J.M. Lynch

Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum populations, soil microbial communities and soil enzyme activities

D.C. NASEBY, J.A. PASCUAL and J.M. LYNCH.2000.Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 8% respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.

Rhizosphere soil enzymes as indicators of perturbations caused by enzyme substrate addition and inoculation of a genetically modified strain of Pseudomonas fluorescens on wheat seed

Original article can be found at: http://www.sciencedirect.com/science/journal/00380717 Copyright Elsevier Ltd. DOI : 10.1016/S0038-0717(97)00061-8

Impact of wild‐type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild‐type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild‐type F113 strain carried a gene encoding the production of the antibiotic 2,4‐diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG‐negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild‐type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY‐6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic‐producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot‐to‐root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β‐glucosidase, β‐galactosidase and N‐acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

Aims: Four well‐described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula.

Effect of cadmium contamination with sewage sludge and phosphate fertiliser amendments on soil enzyme activities, microbial structure and available cadmium

Abstract. The effect of Cd pollution (50xa0mg kg–1), with and without sewage sludge (Sw) and PO43– fertiliser (P) addition, on soil biochemical activity and available Cd was assessed in a 112-day soil incubation experiment. The availability of Cd decreased with incubation time and was reduced by the Sw and P additions resulting in the following order of treatments: Cd>P+Cd>Sw+Cd. With the exception of urease and N-acetylglucosaminidase activities, all enzyme activities were negatively correlated with available Cd. The total culturable bacterial population was significantly higher with the addition of Sw alone than in the control during the incubation period (P<0.05). The number of fluorescent pseudomonads decreased with time, but was significantly increased by the addition of Sw. The total fungal populations decreased with time in all treatments, whilst the addition of Sw and PO43– fertilisers relatively increased the fungal population. Addition of Sw in the presence of Cd increased the fungal populations in relation to the addition of Cd alone. The results support the view that Cd contamination has a large detrimental effect on nutrient cycling and microbial activity and that the effects of Cd are reduced by P and Sw additions.

Effects of Pseudomonas fluorescens F113 on Ecological Functions in the Pea Rhizosphere Are Dependent on pH

A bstractThe aim of this microcosm study was to determine influence of the antibiotic 2,4-diacetylphloroglucinol (DAPG) on the effect of wild-type and functionally modified Pseudomonas fluorescens F113 strains in a sandy loam soil of pH 5.4 planted with pea (Pisum sativum var Montana). The functional modification of strain F113 was a repressed production of DAPG, useful in plant disease control, creating the DAPG negative strain F113 G22; both were marked with a lacZY gene cassette. Lowering the soil pH to 4.4 significantly reduced the plant shoot and root weights and the root length, whereas the bacterial inocula had no significant effect. Both inocula significantly reduced the shoot/root ratio at pH 5.4, but this effect was not evident at the lowered or elevated (6.4) pH levels. The decrease in pH significantly increased the fungal and yeast colony-forming units from the rhizosphere (root extract), but did not affect the total bacterial c.f.u.s. Inoculatioin with strain F113 in the pH 4.4 soil resulted in a significantly greater total bacterial population. The fungal and yeast c.f.u.s were not significantly affected by the inocula at any pH studied. Increasing the pH significantly increased the indigenous Pseudomonas population in comparison to the reduced pH treatment and significantly increased both the introduced and total Pseudomonas populations. The antibiotic producing strain significantly reduced the total bacterial population and the NAGase activity (related to fungal activity) at pH 6.4 where the inocula population was the greatest. Alkaline phosphatase, phosphodiesterase, aryl sulfatase, β-glucosidase, alkaline β-galactosidase, and NAGase activities significantly increased with increasing in pH. The F113 inocula reduced the acid phosphatase activity at pH 5.4 and increased the acid β-galactosidase activity over all the pH treatments. The results presented illustrate the variation in impact with soil pH, with implications for variability in efficacy of Pseudomonas fluorescens biocontrol agents with soil pH.

