Margaret E. Brown

Studies onAzotobacter species in soil: 1. Comparison of media and techniques for counting azotobacter in soil

SummaryMethods for countingAzotobacter species in soil have been examined. The highest counts were obtained from soil suspensions shaken in sterile distilled water containing 10-g glass beads and plated on to glucose agar. Mannitol has been rejected as a suitable substrate in agar media because it gives lower counts of Azotobacter than glucose, an effect which is further enhanced by drying the agar plates. A clear medium free from precipitated phosphate and CaCO3 is recommended for the agar-plate method; the Azotobacter count is affected by the phosphate concentration.The agar-plate and dilution-tube methods were compared; the latter is less accurate but more convenient when many soil samples have to be examined.

Studies onAzotobacter species in soil: II. Populations of azotobacter in the rhizosphere and effects of artificial inoculation

SummaryThe distribution of Azotobacter was studied in the rhizosphere of some crop plants grown in this country. Any rhizosphere effect was very small and frequently Azotobacter was suppressed. No cells were recovered from root washings, indicating absence from the root surface. Azotobacter was absent from the rhizosphere in acid soils. Inoculation of seeds, roots, and soil resulted in establishment of high numbers of Azotobacter in the rhizospheres of plants grown in soil of pH above 6.5 and containing a natural Azotobacter population. Seed inoculation was the most practical and led to good establishment throughout the root system, including adventitious roots of wheat. The final population in the rhizosphere depended on the size and age of the initial inoculum applied to the seed. Best results came from spraying seeds with 14-day-old cultures and sowing the seed on the same day.

Indolyl-3-acetic acid formation byAzotobacter chroococcum

SummarySmall amounts of indolyl-3-acetic acid (IAA) were detected in aerated cultures ofAzotobacter chroococcum grown with or withoutl-tryptophane in the medium, but IAA was detected in agar cultures only whenl-tryptophane was present. Most IAA was found in 7-day-old cultures and less in older cultures. Washed cells did not convert tryptophane enzymically to IAA.The time course of IAA formation byA. chroococcum strain A6 has been described and the effect of adding tryptophane to the medium has been studied. In contrast to results elsewhere strain A6 produced traces of IAA in aerated cultures with or without added tryptophane. IAA was detected only after the end of exponential growth when cells had begun to autolyse. The amount of IAA declined as cultures aged. The slight effect ofl- but not ofd-tryptophane in promoting IAA formation in ageing cultures suggests some kind of biological transformation but it seems unlikely that IAA formation is part of the normal metabolic processes of intact Azotobacter cells.

Nitrate and ammonium in the rhizosphere of wheat crops and concurrent observations of take-all

SummaryIn 1973 the concentration of nitrate nitrogen in the rhizosphere of winter wheat (NO3-Nr, extracted withN-K2SO4 from soil from within 5 mm of the roots) growing in slightly acid, flintly loam decreased progressively from January until early April, when it became less than that of ammonium nitrogen in the rhizosphere (NH4-Nr, extracted withN-K2SO4). An April application of ammonium nitrate (as Nitro-chalk) augmented NO3-Nrmore than NH4-Nr, but because NH4-Nrwas depleted less quickly it exceeded NO3-Nrfrom early June onwards. From January to April pHrremained between 6.9–7.2, but then decreased progressively to a minimum of 6.3 in June. The differences in soil pH and forms of N amongst plots that had grown three, six or fourteen consecutive cereal crops susceptible to take-all were small; a first wheat crop differed only in having less extreme changes in NO3-Nrduring April-June.First winter wheat crops were grown on a similar soil in 1974–5, either with farmyard manure applied in autumn (FYM plot, bulk soil pH 7.4) or Nitro-chalk applied in April (NPK plot, pHb 6.7). Soil nitrate decreased considerably in May and to a lesser extent in June and was always at a lower concentration in the rhizosphere. NH4-Nrwas more than NO3-Nrin water extracts after May in the FYM plot and after June in the NPK plot, but NH4-Nbnever exceeded NO3-Nb.There was, therefore, a tendency for more of the dwindling mineral N to exist as NH4-N close to roots in spring and summer unless temporarily disrupted by spring fertilizer. Take-all developed against this background was mostly slight and never severe. In 1973 it was least in the first wheat, and most in the third, but the differences were small and disease increased steadily in all plots after mid-February. In 1975 it developed quickest and most in the FYM plot. re]19760928

