Kenneth L. Conn

Strain-dependent variability in growth and survival of Escherichia coli in agricultural soil

Abstract This study investigated strain-dependent variability in Escherichia coli survival in soil, and strain-dependent responses to variations in some soil conditions. Collections of E. coli were isolated from swine manure slurry, and from manured soil following 6 days of incubation in the laboratory. The bacteria were fingerprinted by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). During the course of the incubation the composition of the E. coli community changed dramatically suggesting that E. coli phylotypes, distinguishable by ERIC-PCR fingerprinting, varied significantly in their ability to survive in soil under these conditions. A representative isolate from one ERIC group which increased in abundance in soil (designated strain C279) and one which decreased (designated strain C278) were chosen for comparison. These strains persisted comparatively when inoculated into loam soil. However, when added into a loam soil or a sandy soil supplemented with 10% (v/v) swine manure slurry, strain C279 increased in abundance 10-fold, whereas strain C278 did not. At 4 degrees C, or in a clay loam soil, manure slurry did not support the growth of strain C279. These results indicate that the community composition of E. coli populations in manured soils can be very dynamic, and that strains able to proliferate in manured soils can have a selective advantage.

Impact of animal manures on Verticillium wilt, potato scab, and soil microbial populations.

Abstract This study examined the effect of chicken, liquid swine, and solid cattle manures on verticillium wilt and potato scab incidence at two commercial farms near Alliston, Ontario. The manures (chicken, 66 t/ha; swine, 55 hLJha; cattle, 100 t/ha) were spread in the spring of 1996 and incorporated with a Rototiller to a depth of 15 cm. Survival of Verticillium dahliae microsclerotia (MS) was determined by burying nylon mesh bags containing MS in the plots. After 4 weeks, the MS were plated on agaz medium. Soil samples collected after incorporation of manures were brought back to the laboratory and used for concurrent laboratory experiments. Ten-gram amounts of soil were placed into test tubes, bags of MS buried in the soil,and survival measured under controlled conditions. Potatoes were planted in the field 1 month after amendment. Potatoes were planted in 1997 and 1998 without any further treatment. verticillium wilt incidence was determined by isolation of V.dahliae from petioles. Scab incidence was...

Organic amendments as a disease control strategy for soilborne diseases of high-value agricultural crops

Manures and by-products derived from the processing of plants and animals have been used for centuries as sources of fertiliser, but beneficial or detrimental effects on plant diseases were never thoroughly investigated. We found that certain organic amendments controlled a variety of soilborne diseases of potato (including common scab and verticillium wilt), various pests (including plant parasitic nematodes) and weeds at nine field locations in Ontario and Prince Edward Island, Canada. The mechanism of disease control for highnitrogen-containing amendments is the generation of ammonia and / or nitrous acid following degradation of the amendments by microorganisms. The formation of these products to concentrations lethal to pathogens is regulated by the soil pH, organic matter content, nitrification rate, sand content and buffering capacity. Liquid swine manure reduced scab and wilt, but at only three of six locations tested. In acidic soils, swine manure killed Verticillium dahliae within a day after application, but had no effect in neutral or alkaline soils. The toxic components in the manure were identified as volatile fatty acids. Ammonium lignosulfonate reduced scab at all six sites tested. The mechanism of disease control is not yet known. Although many of these amendments reduced pathogen populations, total soil microorganism numbers increased by 10- to 1000-fold after application, indicating that not all organisms were killed. Understanding the mode of action of amendments is essential for the improvement of their effectiveness and assimilation into crop production systems. If costs can be decreased and benefits ensured, organic amendments will have a major role in reducing plant diseases. The discussion on use of these amendments is based on cost/ benefit analysis as well as societal and regulatory considerations.

