Chapter 4
Development and Assessment of Approaches to Physical and Chemical Soil Disinfestation methods for soil disinfestation are discussed, placing special emphasis on the hierarchy of development stages. An effective soil disinfestation method should address the following requirements.
Jaacov Katan Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, The R. H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel
• It must effectively reduce inoculum density at all sites
Abraham Gamliel
in the soil and to the desired depths (usually the cultivated soil layer, namely, 30–50 cm or more in soil and 10–20 cm in soilless culture), which should eventually lead to disease control. • Pathogen control should involve minimal disturbance of the biological, chemical, and physical components of the soil and surrounding environment (DeVay and Katan, 1991; McGovern and McSorley, 1997). This is especially important with regard to the phenomenon of a “biological vacuum,” namely, the drastic reduction of microbial activity that provides a platform for pathogen reinfestation (Chapter 15). Agents that are harmful to plants need to have dissipated by planting time. • It must be environmentally acceptable, with minimal hazards to nontarget living organisms and abiotic components of the environment. • It should be economically acceptable, technologically feasible, and free of safety problems during application.
Laboratory for Research on Pest Management Application, Institute of Agricultural Engineering, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel
Introduction Soil disinfestation was developed and introduced in the early days of modern plant pathology, around 1870 (Wilhelm, 1966). At that time, it was realized that reducing (and preferably eliminating) the inoculum density of a pathogen before planting would enable the production of a healthy crop. In practice, however, such goals were difficult to achieve. Two different approaches to soil disinfestation were developed: physical (by steam) and chemical (fumigation by volatile pesticides) (Newhall, 1955). It was only about 100 years later that a third approach, soil solarization, would be added to the disinfestations arsenal (Chapter 5). Since its introduction, soil fumigation has been, and still is, the major tool for soil disinfestation. Only a few fumigants have been developed and commercialized, however. The narrow spectrum of available measures for soil disinfestation clearly indicates that the development of new approaches or technologies in this field is a difficult and complicated task. Moreover, under the current climate of environmental concerns about pesticides as well as food safety, the potential negative attributes of any new measure are now followed even more closely, with stricter controls (Katan, 1993). Hence, only a few new measures make it to the commercialization field. In this chapter, the challenges and difficulties involved in developing and assessing new
The research that addresses the above requirements, and endeavors to develop and assess new soil disinfestation methods, is complicated and laborious. Moreover, it might produce misleading results, especially when the results are obtained only under laboratory conditions that do not represent real-life agricultural situations. This is especially true with soilborne pathogens since many chemical, physical, and biological processes take place in the soil, affecting both the pathogens and their antagonists (Katan, 1992). Below, an upscaling hierarchical approach is described for the development and assessment of a new or any chemical or nonchemical soil disinfestation method. Technical approaches and tools are discussed, from laboratory studies through field experiments to on-farm implementation, with the ultimate goal of successfully introducing a new approach
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as a practical and commercial tool. Lessons learned from the successful introduction of solarization in various regions can be helpful in developing new disinfestation methods. However, aspects involved in the invention of a fumigant, its registration, or related issues are not dealt with in this chapter.
Principles in Assessing a Soil Disinfestation Method Laboratory Studies Versus Field Work Laboratory studies for the development of a soil disinfestation method (or any pest-management tool) are less expensive and produce results much more quickly than do field studies. This is because the distribution of inoculum in field plots is not uniform and the plots are exposed to changing ambient conditions, parameters that do not affect laboratory studies. Most field studies can only be carried out at certain times of the year and they are quite expensive. On the other hand, the reliability of the results obtained under laboratory conditions, namely, the extent to which they represent real-life conditions, should be questioned and examined in every case. Thus, results obtained through studies carried out under regular agricultural conditions (open field or commercial greenhouse) should be the ultimate goal, but because they are more difficult and expensive to obtain, a proper combination of laboratory and field studies, as detailed below, can provide testing at a reduced cost, while maintaining reasonable reliability and validity.
