the soil extract replaced by water were as high as those on the soil-extract media. Different levels of potassium phosphate were tested in each of the above media. In each medium for bacteria, and in each for fungi, counts varied inversely as the amount of potassium phosphate. The same held true when sodium phosphate was used instead of potassium phosphate in each medium.

Soil extract in soil microbiology.

Soil extract contains unidentified growth factors not present in other materials. In this study the extract was considered critically in so far as various treatments affect numbers of bacteria developing on soil-extract agar. Dilution of the extract to 1/10th concentration produced lower counts in most soils. Calcium sulphate and calcium carbonate, used as precipitants, failed to produce more or clearer extracts. Unheated extract provided the nutritional requirements for a considerable fraction of the bacteria in soil, but not for as large a fraction as did heated extract. Dried extract produced lower counts than did freshly prepared extract. The addition of 0.02% dipotassium acid phosphate to soil extract increased counts significantly. The use of the proportion of soil and water that gave the amount of extract desired, without the addition of water after filtration, produced the maximum amount of extractives from the amount of soil used.

Notes on Fungi from North-East India. I. A New Genus of Tuberculariaceae

Comprehensive accounts of the principal diseases of tea in northeast India have been published in the past by Tunstall (1940) and Tunstall and Sarmah (1948). During the routine examination of diseased specimens of tea and its ancillary crops, the writer had occasion to isolate many interesting fungi and a few of them have already been reported elsewhere (Agnihothrudu and Barua, 1957a, b and Agnihothrudu, 1958). In view of the rich fungal flora, parasitic or saprophytic, associated with the tea gardens in northeast India, it is now proposed to record, describe or discuss fungi occurring in this area in a series of papers, of which the present is the first. In the course of investigations on the fungi associated with withering floral parts of tea Camellia sinensis (L.) 0. Kuntze, on three occasions an interesting tuberculariaceous fungus was isolated which was found to be hitherto undescribed. The fungus was first observed on dead calyces of tea flowers collected from the Tocklai Experimental Station campus, the flowers having been incubated on sterilized soil in glass trays under bell-jars. On two other occasions it was found to grow from petals plated on soil-extract agar. The fungus was seen in association with a variety of other fungi-Pestalozzia sp., Fusarium semitectum Berk. & Rav., Pullularia pullulans (de Bary) Berk., Botrytis sp., Hormodendrum resinae Lindau, Cladosporium herbarum Link ex Fr. and Helicomyces sp. Its ability to infect tea flowers has not been investigated. THE FUNGUS IN NATURE

The isolation of yeasts from soil.

Summary: A series of comparative studies was made of soil yeasts in a yellow-brown earth originally under forest but now under pasture. Of six solid acid media used for primary isolations, an agar medium containing 4 % (w/v) glucose and 1 % (w/v) peptone gave the highest yeast counts. Two soil-extract agars and a modified Czapek-Dox agar gave slightly lower counts; a third soil-extract agar inhibited all but one yeast species and gave a much decreased count. The addition of a surface active agent to glucose peptone agar did not increase the count. Acid broth enrichment cultures gave a distorted picture of the species pattern shown by primary cultures on solid media. Prolonged mechanical shaking of soil dilutions before culturing, and the addition of a surface active agent to the diluent did not affect the yeast pattern seen, quantitatively or qualitatively.

EFFECT OF NUTRITION ON GROWTH AND CHLAMYDOSPORE FORMATION IN BROWN AND GRAY CULTURES OF THIELAVIOPSIS BASICOLA

Physiological differences between brown and gray wild type cultures of Thielaviopsis basicola were sought in order to develop selective media for use in mutational studies, and as a basis for pathogenicity and ecological studies. The gray wild type was more thiamine-deficient, yielded less mycelium, and grew more slowly than the brown on all media tested regardless of composition and thiamine content. In liquid media both types were more thiamine-deficient on sucrose than on glucose, but the reverse was true on agar media. The gray type was inhibited more than the brown by malachite and methyl green. A two-layered thiamine-deficient agar medium, and one containing dyes, was selective for the brown type. Gray and brown types grew better on thiamine-deficient water agar and soil extract agar than on thiamine-deficient agar media containing 20 gm/l. of sugar and 1 gm./l. of nitrogen compounds. On thiamine-deficient media, distortion and extrusion of the contents of endoconidia and hyphae were more common with the gray wild type than the brown.Endoconidia, but not chlamydospores, were formed in varying amounts on all thiamine-deficient media. The addition of thiamine resulted in the abundant production of chlamydospores and greatly increased growth. Chlamydospore production was also influenced by the source of nitrogen. Certain amino acids were more inhibitory to chlamydospore formation by the brown than gray type and vice versa. Asparagine, for example, blocked chlamydospore formation by the brown but not the gray type, even though growth and endoconidial production were abundant. Mutants capable of overcoming this blockage were readily produced in culture.

