F.A.A.M. De Leij

The use of colony development for the characterization of bacterial communities in soil and on roots

A simple agar plating method for the description of microbial communities is described. This method is based on the quantification of the numbers of bacterial colonies in 6–7 age-based classes as they appear on agar media over a period of 6–10 days. The method can be used to quantify microbial communities in different habitats (roots and soil) and can be related to the ecophysiology of the microbial communities present. Significant differences in distribution patterns were found in time and depth on the roots. In general, as roots matured, the microbial communities changed from one dominated by r-strategists to one that was more distributed towards K-strategists. The soil had the greatest percentage of organisms that could be characterized as K-strategists. The method was also used to compare microbial communities on wheat roots and in soil in both the field and in microcosms in the glasshouse. In general, the method enabled differentiation between r- and K-strategists in environmental samples, something that could not be done using an ecophysiological index (a modification of the Shannon diversity index) or total bacterial numbers alone.

Effect of copper on the degradation of phenanthrene by soil micro‐organisms

Aims: The effect of copper on the degradation by soil micro‐organisms of phenanthrene, a polycyclic aromatic hydrocarbon, was investigated.

Long-term suppression of Pythium ultimum in arid soil using fresh and composted municipal wastes

Abstract The effect of addition of municipal solid waste (MSW) at different degrees of stabilisation on the biological properties of an arid soil was studied 24 months after application. This included effects on the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium ultimum. The addition of organic waste (fresh or composted) reduced populations of culturable bacteria and fungi and disease symptoms caused by P. ultimum, and resulted in heavier plants with longer and more extensively branched roots. Addition of organic waste increased the population size of culturable bacteria and fungi, while enzymatic activity of the soil was higher in soil amended with organic matter than in non-amended soil. Populations of biological control agents, such as Trichoderma and fluorescent pseudomonads, were larger in soil amended with organic matter. The addition of urban waste could therefore be a suitable technique with which to restore soil quality by stimulating biological control against plant pathogens such as P. ultimum.

Strategies for the isolation of cellulolytic fungi for composting of wheat straw

It was shown that inoculation of straw with cellulolytic fungi offers potential for manipulating and improving the composting of cellulose waste, where the C:N ratio is not optimal for composting. In this paper we report on a screening strategy used to isolate novel cellulolytic fungi from field samples. The screen comprised of two phases. In phase I, 300 cellulolytic fungi were isolated to pure culture from field samples collected from Hawaii, China and the UK. Isolates were selected on the basis of high cellulolytic activities and growth rates on cellulose agar. A total of 137 lead isolates progressed to an unreplicated phase II screen to rapidly identify strains that improved quality of the resulting compost over and above that of the uninoculated control. Compost quality was assessed by measuring C:N ratio, water holding capacity, water content and potential and polysaccharide content of the resulting compost. Effect on the aggregate stability of soil and the growth of wheat seedlings was assessed when compost was added to a sandy loam soil. Performance of each isolate was quantified by allocating a utility score for each compost analysed. Utility scores were based on the sum of the logged ranked score in each assay. The 10 highest scoring isolates were subsequently processed through a replicated phase II screen and the best performing isolates identified by calculating utility scores as before. Significantly lower C:N ratios, higher water-holding capacities and improved aggregate stabilities were obtained with some inoculated treatments compared to the uninoculated control, whilst the results obtained for polysaccharide content and plant growth showed no significant differences. Isolate 304, isolated from decomposing vegetation obtained from Egham, Surrey, UK, and identifed as a Trichurus sp., appeared the most effective inoculant, significantly decreasing the C:N ratio by 36% and increasing the aggregate stability of soil by 54% compared to the uninoculated control. As a result of adopting this screening strategy, it has been possible to identify cellulolytic fungi that can, under non-sterile (laboratory) conditions, significantly improve the quality of compost. This screening approach therefore offers real possibilities for selecting microbial inoculants in low-tech agricultural practices.

Effect of Introduced Pseudomonas fluorescens Strains on Soil Nematode and Protozoan Populations in the Rhizosphere of Wheat and Pea

A bstractPrevious studies have shown that inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 increased mineralization of organic nitrogen in the rhizosphere. In contrast, inoculation of the same strains onto wheat seeds reduced mineralization of N from organic residues incorporated into soil. In the present study, we report on a likely explanation of this phenomenon, which appears to be governed by the effect of plant-microbe interactions on bacterial-feeding nematodes and protozoa. In soil microcosm tests, inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 resulted in an increase in the number of nematodes and protozoa in the rhizosphere as compared to noninoculated controls. This trend was repeated using a model sand system into which the bacteriophagous nematode Caenorhabditis elegans was introduced. It was subsequently found that non-inoculated germinating pea seeds exerted a nematicidal effect on C. elegans, which was remedied by inoculation with either strain F113lacZY or F113G22. This suggests that nematicidal compounds released by the germinating pea seeds were metabolized by the microbial inoculants before they affected nematode populations in the spermosphere or rhizosphere of pea. In contrast, inoculation of wheat plants resulted in significantly lower nematode populations in the rhizosphere, whereas protozoan numbers were unaffected. No nematicidal effects of inoculated or noninoculated wheat seeds could be found, suggesting that microfaunal populations were affected at a later stage during plant growth. Because of their key roles in accelerating the turnover of microbially immobilized N and organic matter, plants that support a larger microfaunal population are likely to benefit from a higher availability of inorganic nitrogen. Therefore, an understanding of plant-microbe interactions and their effects on soil microfaunal populations is essential in order to assess the effects of microbial inocula on plant mineral nutrition.