Volatile Fatty Acids in Liquid Swine Manure Can Kill Microsclerotia of Verticillium dahliae

ABSTRACT Liquid swine manure added to acidic soils killed microsclerotia of the wilt fungus Verticillium dahliae. We investigated whether volatile fatty acids (VFAs) in the manure were responsible for this toxicity. The survival of microsclerotia was determined after exposure to various dilutions of manure or its VFA components. Acetic, propionic, and isobutyric acids constituted the major VFAs in the manure, while n-butyric, n-valeric, iso-valeric, and n-caproic acids were present in lesser amounts. Formic acid was not detected. The individual VFAs were more toxic to microsclerotia as the solution pH was decreased, indicating that the protonated forms of the VFAs were toxic (e.g., acetic acid and not acetate). The effective concentration reducing germination of microsclerotia by 95% (EC(95)) for formic and n-caproic acids was approximately 4 mM, the most toxic of the acids tested; for n-valeric, the EC(95) was 9.2 mM, isovaleric was 16.1 mM acids, and acetic, propionic, n-butyric, and isobutyric acids were approximately 30 mM. The toxicity of acetic acid, and likely all the others, was directly related to the duration of exposure. Inhibition of microsclerotia germination followed identical trends in solutions of the manure or in a mixture of VFAs with equivalent concentrations of the individual acids found in the manure. Similarly, germination declined to the same extent in the atmosphere above the manure or the VFA mixture, confirming the toxicity of VFAs to microsclerotia. Thus, under acid conditions, VFAs in liquid swine manure can kill microsclerotia of V. dahliae.

Liquid Swine Manure Can Kill Verticillium dahliae Microsclerotia in Soil by Volatile Fatty Acid, Nitrous Acid, and Ammonia Toxicity

ABSTRACT In previous studies, liquid swine manure (LSM) was sometimes shown to reduce Verticillium wilt of potato caused by Verticillium dahliae. We also observed that microsclerotia of this fungus died within 1 day, or between 3 and 6 weeks, after addition of LSM to some acid soils and within 1 week in some alkaline soils. In this study, we demonstrated that a volatile fatty acid (VFA) mixture with an identical concentration of VFAs as that found in an effective LSM reduced germination in an acid soil (pH 5.1) to the same extent as the LSM after 1 day of exposure. Germination was reduced by 45, 75, and 90% in the 10, 20, and 40% ([wt/wt] soil moisture) treatments, respectively, with the latter being equivalent to an application of 80 hl/ha. Addition to this acid soil of 19 LSMs (30% [wt/wt] soil moisture) collected from different producers resulted in complete kill of microsclerotia with 12 manures. Effective manures had a total concentration of nonionized forms of VFAs in soil solution of 2.7 mM or higher. In some acid soils (pH 5.8), addition of LSM (40% [wt/wt] soil moisture) did not kill microsclerotia until 3 to 6 weeks later. Here, a reduction in viability of microsclerotia was attributed to the accumulation of 0.06 mM nitrous acid in the soil solution at 4 weeks. When an LSM was added (40% [wt/wt] soil moisture) to an alkaline soil (pH 7.9) where VFAs are not toxic, microsclerotia germination was reduced by 80% after 1 week. Here the pH increased to 8.9 and the concentration of ammonia reached 30 mM in the soil solution. An ammonium chloride solution having an equivalent concentration of ammonium as the manure was shown to have the same spectrum of toxicity as the manure in assays ranging from pH 7 to 9, both in solutions and above the solutions. At pH 9, the concentration of ammonia reached 18 mM and 100% mortality of microsclerotia occurred. Thus, in acid soils, LSM can kill microsclerotia of V. dahliae by VFA and/or nitrous acid toxicity and in alkaline soils by ammonia toxicity. In order to take advantage of these mechanisms for disease reduction, the manure chemical composition, rate of addition, and soil characteristics need to be determined for each instance of use.

Reduction of potato scab, Verticillium wilt, and nematodes by soymeal and meat and bone meal in two Ontario potato fields.