Changes in Pathogen Populations During Soil Disinfestation: Naturally Existing Versus Added Inoculum as an Assessment Tool One of the major parameters in assessing control effectiveness is changes in inoculum density in the soil during soil disinfestation. Thus, inoculum density is determined before, during, and after disinfestation; in addition, its mortality rate is ascertained. Inoculum density can be determined either directly in samples taken from naturally infested and treated soils or in samples taken from external inoculum incorporated into the soil to various depths before disinfestation. Each approach has its pros and cons. Results from naturally infested soils are the most relevant and representative. This approach, however, requires the use of highly sensitive enumeration methods (since inoculum density in field soils is generally low) and numerous samplings (since variation in natural populations is high). Moreover, natural inoculum is genetically heterogeneous; for example, it may consist of several biotypes with different heat or pesticide sensitivities. Assessing external inoculum that has been introduced into the soil is more convenient but not always representative of the natural population, especially with regard to its sensitivity to control agents (e.g., heat, chemicals, antagonists,
and other biotic and abiotic factors). The incorporation of a representative inoculum containing naturally produced propagules (e.g., soil containing chlamydospores, oospores, sclerotia, or pieces of infected tissue, but not conidia from cultures) is recommended (Katan, 1992). Monitoring the rate of control by solarization over time, for example, the rate of killing sclerotia of Verticillium dahliae Kleb. in the soil, showed that extending the length of the solarization kills the pathogen down to the deeper layers (Fig. 4.1). This observation served as one of the guidelines in developing soil solarization in its initial stages, achieved through a combination of field study and monitoring of a pathogen population. A more complicated question is the relationship between the reduction of inoculum density and the reduction in disease incidence (ID-DI) with a reduction in disease incidence being the main goal. This issue is discussed in Chapter 1. Measuring only inoculum density might result in an under- or overestimation of the control method’s effectiveness (Martyn and Hartz, 1986); for example, if biocontrol processes, induced soil suppressiveness, or weakening of the propagules takes place after termination of the treatment as well as during the treatment, as shown in other studies (Adams, 1990; Eshel et al., 2000; Greenberger et al., 1987; Stapleton and DeVay, 1984; Chapter 15), these beneficial effects will not be reflected in the inoculum reduction that was only evaluated during the disinfestation process or shortly after its termination, although they might be reflected in disease reduction. On the other hand, with certain pathogens, even a drastic reduction in inoculum might not be sufficient to reduce the disease from an economical standpoint or other considerations (Campbell and Benson, 1994).
Figure 4.1. Effect of soil mulching with transparent polyethylene sheets (soil solarization) on the temporal control of Verticillium dahliae at soil depths of 5–25 cm. Control percentage was calculated by comparing with the respective nonmulched control. Incorporation means introduction of the inoculum in the soil before mulching, namely, length of the solarization period. (Reproduced from Katan et al., 1976)
Physical and Chemical Soil Disinfestation
Therefore, data on changes in inoculum density during soil disinfestation can be very useful for comparative studies, for studies on optimal application, or as mechanisms of control or ecological studies, but the data cannot be used as a single tool for predicting the examined soil disinfestation method’s effectiveness at controlling diseases.
Assessing the Involvement of Physical, Chemical, and Biological Changes in Soil Such assessments are usually performed in the laboratory under controlled conditions and include generationdissipation curves of the active ingredients of chemical control agents and their movement in packed soil columns, as well as changes in pathogen and microbial populations in the soil (Gan et al., 1996; Guo et al., 2003). However, the behavior of a disinfestant, either chemical or physical, in a small container in the laboratory under uniform conditions might be very different from its behavior in a large plot in the field under ambient conditions. Therefore, such tests are also carried out at a later stage under realistic agricultural conditions. Nevertheless, the data obtained in the laboratory can be very helpful, as further discussed below.