Soil enzyme activities in the rhizosphere of field-grown sugar beet inoculated with the biocontrol agent Pseudomonas fluorescens F113

Pseudomonas fluorescens F113, which produces the antimicrobial compound 2,4-diacetylphloroglucinol, is a prospective biocontrol agent. Soil enzyme activities were used to investigate the ecological impact of strain F113 in the rhizosphere of field-grown sugar beet. There were distinct trends in rhizosphere enzyme activities in relation to soil chemistry [determined by electro-ultrafiltration (EUF)]. The activities of enzymes from the P cycle (acid phosphatase, alkaline phosphatase and phosphodiesterase) and of arylsulphatase were negatively correlated with the amount of readily available P, whereas urease activity was positively correlated with the latter. Significant correlations between nutrient levels determined by EUF and enzyme activities in the rhizosphere were obtained, highlighting the usefulness of enzyme assays to document variations in soil nutrient cycling. Contrary to previous microcosm studies, which did not investigate plants grown to maturity, the biocontrol inoculant had no effect on enzyme activities or on soil chemistry in the rhizosphere. The results showed the importance of using homogenous soil microcosm systems, also employed in previous work, for risk assessment studies, whereby the effects of inherent soil variability were minimised, and effects of the pseudomonad on soil enzymology could be detected.

Establishment and impact of Pseudomonas fluorescens genetically modified for lactose utilization and kanamycin resistance in the rhizosphere of pea

The impact of a Pseudomonas fluorescens strain, genetically modified for kanamycin resistance and lactose utilization (the GMM), could be enhanced by soil amendment with lactose and kanamycin. Lactose addition decreased the shoot to root ratio of pea, and both soil amendments increased the populations of total culturable bacteria and the inoculated GMM. Only kanamycin perturbed the bacterial community structure, causing a shift towards slower growth organisms. The community structure with the GMM inocula in the presence of kanamycin showed the only impact of the GMM compared to the wild type inocula. The shift towards K strategy (slower growing organisms), found in the other kanamycin‐amended treatments, was reduced with the GMM inoculation. Lactose amendment increased the acid and alkaline phosphatase, the phosphodiesterase activity and the carbon cycle enzyme activities, whereas the kanamycin addition only affected the alkaline phosphatase and phosphodiesterase activities. None of the soil enzyme activities was affected by the GMM under any of the soil amendments.

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, kmR marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strain’s strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21’s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.

Carbon fractions in the rhizosphere of pea inoculated with 2,4 diacetylphloroglucinol producing and non-producing Pseudomonas fluorescens F113

The aim of this work was to determine the effect of wild type and functionally modified Pseudomonas fluorescens strains on C fractions in the rhizosphere of pea. The lacZY marked F113 strain produces the antibiotic 2,4 diacetylphloroglucinol (DAPG) useful in plant disease control. The modified strain of F113 was represented in production of DAPG, creating the DAPG negative strain F113 G22. The F113 treatment resulted in a significantly lower shoot/root ratio. The F113 G22 treatment had a significantly greater indigenous and total fluorescent Pseudomonas population than the control and F113 (DAPG+) treatment. Both strains significantly increased the water soluble carbohydrates and the total water soluble carbon in the pea rhizosphere soil. Strain F113 significantly increased the soil protein content relative to the control, but not in relation to the F113 G22 treatment. The F113 treatment had a significantly greater organic acid content than the control and F113 G22 treatments, whilst the F113 G22 treatment was also significantly greater than the control. Both inocula resulted in significantly lower phosphate contents than the control. The F113 inocula significantly increased alkaline phosphatase, sulphatase and urease activities, and reduced β glucosidase activities indicating increased carbon availability. Both inocula increased C availabilityu2003; however, antibiotic production by strain F113 reduced the utilisation of organic acids released from the plant resulting in differing effects of the two strains on nutrient availability, plant growth, soil enzyme activities and Pseudomonas populations.