Interactions of azotobacter with rhizosphere and root-surface microflora

SummaryActinomycetes isolated from soil and from the rhizosphere of seedlings, some inoculated with Azotobacter and others not, were tested for antagonism againstAzotobacter chroococcum. Antagonistic actinomycetes were initially fewer in the rhizosphere of inoculated than of uninoculated seedlings. In the rhizospheres of inoculated and uninoculated seedlings, the number of antagonists increased with time, but not in the soil. The proportion of isolates that showed antagonism was same in soil and rhizosphere. Fungi isolated from root surfaces showed two types of antagonism against Azotobacter; bactericidal and bacteriostatic. When inoculated to sterilized soil, only the strongly antagonistic actinomycete greatly checked initial Azotobacter multiplication. The slow decline in the numbers of Azotobacter in the rhizosphere of older plants may reflect the increasing population of antagonists.

Effects of nitrate and ammonium on wheat roots in gnotobiotic culture: Amino acids, cortical cell death and take-all (caused by Gaeumannomyces graminis var. Tritici)

Abstract Aseptically-grown wheat seedlings were supplied with mineral nutrients containing nitrogen as calcium nitrate or ammonium phosphate. Larger numbers and greater concentrations of amino acids occurred in root exudates and there were slightly greater concentrations of amino acids in root extracts of plants given ammonium. Cortices of proximal and middle regions of roots on seedlings which were 5–18 days old had smaller percentages of nucleate cells when nitrogen was supplied as ammonium than when nitrogen was supplied as nitrate or a mixture of the two forms. Nitrate prolonged the survival of the nucleate cells in the outer layers of the cortex. When suspensions of mycelial fragments of Gaeumannomyces graminis var. tritici were poured over roots growing down agar slopes, proximal and distal regions of the root, but not the middle region, developed lesions which were larger where the agar contained ammonium nitrogen rather than nitrate nitrogen. Irrespective of the form of nitrogen the length of lesions decreased with increasing distance from the seed. The association of these effects seemed strongest in the proximal region of the root and the observations are discussed in relation to hypotheses of lesion severity and distribution in seedling-infection tests.

Microbiology of roots infected with the take-all fungus (Gaeumannomyces graminis var. tritici) in phased sequences of winter wheat

Abstract Bacteria with possible relevance to the growth of the take-all fungus were counted from surfaces of lesioned and healthy roots of wheat growing in soil from a field monoculture system. Numbers showed short-term seasonal and long-term monocultural changes, which seemed to be genuinely associated with the monoculture. Bacteria were more numerous on lesioned than healthy roots. Only bacteria inhibitory to growth of Gaeumannomyces graminis on agar and Pseudomonas spp showed consistent changes irrespective of the source of the roots. Relationships were considered between the microflora on lesioned tissue and (a) severity of disease on roots supplying the lesions, and (b) infection produced on axenic seedlings inoculated with the lesioned tissue. Only total bacterial counts on the lesions from tillering and mature plants were positively correlated with disease on the donor roots. Only inhibitory bacteria on lesions from tillering and mature plants were positively correlated with disease on test seedlings. Pseudomonas spp showed no correlations. Interpretation of data differed with age of plant and the sequence in the monoculture from which plants or soil came.

Infectivity of lesioned wheat root tissue related to the presence of dehydrogenase enzymes in hyphae of Gaeumannomyces graminis var. Tritici in the lesions

Abstract Transverse sections of lesioned tissue taken from wheat roots grown in soil naturally infested with Gaeumannomyces graminis var, Tritici were stained with trypan blue and the area of stele occupied by hyphae or by brown host deposits was measured. The area of mycelium in lesioned pieces taken from seedling or tillering plants and used as inoculum in host infectivity tests was positively correlated with the disease produced and the area of brown deposits in lesioned pieces taken from tillering or mature plants was negatively correlated. Whole pieces of lesioned tissue were examined cytochemically for glutamic and succinic dehydrogenases in the invading hyphae. Groups of host cells in the endodermal region were filled with hyphae showing positive reactions for both dehydrogenases (active) and separated by areas of brown discoloured host tissue containing few active hyphae. Less than half the discoloured lesion was occupied by active hyphae. The area of lesion containing hyphae with dehydrogenase enzymes was positively correlated with the measure of disease severity of the roots and with the infectiveness of the lesioned tissue when inoculated on to axenic wheat seedlings. The progress of infection in axenie seedlings inoculated at 3 or 8 cm from the seed differed with the two placements, notably in the host response and the growth of active hyphae in the cortex.