Stages in the Development of Soil Disinfestation Methods Developing a new disinfestation method dictates a series of steps that provide a platform for effective and efficient assessment of the wide spectrum of parameters. These steps include the following, in hierarchical order. 1. Laboratory studies under controlled conditions: These studies provide the basic database with regard to the spectrum of controlled pathogens, required dosages of chemicals or heating levels, and the various parameters of generation-dissipation behavior of fumigants in different soils and under various conditions. Special care should be given, however, to the methodology and experimental design of the experiments so that they reflect, as closely as possible, conditions in the field. 2. Small-plot studies: These studies involve testing the application method, biological activity, especially changes in pathogen populations, and when appropriate, chemical and physical analyses under field conditions. This stage does not involve crop growing. 3. Field experiments, in which the relevant crop is included: This step of the study includes assessment of application methods as well as pathogen mortality, along with assessments of disease control (and, in some cases, changes in pathogen populations) and crop yield and quality. 4. Large-scale on-farm field application: This is designed to validate the suitability of a new method for commercial crop production practices. A new technology
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can be implemented only if its application is feasible and cost effective, with minimal and acceptable risk to the environment and operator. Moreover, the application technology should align with common agricultural practices. Economic evaluations should also be included (Chapter 20). Below are detailed descriptions of the various stages involved.
Controlled-Environment Studies Dose-Response Studies These studies are usually carried out in controlled-environment systems in which the relationships between dosage of the disinfestant (fumigant or heating) and viability of the pathogen are determined (Munnecke and Van Gundy, 1979; Pullman et al., 1981; Shlevin et al., 2003; Chapter 17). The dose-response studies examine the rate at which the agent is optimally effective, in a standardized and accurate manner. Furthermore, with standardized controlled systems, the effect of various conditions on pathogen control, separately or combined (e.g., soil type, moisture, temperature, and interactions with other chemicals), can be assessed. These systems are especially useful for comparing to a reference control agent. For example, the relative potency values of methyl iodide to methyl bromide were found to be 2.7-fold higher for most fungi (Hutchinson et al., 2000). The toxic effect of a disinfestant is affected by two dosage components: concentration of the control agent (C ) and duration of its exposure (T ). The lethal effect increases with increasing exposure time or dose (Gamliel, 2005; Rozman and Doull, 2000). The C×T product is a standard value that is used to quantify the effect of chemicals or heat on pathogen viability (Eshel et al., 1999). It is calculated, for each dosage, as the area under the exposure time curve (Gamliel et al., 1998b; Triky-Dotan et al., 2007). The results from a controlled study can reflect field conditions only if the following items are considered.
• The tested propagules should be relevant for survival in
the soil (e.g., resting structures such as sclerotia, infected plant tissues, and eggs of root-knot nematodes in infected roots). In contrast, cultures of fungi or bacteria can only provide relative data that are not necessarily relevant to field conditions. Thus, it was demonstrated by Munnecke and Van Gundy (1979) that the C×T of methyl bromide required to control sclerotia of Sclerotium rolfsii Sacc. is 1.35 times higher than that needed to eliminate the viability of mycelium of the fungus. In contrast, the C×T of methyl bromide required to control mycelium of Phytophthora cinnamomi Rands is 1.51 times higher than that needed to eliminate chlamydospores of this pathogen. • The conditions in a laboratory study should reflect, as closely as possible, realistic conditions. Closed containers create anoxic and anaerobic conditions within a
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Chapter 4
few hours of sealing. Hence, testing the lethal effect of an agent may overlook the lack of oxygen as a control factor. Creating a soil atmosphere that emulates field conditions—such as oxygen content and fluctuations in temperature—is complicated, yet both feasible and important (see Simulation Systems).