A New Genus of Moniliaceae

The soil was collected in September, 1952, from a depth of 2-4 inches with a sterile spatula, packed in a sterile container and brought to the laboratory. The soil was plated in three different dilutions in Petri dishes of several media. The fungus first appeared on potato dextrose agar. Further isolation and purification was done on Czapek's agar and detailed observations have been recorded on the same. The fungus was later tried on various other media, viz. soil extract agar, malt agar, potato dextrose agar and Waksman's agar for recording the behavior and cultural characteristics.

Soil-extract agar for Candida albicans.

SPECIES of Candida isolated from patients can be differentiated morphologically when grown on a nutrient-poor medium such as cornmeal agar. I recently described a yellow cornmeal agar medium for such purposes and showed that on it Candida albicans can usually be identified by its chlamydospores in one to three days. 1 In this laboratory a soil-extract agar is used as an alternative medium to the yellow cornmeal agar. Results obtained in the identification of 424 yeast cultures from Alberta patients show this medium to be as satisfactory as the yellow cornmeal medium for the identification of C. albicans and other species of Candida. MATERIALS AND METHODS Preparation of Soil-Extract Agar Medium. —Approximately 1 liter of fine, sandy loam 2 is added to 1 liter of distilled water in an enamel pot, and the mixture is autoclaved for 15 minutes at 15 lb. pressure. The supernatant upon cooling is filtered through

QUALITATIVE STUDIES OF SOIL MICROORGANISMS: XI. FURTHER OBSERVATIONS ON THE NUTRITIONAL CLASSIFICATION OF BACTERIA

A review of the method developed in this laboratory in 1943 for the nutritional classification of soil bacteria has suggested slight amendments in certain differential media: (1) the substitution of vitamin-free casamino acids for a combination of amino acids, and (2) the addition of vitamin B12 to the growth factor media. In a comparative study with a newly proposed scheme of classification, the more selective plating medium advocated was found to be less suitable for the isolation of soil bacteria than the nonselective soil extract agar in the original method. Furthermore, the replacement of potassium nitrate with diammonium phosphate as source of inorganic nitrogen in the basal medium failed to cause any significant change in the nutritional grouping. Results from the nutritional classification of some 600 isolates by the two methods showed that the new procedure represents only a slight modification of the original system.

Nature of the Factor in Soil-extract Responsible for Bacterial Growth-stimulation

SOIL-EXTRACT is a valuable constituent of media for the growth of many soil bacteria. A medium containing only agar and aqueous soil-extract will give much higher plate counts from soil than any other medium in common use. Lochhead and Chase1 showed that one-third of the bacterial cultures isolated from soil in soil-extract agar were unable to grow in a liquid medium containing a wide selection of amino-acids and vitamins, unless soil-extract was also added. They tentatively concluded that several different growth-promoting substances were involved. Some work on the nature of the soil-extract effect is briefly summarized here.

THE CONTROL OF DISEASES OF LETTUCE BY THE USE OF ANTAGONISTIC ORGANISMS II. THE CONTROL OF RHIZOCTONIA SOLANI KUHN.

The antagonism of a number of bacteria, actinomycetes and fungi in pure culture on soil extract agar has been investigated. A considerable number of each of the three groups were highly antagonistic at 25° C., fewer were active at 15° C. and only a very limited number were effective at 5° C.In greenhouse experiments damping‐off of lettuce seedlings was substantially controlled by selected antagonists in sterile sand, soil or in sand/soil mixtures. Under similar conditions in unsterile soil, forms highly antagonistic in pure culture were generally ineffective in controlling disease. In one experiment control of disease in natural soil was obtained by the use of soil amendments, the effect being enhanced by the inoculation of one of these amendments with suspensions of antagonists.Attempts to control disease in the field by the use of soil amendments are described. In one year a substantial increase in the stand of healthy seedlings was obtained with certain treatments.