A simple most probable number technique for the sensitive recovery of a genetically-modified Pseudomonas aureofaciens from soil

A strain of Pseudomonas aureofaciens (SBW25EeZY‐6KX) that was chromosomally marked with a lacZY and a kmr‐xylE cassette could be recovered from non‐sterile soil by a selective Pseudomonas enrichment broth amended with 100 ppm kanamycin and 50 ppm X‐gal in an MPN (most probable number) assay. The assay was sensitive and reliable, allowing detection of as few as one recombinant cell in a 1% (w v) soil suspension. The soil used contained a large (ca 5% of total culturable bacteria) background of indigenous bacteria that were either able to utilize lactose, were resistant to kanamycin, or both.

Effect of introduced Pseudomonas fluorescens strains on the uptake of nitrogen by wheat from 15N-enriched organic residues

The effects of an antibiotic-producing Pseudomonas fluorescens strain (F113) carrying the marker gene cassette lacZY and a marked, non-producing strain (F113G22) on the uptake of nitrogen from 15N-enriched organic residues incorporated into a sandy soil were investigated in microcosm studies. Strain F113 produces the antibiotic 2,4-diacetylphloroglucinol (DAPG), whilst its modified derivative strain F113G22 has DAPG production deleted by Tn5 mutagenesis. Uptake of nitrogen by wheat (Triticum aestivum) from 15N-enriched organic residues was estimated using stable isotope-ratio mass spectrometry of shoot and root material of 17-day-old plants. In addition, plant growth and active microbial biomass in soil were monitored. In contrast to results obtained in our previous study on pea (Pisum sativum), it was found that in wheat, inoculation with either strain F113 or F113G22 decreased the proportion of nitrogen derived from 15N-labelled organic residues incorporated into soil as compared to non-inoculated controls. It is therefore suggested that these strains decreased mineralization of organic residues in the rhizosphere of wheat, making less inorganic N (15N) available for plant uptake. The results of this study indicate that the effects of introduced Pseudomonas fluorescens strains on nitrogen mineralization in the rhizosphere are plant-species dependent, and highlight the importance of testing microbial inocula on a range of plant species.

Spread and survival of a genetically modified Pseudomonas aureofaciens in the phytosphere of wheat and in soil

Abstract An isolate of Pseudomonoas aureofaciens indigenus to the UK, which was chromosomally modified by the insertion of two gene cassettes ( xy / E in combination with the gene for kanamycin resistance and lacZY ) for the sensitive monitoring purposes was provided by IVEM (Oxford). Growth of this organism and the non-modified wildtype strain were not significantly different in sterile tryptic soy broth. Spread and survival of the marked bacterium in the phytosphere of spring wheat and in soil was quantified in a number of glasshouse and laboratory experiments. Six days after vacuum infiltration of wheat seeds (growth stage (GS) 11; seedling) with bacteria, most (approximately 99%) of the bacteria stayed associated with the seed, while a small proportion was recovered from the roots and leaves (0.8% and 0.2%, respectively). When seeds were inoculated with 5 × 10 8 recombinant colony forming units (cfu) per seed the bacterium was able to survive up to levels of 10 5 cfu g −1 root during the whole of the plant cycle (4 months), but the bacterium established on the first emerging leaves only in low number (approximately 1000 cfu g −1 tissue). The bacterium could be recovered from roots extracted from a depth of 60 cm, but most (> 95%) of the bacteria stayed associated with roots in the top 15 cm the soil profile. When bacteria were sprayed onto wheat plants at GS 22 (tillering), they could be recovered 1 month later (GS 69; flowering) from all leaves at levels greater than 10 5 cfu g −1 tissue, but not from the ear. Seventy days after spray application (GS 92; ripening) most of the bacteria had disappeared from the phylloplane. After incorporation in soil at rates of 10 6 recombinant cfu g −1 soil, bacterial numbers of P. aureofaciens declined by 0.028 log units/d at a temperature of 5°C and 0.07 log units per day at 18°C. The isolate of P. aureofaciens used in this study was able to survive desiccation at 60% r.h. for 1 month, but was unable to survive for more than 1 month when exposed to high levels of microbial competition at 100% r.h.

Production of Trichurus spiralis to enhance the composting of cellulose-containing waste

In this study, we investigated the effect of three substrate types (wheat straw, white rice and grit-bran) on conidia production of the cellulolytic fungus Trichurus spiralis. Besides the number of conidia produced per gram substrate, enzyme activity and spore vigour of the produced inoculum were assessed. Subsequently, conidia produced on the different substrates were inoculated into non-sterile wheat straw to assess the competitive ability of the different inocula during composting. Grit-bran showed significantly higher conidial yields per gram substrate than the other substrates. However, cellulolytic activity of inoculum produced on grit-bran was significantly lower compared to that of the inoculum produced on either rice or wheat straw. Conidia produced on both rice and grit-bran had a germination rate of around 80%, while conidia produced on wheat straw had a significantly lower germination rate of around 50%. Spores produced on wheat straw and grit-bran had larger food reserves than those produced on rice. Microbial activity in non-sterile straw was significantly enhanced with conidia produced on grit-bran but not in straw inoculated with conidia produced on either rice or wheat straw. Our results suggest that inoculum produced on grit-bran was the most effective in enhancing composting of wheat straw. The results of this study indicate that substrate type can affect the quantity and quality of the resulting inoculum. The data obtained suggest that nutrient-rich substrates, such as wheat bran, not only gave high conidial yields, but that the conidia produced had sufficient vigour to effectively colonise non-sterile substrates.

Desorption of zinc by extracellularly produced metabolites of Trichoderma harzianum, Trichoderma reesei and Coriolus versicolor

Aims:  To determine the role of fungal metabolites in the desorption of metals.