Soymeal (SM) and meat and bone meal (MBM) were incorporated into soil to a depth of 15 cm at a rate of 37 t/ha at two commercial potato (Solatium tuberosum) fields in Ontario in the spring of 1996. The incidence of verticillium wilt and potato scab, the population of plant parasitic nematodes, and the soil chemistry and microbiology were monitored in three subsequent crops of potatoes in 1996-1998. Verticillium dahliae microsclerotia (MS) were buried in the soil immediately after incorporation of the amendments, both in the field and in a concurrent laboratory assay using soil from the field placed in test tubes, and the viability of the MS was determined 4 weeks later. Both treatments reduced the incidence of verticillium wilt, potato scab, and the nematode population to near zero levels in 1996. Reduced disease levels were still evident in the 1997 crop, but by 1998, disease levels were equal to or higher than those in the control treatments. Nematode numbers remained below control levels for the 3 year...

Soil factors influencing the efficacy of liquid swine manure added to soil to kill Verticillium dahliae

Addition of liquid swine manure (SwM) to field soils killed Verticillium dahliae microsclerotia (MS) and reduced verticillium wilt of potato, but only at one (site B) of several fields tested. This study examined what factors in soil influence the capability of SwM to kill V. dahliae MS. When added to soil from site B in a microcosm assay, the SwM used in the field study killed MS within 1 day after application. Also, efficacy increased as the concentration of SwM increased, indicating that one or more components were directly toxic to MS. Toxicity of SwM to MS was reduced with increasing soil moisture, indicating that the active product(s) was (were) undergoing dilution. Adjusting the pH of site B soil from 5.0 to 6.5 eliminated the toxicity of the SwM. Conversely, when soil from a location where SwM had no effect was reduced from its initial value of 7.5 to below 6, MS mortality occurred. Adding increasing concentrations of SwM at pH 7.7 to soils commonly raised the pH of the mixture to levels where efficacy of kill became progressively less effective. In soils with high buffering capacity, the pH did not immediately rise and MS kill increased with higher rates. Liquid swine manure killed MS to the same extent in soils ranging from sand to loam when these were made equal with respect to pH and SwM concentration. It was also equally effective in soils with organic carbon contents ranging from 1.4 to 6% when soil pH and moisture levels were made equal. Increasing soil temperature slightly improved the toxicity of the SwM. The results demonstrate that the efficacy of SwM in soil to kill V. dahliae MS is most influenced by soil pH and, to a lesser extent, by soil moisture level, buffering capacity, and temperature.

SUPPRESSION OF RHIZOCTONIA AND PYTHIUM DAMPING-OFF OF RADISH AND CUCUMBER SEEDLINGS BY ADDITION OF FISH EMULSION TO PEAT MIX OR SOIL

The efficacy of fish emulsion in enhancing plant growth and suppressing seedling damping-off diseases caused by Rhizoctonia solani and Pythium aphanidermatum was investigated on peat mix and soil. Fish emulsion (1%–4%; m/m peat mix) or equivalent inorganic fertilizer (N–P–K) was incorporated into pathogen-infested peat mix and incubated in plastic bags for 1, 7, 14, and 28 days prior to planting radish or cucumber seeds. Plants were rated 14 days later for incidence and severity of damping-off. Negligible protection of seedlings from damping-off occurred in peat mix incubated for 1 day with fish emulsion. After 7 days incubation, however, 70%–80% of the seedlings remained disease-free in peat mix amended with 4% fish emulsion. After 28 days, equivalent levels of disease protection were found with all concentrations of fish emulsion. As the inorganic N–P–K treatment was adjusted to reflect N–P–K levels in the fish emulsion, no disease control was obtained, indicating that disease protection was not due to increased plant nutrition. Incorporation of 0.5% (m/m soil) fish emulsion into soil 5 days before planting radish provided effective control of damping-off disease. Fish emulsion (2% and 4%; m/m muck soil) also effectively and consistently suppressed damping-off of cucumber seedlings in muck soil naturally infested with damping-off pathogens. Pasteurization of the peat mix followed by re-infestation with R. solani resulted in a much higher level of disease than that obtained in unpasteurized infested peat mix. Addition of fish emulsion resulted in restoration of disease suppression within 7 days. These results suggest that fish emulsion may not be toxic to the pathogens but may create a biological climate in peat substrate or soil that is suppressive to the disease. Fresh and dry mass measurements of plants produced in 4% fish emulsion were 2 to 3 times greater than in nonfertilized peat but were comparable with those receiving equivalent N–P–K. The results suggest that fish emulsion has both nutritive value for plant growth as well as disease suppressive properties. Thus, it may be an ideal product for use in organic or conventional transplant production.

