George Lazarovits

Ammonia and Nitrous Acid from Nitrogenous Amendments Kill the Microsclerotia of Verticillium dahliae

ABSTRACT This study examined the mechanisms by which nitrogenous amendments such as meat and bone meal kill the soilborne plant pathogen Verticillium dahliae. The effect of nitrogen products from the amendments on the survival of microsclerotia of V. dahliae was examined by solution bioassay and soil microcosm experiments. Ammonia and nitrous acid but not their ionized counterparts, ammonium and nitrite, were toxic to microsclerotia in bioassays. In microcosms, addition of meat and bone meal (2.5%) to an acidic loamy sand resulted in the accumulation of ammonia and death of microsclerotia within 2 weeks. At lower concentrations (0.5 and 1%), microsclerotia were killed after 2 weeks when nitrous acid accumulated (>0.03 mM). In an alkaline loam soil, microsclerotia survived at 3% meat and bone meal and neither ammonia nor nitrous acid accumulated. The toxicity of ammonia to the pathogen was verified by increasing the concentration of meat and bone meal to 4% or addition of urea (1,600 mg of N per kg) to the loam soil resulting in the accumulation of ammonia (>35 mM) and death of microsclerotia. The toxicity of nitrous acid was verified by adding ammonium sulfate fertilizer to an acidic sand soil. Inhibiting nitrification with dicyandiamide revealed that nitrous acid was generated as a result of the accumulation of nitrite and an acidic pH. Thus, levels to which the toxins accumulated and the effective concentration of amendment were dependent upon the soil examined. Of the two mechanisms identified, accumulation of nitrous acid is the more promising strategy to control plant diseases in acidic soil because it is more toxic than ammonia and is formed at lower concentrations of amendments.

Inoculation Effects of Pseudomonas putida, Gluconacetobacter azotocaptans, and Azospirillum lipoferum on Corn Plant Growth Under Greenhouse Conditions

Alcohol production from corn is gaining importance in Ontario, Canada, and elsewhere. A major cost of corn production is the cost of chemical fertilizers and these continue to increase in price. The competitiveness of alcohol with fossil fuels depends on access to low-cost corn that allows growers to earn a sustainable income. In this study we set out to determine if we can identify root-associated microorganisms from Ontario-grown corn that can enhance the nutrient flow to corn roots, directly or indirectly, and help minimize the use of extraneous fertilizer. Bacteria were isolated from corn rhizosphere and screened for their capacity to enhance corn growth. The bacteria were examined for their ability to fix nitrogen, solubilize phosphate, and produce indole acetic acid (IAA) and antifungal substances on potato dextrose agar. Bacterial suspensions were applied to pregerminated seed of four corn varieties (39D82, 39H84, 39M27, and 39T68) planted in sterilized sand and unsterilized cornfield soil. The plants were grown under greenhouse conditions for 30 days. Three isolates were identified as having growth-promoting effect. These bacteria were identified as to species by biochemical tests, fatty acid profiles, and 16S rDNA sequence analysis. Corn rhizosphere isolates, Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, and Azospirillum lipoferum N7, provided significant plant growth promotion expressed as increased root/shoot weight when compared to uninoculated plants, in sand and/or soil. All strains except P. putida CQ179 were capable of nitrogen fixation and IAA production. Azospirillum brasilense, however, produced significantly more IAA than the other isolates. Although several of the strains were also able to solubilize phosphate and produce metabolites inhibitory to various fungal pathogens, these properties are not considered as contributing to growth promotion under the conditions used in this study. These bacteria will undergo field tests for their effect on corn growth.

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...

Differential Colonization of Tomato Roots by Nonpathogenic and Pathogenic Fusarium oxysporum Strains May Influence Fusarium Wilt Control

ABSTRACT Histochemical staining, beta-glucuronidase (GUS) activity, or placing roots on agar were methods used to characterize interactions between the pathogenic fungus, Fusarium oxysporum f. sp. lycopersici, and the nonpathogenic biocontrol F. oxysporum strain 70T01 with respect to colonization behaviors, interaction sites, and population densities on tomato roots. Mycelia of strain 70T01, a genetic transformant expressing stable GUS activity, hygromycin B resistance, and effective disease control, were localized in epidermal and cortex cell layers of tomato roots in a discontinuous and uneven pattern. In contrast, mycelia of F. oxysporum f. sp. lycopersici were found in the vascular bundles. Thus, direct interactions between the two fungi likely happen in the root surface cell layers. Colonization density of strain 70T01 was related to the inoculation density but decreased with distance from the inoculation site. Host defense reactions, including increased cell wall thickness or papilla deposits, were adjacent to 70T01 hyphae. Experiments done in soil showed that strain 70T01 densities in roots were highest at inoculation zones and barely detectable for root segments more than 2 cm away from the inoculation sites. F. oxysporum f. sp. lycopersici densities were lowest at 70T01 inoculation zones and highest (>10 times) where strain 70T01 was not directly applied. Newly elongating roots where strain 70T01 did not reach were available for infection by the pathogen. The higher strain 70T01 density was always found when the plants were simultaneously infected by F. oxysporum f. sp. lycopersici, suggesting that F. oxysporum f. sp. lycopersici has as much influence in predisposing the plant to colonization by strain 70T01 as strain 70T01 has on providing disease protection against the pathogen.

A mail survey of growers to estimate potato common scab prevalence and economic loss in Canada

A mail-out survey of Canadian potato growers was conducted in February 2003 to determine common scab prevalence and extent of economic loss resulting from scab for their 2002 yield. A survey of this type is feasible given that the disease is readily observed on the surface of the tubers. A questionnaire was sent to grower associations for distribution to a sample of their membership. All provinces except British Columbia and Newfoundland and Labrador participated. The overall response rate (24%) was comparable to other mail surveys sent to growers. Response bias may therefore influence estimates. The mean prevalence of scab was 82%, representing a mean economic loss of Can