A Comparison of the effects of several concentrations of oxgall in platings of soil fungi

A study was made of the effect of adding varying concentrations of dehydrated bile to soil extract agar and potato dextrose agar in platings from two Ontario soils. The development of colonies of bacteria and actinomycetes was retarded, but no decrease was noted in the number of fungus colonies that appeared in the plates. The crowding effect of certain bacteria and fungi was lessened, facilitating the counting and isolation of individual fungus cultures.

Spore Prints from Individual Phialides of Verticillium spp

STERILE soil-extract agar pH. 4 (without mineral or organic additions) is allowed to set in a Petri dish. Using a fine rod finished off at the tip into a small glass bead, the agar surface is streaked with the given spore suspension or smear, care being taken to avoid breaking the surface. When phialospores are being produced in appreciable numbers, rectangular pieces of coverslip are placed flat on the surface of the agar close to and on opposite sides of the streak, and entire coverslips are then rested on them so as to cover the streak. Using a 1/5 obj. and ocular × 10 the upper surface of the coverslip is first brought into focus. Downward racking with the fine adjustment is then continued until phialides and spores are observed at or near the underside of the coverslip. Many upwardly directed phialides will have been caused to deposit their spores against the coverslip by this means, and in many cases the points of the phialides may be observed beside their own spore or spores. Phialides at a lower focus can also be caused to deposit their spores if the lens is pressed gradually down on the coverslip by means of the fine adjustment. In fact, it is quite possible, first to observe individual phialides bearing a spore mass, and then to pick off these spores by pressing the coverslip against them. The spore or spore masses may then be observed at a higher focus lying on the coverslip, and by again focusing downwards their previous relation to the now sporeless phialide-tips may be checked.

Differentiation of the Vegetative and Sporogenous Phases of the Actinomycetes: 2. Factors affecting the Development of the Aerial Mycelium

SUMMARY: When first isolated on soil extract agar, soil actinomycetes consistently produce aerial spores in surface colonies. They retain this property when maintained in sterile soil, or when grown on washed suspensions of common soil bacteria, living or dead, in a water agar medium. In soil, when the composition, moisture content and temperature are kept constant, the initial stimulus towards the production of aerial mycelium is free access of air: the quantity and nature, vegetative or sporogenous, of the inoculum, and, within a broad range, the pH of the soil are of minor importance. Once growth is established, the next most important factor stimulating sporulation in the soil is also physical, namely dehydration. In natural and sterilized soils of different origins, and in a ‘Synthetic’ soil containing 250 p.p.m. of nitrogen as nitrate, the modes of growth of different actinomycetes strains are similar, and generally uncharacteristic of their species.

The Protozoa of Some East Greenland Soils

A SERIES of soil samples illustrating stages of plant colonization was collected by Dr and Mrs H. G. Wager on the British East Greenland Expedition (2) in the summer of 1936 from Kangerdlugssuak, Lat. 68? 30' N. Previous work on the Protozoa of Greenland soils was done by Sandon (3) who examined nine samples collected by Dr Morten Porsild at Disko in the neighbourhood of the Danish Arctic Station. His samples were a variety of types, including among them two black peaty soils. On the whole, these samples were rather poor in Protozoa, and in some respects their distribution was unexpected, e.g. one heath soil yielded 15 species of Protozoa-more than soils which were supporting a more luxuriant vegetation. Sandon's findings show that the climatic conditions do not make the soils of this region unsuitable for the development of Protozoa, since, among these samples, a garden soil enriched every year with poultry manure yielded 46 species, this being the highest number in any of the samples from his world's survey. This garden sample was very rich in Ciliates and Flagellates, but the high numbers were not made up by the testaceous Rhizopoda, as is the case in the soils described here. The British East Greenland Expedition's samples were taken to a depth of 4 in. and placed in sterile jars which were subsequently sent to Rothamsted for an examination of the protozoan fauna. The observations made were only qualitative, since the time taken in transport precluded any useful quantitative work. Four media were used for cultivating the Protozoa: peptone agar, soil extract agar, soil extract and hay infusion, and the cultures were kept under observation for four weeks (1).