Chemical Analyses Detection and quantification of the active molecules in a disinfestation agent are essential factors in developing and assessing a new chemical soil disinfestation method, such as fumigation and biofumigation. Dissipation studies of a chemical control agent allow its mode of action to be investigated and effective means for its application to be developed. Leaching and volatilization are unlikely to occur in sealed containers. Hence, the dissipation curves of a chemical disinfestant only reflect its chemical or biological degradation or adsorption to soil particles. These can be assessed in soil columns (Guo et al., 2003). The dissipation curves can be used to calculate the C×T products and assess the length of time the disinfestant is effectively retained in the soil (Gamliel et al., 1998a, 1998b). In addition, the information provided is important for safely planting without phytotoxic effects. A simple procedure for rapid sampling and analysis, which can also be used later in field studies, is essential for obtaining valid conclusions. A chemical analyzer based on pumping the soil atmosphere through a thermal conductivity cell is a good example of a practical and useful tool (De Groot et al., 1974). It was used to monitor methyl bromide and served as an important tool in developing an improved use of this fumigant with impermeable films at reduced dosage (Gamliel et al., 1997b, 1998b). However, it cannot be used with other volatile compounds because of inadequate sensitivity to other fumigants, such as methyl iodide and dimethyl disulfide, and the interference of other gases, such as carbon dioxide. With volatile compounds, gas chromatography is an accurate and reliable procedure for such purposes, although it carries with it the logistics of delivering samples from the field to the laboratory; thus, it can only be performed for fields that are in close proximity to the laboratory (Klein et al., 2007). Alternatively, portable gas chromatography equipment can be used, but this equipment is expensive and, therefore, not frequently available. Another difficulty in interpreting the results from controlled systems is that these studies are usually carried out at constant temperatures. Naturally fluctuating ambient temperatures are very difficult to simulate. This issue is further discussed below and in Chapter 17.
deeper layers; and in artificial growth substrates, such as perlite, which involves shallow layers. Each situation has its own requirements for controlling soil pests. Soil columns provide the most rapid method of testing applications for each particular objective and for assessing the movement of a chemical disinfestant and its breakdown products in soil, which is packed within the column. Soil columns also serve to test the C×T values at various depths, as well as the control of bioassay organisms. Various soil columns have been designed and tested based on specific requirements; their size can also be modified as needed. Inocula of the tested organisms are placed at different depths from the top of the column during packing. The disinfestant is applied to the soil in the column to the desired location (depth), and additional actions (such as irrigation) are applied to further distribute the disinfestant and assess its movement. Sealing the column with various tarps can also provide information on disinfestant escape through volatilization. The movement of a disinfestation agent within the soil, and its vertical and horizontal distribution, are important factors in determining the optimal formulation (in the case of chemicals) and method of application. Soil columns can be used to assess the movement and retention of a chemical in soil, as well as its adsorption, volatilization, and degradation. The soil moisture must be maintained at optimum levels, however, and special care should be given to the methodology of soil packing in the columns to maintain soil compaction conditions that reflect, as closely as possible, those found in the field.
Small Field Plots Small-field-plot studies are an intermediate stage between laboratory studies and field trials. This system provides an appropriate and realistic platform, on a small-field scale, for the relatively rapid assessment of biological, physical, and chemical variables, some of which cannot be tested in the laboratory or are not yet ready enough for large trials
Movement of Pesticides in a Soil Column (Soil Microcosm) Disinfestation can be applied to different field situations, such as in soil for annual crops; in potting soil for use in pots or containers, which is high in organic content; in soil for perennial crops, which involves disinfestation to
Figure 4.2. Assessment of various soil fumigants in small field plots (3 × 3 meters each). Note the different plastic films being tested and the shading that is applied in some plots to prevent a solarization effect.