A quantitative method for determining soil populations of Streptomyces and differentiating potential potato scab-inducing strains

A procedure is described for estimating Streptomyces populations in soil. Soils are air-dried, 10g quantities are shaken in plastic bags containing 0.1% water agar and homogenized with a Stomacher homogenizer, serial dilutions are plated on a semi-selective culture (STR) medium and incubated for 2 weeks at 22°C, and the Streptomyces colonies are enumerated. Use of STR medium reduced the bacterial and fungal colonies recovered from soil to levels below that of the Streptomyces spp. while not affecting the number of Streptomyces colonies compared with those enumerated on yeast malt extract medium. A procedure for screening large numbers of Streptomyces strains for thaxtomin production, a phytotoxin recognized as a virulence marker in S. scabies, is also described. Strains are grown on oatmeal medium, and the thaxtomin is extracted from the medium by facilitated diffusion and detected by miniature thin layer chromatography. S. scabies and S. acidiscabies strains (approximately 130 from Ontario and 70 from other locations in North America) that produced thaxtomin did not form aerial mycelia or sporulate on STR medium within 2 weeks at 22°C. Ontario S. scabies strains that produced thaxtomin A also produced melanin on STR medium. All S. scabies strains from scab lesions that produced thaxtomin A had this colony morphology, whereas only 4 to 9% of strains from soil with this colony morphology produced thaxtomin A. Using these procedures, we determined that the population of thaxtomin-producing S. scabies in soil from a potato field in Ontario with a history of potato scab was about 20,000 CFU/g soil.

Edaphic soil levels of mineral nutrients, pH, organic matter, and cationic exchange capacity in the geocaulosphere associated with potato common scab

ABSTRACT In order to determine possible relationships between geocaulosphere soil properties and severity of common scab of potato caused by Streptomyces scabies, soils were collected from representative commercial potato fields in Canada: in Simcoe and Dufferin Counties, Ontario and across Prince Edward Island (PEI) in August 2004. Soils immediately adjacent to tubers were sampled and analyzed for select edaphic factors and for pathogen presence using polymerase chain reaction (PCR) tests with primers that amplify a region of the TxtA gene involved in regulating the biosynthesis of the thaxtomin toxin family. Individual tubers were assessed visually for scab severity. The relationships between soil chemical factors and disease severity were investigated for each region to detect the strongest relationships. Principal component analysis revealed a distinctive clustering of samples with respect to disease severity in PEI but not in Ontario soils. Total and percent saturation of K (%K) were the only factors found associated with high disease severity in soils from both provinces. In PEI soils, pH, Mg, Ca, Cu, and %K, %Mg, %Ca, and %Na were associated with high disease severity, whereas cation exchange capacity (CEC) and Al were correlated with low disease severity soils. In Ontario, high Mn content was strongly correlated with low disease severity soils, whereas %K and organic matter content were correlated with disease severity. Partitioning samples into presence or absence of the TxtA PCR product with corresponding high or low severity showed further significant relationships in the data. There was an excellent correlation between Streptomyces spp. presence as detected by PCR and disease severity in PEI soils; however, the relationship was not as clear in Ontario soils, where many PCR-positive soils had low disease incidence. Principal component and partial least square analysis indicated that disease severity was predicted by soil factors such as organic matter, CEC, pH, Al, %Ca, %Mg, and %K for PEI but not for Ontario soils. The data reveal that the relationship between scab severity and soil chemical components is complex and potentially soil specific.