A.J. Termorshuizen

Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping.

ABSTRACT A new method for the control of soilborne plant pathogens was tested for its efficacy in two field experiments during two years. Plots were amended with fresh broccoli or grass (3.4 to 4.0 kg fresh weight m(-2)) or left nonamended, and covered with an airtight plastic cover (0.135 mm thick) or left noncovered. In plots amended with broccoli or grass and covered with plastic sheeting, anaerobic and strongly reducing soil conditions developed quickly, as indicated by rapid depletion of oxygen and a decrease in redox potential values to as low as -200 mV. After 15 weeks, survival of Fusarium oxysporum f. sp. asparagi, Rhizoctonia solani, and Verticillium dahliae in inoculum samples buried 15 cm deep was strongly reduced in amended, covered plots in both experiments. The pathogens were not or hardly inactivated in amended, noncovered soil or nonamended, covered soil. The latter indicates that thermal inactivation due to increased soil temperatures under the plastic cover was not involved in pathogen inactivation. The results show the potential for this approach to control various soilborne pathogens and that it may serve as an alternative to chemical soil disinfestation for high-value crops under conditions where other alternatives, such as solarization or soil flooding, are not effective or not feasible.

Manure-amended soil characteristics affecting the survival of E. coli O157:H7 in 36 Dutch soils

The recent increase in foodborne disease associated with the consumption of fresh vegetables stresses the importance of the development of intervention strategies that minimize the risk of preharvest contamination. To identify risk factors for Escherichia coli O157:H7 persistence in soil, we studied the survival of a Shiga-toxin-deficient mutant in a set of 36 Dutch arable manure-amended soils (organic/conventional, sand/loam) and measured an array of biotic and abiotic manure-amended soil characteristics. The Weibull model, which is the cumulative form of the underlying distribution of individual inactivation kinetics, proved to be a suitable model for describing the decline of E. coli O157:H7. The survival curves generally showed a concave curvature, indicating changes in biological stress over time. The calculated time to reach the detection limit ttd ranged from 54 to 105 days, and the variability followed a logistic distribution. Due to large variation among soils of each management type, no differences were observed between organic and conventional soils. Although the initial decline was faster in sandy soils, no significant differences were observed in ttd between both sandy and loamy soils. With sandy, loamy and conventional soils, the variation in ttd was best explained by the level of dissolved organic carbon per unit biomass carbon DOC/biomC, with prolonged survival at increasing DOC/biomC. With organic soils, the variation in ttd was best explained by the level of dissolved organic nitrogen (positive relation) and the microbial species diversity as determined by denaturing gradient gel electrophoresis (negative relation). Survival increased with a field history of low-quality manure (artificial fertilizer and slurry) compared with high-quality manure application (farmyard manure and compost). We conclude that E. coli O157:H7 populations decline faster under more oligotrophic soil conditions, which can be achieved by the use of organic fertilizer with a relatively high C/N ratio and consequently a relatively low rate of nutrient release.

Integrated approaches to root disease management in organic farming systems

Conventional agriculture has had major environmental impacts, in particular with respect to soil degradation. Soil structure, fertility, microbial and faunal biodiversity have declined, and root diseases are common unless genetic resistance, soil fumigation and/or seed treatments are used. Primarily for environmental reasons and increasing demands for safe and healthy food from the public, farmers have switched over to organic production at an increasing rate. During a transition period of about 5 years, organic farmers may face problems with yield losses and pest or disease problems. However, in well-managed, long-term organic farms, soilborne diseases need not be a problem. Several studies in which disease severity was compared in organic and conventional farming systems (or with soils from those systems) showed that root diseases are generally less severe in organically than conventionally managed soils. The reasons for reduced root disease severity have seldom been investigated, although relationships with nitrogen supply or microbial diversity have sometimes been found. Crop protection in organic farming is generally not directed at controlling particular pathogens or pests but at management of the environment so that plants are able to withstand potential attacks. Resistant cultivars adapted to the local conditions are in demand among organic farmers. However, the main practices that contribute to disease control are long, balanced rotations, organic amendments and reduced tillage, all geared towards maintenance of the soil organic matter content and fertility. Organic farmers can make use of biological control agents and natural toxic compounds in plant extracts. However, these practices are methods of last resort.Conventional agriculture has had major environmental impacts, in particular with respect to soil degradation. Soil structure, fertility, microbial and faunal biodiversity have declined, and root diseases are common unless genetic resistance, soil fumigation and/or seed treatments are used. Primarily for environmental reasons and increasing demands for safe and healthy food from the public, farmers have switched over to organic production at an increasing rate. During a transition period of about 5 years, organic farmers may face problems with yield losses and pest or disease problems. However, in well-managed, long-term organic farms, soilborne diseases need not be a problem. Several studies in which disease severity was compared in organic and conventional farming systems (or with soils from those systems) showed that root diseases are generally less severe in organically than conventionally managed soils. The reasons for reduced root disease severity have seldom been investigated, although relationships with nitrogen supply or microbial diversity have sometimes been found. Crop protection in organic farming is generally not directed at controlling particular pathogens or pests but at management of the environment so that plants are able to withstand potential attacks. Resistant cultivars adapted to the local conditions are in demand among organic farmers. However, the main practices that contribute to disease control are long, balanced rotations, organic amendments and reduced tillage, all geared towards maintenance of the soil organic matter content and fertility. Organic farmers can make use of biological control agents and natural toxic compounds in plant extracts. However, these practices are methods of last resort.

Estimating the stability of Escherichia coli O157:H7 survival in manure‐amended soils with different management histories

The objective of this study is to describe survival of Escherichia coli O157:H7 populations in manure-amended soils in terms of population stability, i.e. the temporal variation around the decline curve, in relation to soil characteristics indicative of soil health. Cow manure inoculated with E. coli O157:H7 was mixed with 18 pairs of organically and conventionally managed soils (10% of manure, kg kg(-1)). For four of the soil pairs, also three different manure densities (5%, 10% and 20%) were compared. All soil-manure mixtures were incubated for 2 months, and population densities of E. coli O157:H7 were quantified weekly. De-trending of survival data was done by modified logistic regression. The residual values were used to assess variation in the changes of E. coli O157:H7 populations by performing the approximate entropy (ApEn) procedure. The term irregularity is used to describe this variation in ApEn literature. On average, the decline of E. coli O157:H7 was more irregular in conventional and loamy soils than in organic and sandy soils (P < 0.05). Multiple regression analysis of irregularity of E. coli O157:H7 survival on 13 soil characteristics showed a positive relation with the ratio of copiotrophic/oligotrophic bacteria, suggesting greater instability at higher available substrate concentrations. Incremental rates of manure application significantly changed the irregularity for conventional soils only. Estimation of temporal variation of enteropathogen populations by the ApEn procedure can increase the accuracy of predicted survival time and may form an important indication for soil health.

Long-Term Effect of Biological Soil Disinfestation on Verticillium Wilt

Biological soil disinfestation (BSD), involving incorporation of grass combined with plastic mulching, eliminates many soilborne pests and diseases through the creation of anaerobic conditions. BSD was compared at two locations with a nontreated control, Italian ryegrass amendment alone, and plastic mulch alone. After the soil treatments, plots were cropped with Acer platanoides and Catalpa bignonioides and grown for 4 years. Relative to the control, soil inoculum levels of Verticillium dahliae were reduced by 85% after BSD and did not increase for 4 years. Populations of Pratylenchus fallax, known for their interaction with V. dahliae, in the soil and in roots were reduced by 95 to 99%. The incidence of infection by V. dahliae was reduced by 80 to 90%. Verticillium wilt severity was significantly reduced in A. platanoides in all 4 years at one location and in the first 2 years at the other location, and significantly fewer plants died at one location. Shoot length and trunk width were larger after BSD compared with the control at one location. Market value of the crop in BSD plots was up to € 140,000 ha-1 higher for A. platanoides and up to € 190,000 ha-1 higher for C. bignonioides than in the untreated control. BSD is an effective, economically profitable, and environmentally friendly control method for tree nurseries.

Prevalence of Shiga Toxin-Producing Escherichia coli stx1, stx2, eaeA, and rfbE Genes and Survival of E. coli O157:H7 in Manure from Organic and Low-Input Conventional Dairy Farms

ABSTRACT Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx1 (encoding Shiga toxin 1), stx2 (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx1, and stx2, which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.

Effect of organic management of soils on suppressiveness to Gaeumannomyces graminis var. tritici and its antagonist, Pseudomonas fluorescens

Organic management of soils is generally considered to reduce the incidence and severity of plant diseases caused by soil-borne pathogens. In this study, take-all severity on roots of barley and wheat, caused by Gaeumannomyces graminis var. tritici, was significantly lower in organically-managed than in conventionally-managed soils. This effect was more pronounced on roots of barley and wheat plants grown in a sandy soil compared to a loamy organically-managed soil. Fluorescent Pseudomonas spp. and in particular phlD+ pseudomonads, key factors in the take-all decline phenomenon, were represented at lower population densities in organically-managed soils compared to conventionally-managed soils. Furthermore, organic management adversely affected the initial establishment of introduced phlD+P. fluorescens strain Pf32-gfp, but not its survival. In spite of its equal survival rate in organically- and conventionally-managed soils, the efficacy of biocontrol of take-all disease by introduced strain Pf32-gfp was significantly stronger in conventionally-managed soils than in organically-managed soils. Collectively, these results suggest that phlD+Pseudomonas spp. do not play a critical role in the take-all suppressiveness of the soils included in this study. Consequently, the role of more general mechanisms involved in take-all suppressiveness in the organically-managed soils was investigated. The higher microbial activity found in the organically-managed sandy soil combined with the significantly lower take-all severity suggest that microbial activity plays, at least in part, a role in the take-all suppressiveness in the organically-managed sandy soil. The significantly different bacterial composition, determined by DGGE analysis, in organically-managed sandy soils compared to the conventionally-managed sandy soils, point to a possible additional role of specific bacterial genera that limit the growth or activity of the take-all pathogen.

Quality of methods to quantify microsclerotia of Verticillium dahliae in soil

Existing methods used to quantify microsclerotia of Verticillium dahliae in soil are reviewed. Most quantification methods are soil-type dependent, but are useful for disease prediction within certain soils. The major factor determining the accuracy of dry plating methods is the amount of soil plated per Petri dish. Wet plating methods are less sensitive to higher amounts of soil, especially when the fraction smaller than 20 um is removed by wet sieving. Despite general assumptions, wet plating methods do not have lower detection limits than dry plating methods. Dry plating methods are less variable at higher inoculum levels, but more variable at low inoculum levels. Bioassays are helpful tools in answering specific research questions, but are not convenient for large scale use. Molecular quantification techniques are promising, because they are not hampered by antagonistic effects, but data on their disease predictive abilities are still largely lacking. Suggestions are given for a better comparison of techniques, and some original results are presented to illustrate certain arguments.

Influence of aerobic and anaerobic conditions on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in Luria–Bertani broth, farm-yard manure and slurry

The influence of aerobic and anaerobic conditions on the survival of the enteropathogens Escherichia coli O157:H7 and Salmonella serovar Typhimurium was investigated in microcosms with broth, cattle manure or slurry. These substrates were inoculated with a green fluorescent protein transformed strain of the enteropathogens at 10(7) cells g(-1) dry weight. Survival data was fitted to the Weibull model. The survival curves in aerobic conditions generally showed a concave curvature, while the curvature was convex in anaerobic conditions. The estimated survival times showed that E. coli O157:H7 survived significantly longer under anaerobic than under aerobic conditions. Survival ranged from approximately. 2 weeks for aerobic manure and slurry to more than six months for anaerobic manure at 16 °C. On average, in 56.3% of the samplings, the number of recovered E. coli O157:H7 cells by anaerobic incubation of Petri plates was significantly (p < 0.05) higher in comparison with aerobic incubation. Survival of Salmonella serovar Typhimurium was not different between aerobic and anaerobic storage of LB broth or manure as well as between aerobic and anaerobic incubation of Petri dishes. The importance of changes in microbial community and chemical composition of manure and slurry was distinguished for the survival of E. coli O157:H7 in different oxygen conditions.

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.