Physical and Chemical Soil Disinfestation
(Fig. 4.2). Small field plots are less expensive than field trials and do not include the complexity of crop growing and long cropping seasons. They should not be regarded as an extended soil-column design since the plot size, application method, and application rates of the disinfestant more closely resemble field conditions and are relevant for later application in larger field plots. The assessed disinfestation agent is applied to the soil and exposed to ambient conditions (Eshel et al., 1999; Gamliel et al., 1997b). The plots must be large enough (at least 3 × 3 m) to reduce border effects (Grinstein et al., 1995; Chapter 16), namely, a decreasing gradient of control agent dosage toward the edges of the plot, including rapid dissipation of the chemical or a decrease in soil temperature (in heating processes). However, it might be difficult to apply small amounts of a chemical under field conditions. The small-plot approach also enables testing the new method with different soil types, at different times of the year, and under different conditions (Gamliel et al., 1998b; Ramirez-Villapudua and Munnecke, 1988). Small field plots allow, under field conditions, the temporal reduction in inoculum density (naturally occurring or introduced) at various depths to be followed; the concentration of the disinfestant, as well as the C×T, to be monitored over time; and the microbial, physical, and chemical changes in the soil to be recorded (Becker et al., 1998). To achieve relevant and reliable results, the plots must be prepared properly, including the installation of measurement tools for pathogen inoculum, headspace or tubing to sample volatile compounds during the course of the process, and probes to monitor soil temperature at different depths. The small-field-plot approach has been used successfully by many researchers to explore the effectiveness of soil solarization under local conditions. For example, this approach allows, in a relatively inexpensive manner, the optimal time for solarization in a particular region to be determined and, at the next stage, detailed field experiments to be carried out during that optimal period. It is also a very useful method for rapidly testing the effectiveness of new films in elevating soil temperatures and enhancing biological activity.
Delivery and Distribution of the Agent in Soil The distribution of the disinfestant in the soil can be controlled by the application system and its execution. The primary distribution can be precisely controlled by manual injection (using special equipment for small plots) to the desired depth. It can be applied using a drip-irrigation system that is installed with the appropriate flow rate and spacing between drip lines and emitters. The primary distribution of solid material can be applied via its incorporation into the soil by the appropriate (or tested) methods (e.g., rotovation). The secondary distribution is monitored at different depths, preferably by sampling with special instruments that have been installed prior to its application. Alternatively, soil samples can be taken and the disinfestant extracted. The latter method, however, is destructive and may affect the continuity of the experiment.
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Chemical and Physical Changes Sampling devices, such as gas tubing and charcoal traps, are buried at the desired depths in the soil, with the end of the tubing extended to the soil surface. The concentration of the tested disinfestant in the soil atmosphere at each depth is recorded and quantified using the appropriate method. Concentration measurements are taken until the disinfestant decreases to below detectable levels. The data are used to calculate C×T and relate it to reduction in pathogen viability. Soil temperature at various depths is measured using the appropriate measuring device during the relevant period and intervals (Eshel et al., 1999; Gamliel et al., 1998a; Klein et al., 2007; Triky-Dotan et al., 2007).
Inoculum Viability In addition to assessing the effectiveness of pathogen control, viability tests can serve as an indirect indicator of the disinfestant’s distribution. This is useful as a bioassay, especially when the experimental conditions, or lack of appropriate means, prevent direct measurements (chemical or physical) of the disinfestant. The inoculum-viability assay must include the appropriate preconditioning, such as wetting, to avoid experimental errors. The inoculum should be placed in net bags, at different spacing, attached to a string, which allows for accurate placement in the soil and easy retrieval. The inoculum propagules must be in a form that allows reliable recovery using the appropriate microbiological methods. Alternatively, these bags can be attached to a mesh grid that is buried vertically during plot preparation. Methods for assessing viability are described in Chapter 2.
Phytotoxic Effect The residual effect of the disinfestant is easily and rapidly assessed in small-field-plot systems. The assessment can be performed by planting a bioassay plant (seed or transplant) directly into the plots. Alternatively, soil samples are taken from different depths and the bioassay plant is grown in the sampled soil in containers under controlled conditions. The assessment can be performed at different intervals from termination of the treatment to identify the critical time for planting (Semer, 1987).
Field Experiments The purpose of a field experiment is to assess, in infested soil, the effect of the tested soil disinfestation method on pathogen viability, disease control, crop development, and yield components, as well as side effects, under practical agricultural conditions with representative open-field or protected crops. Field experiment parameters (e.g., disinfestant rate and application method) are based on the results obtained in the controlled studies and the small-field-plot trials. The research team performs the disinfestation treatments, while the farmer, or staff member of a research unit, carries out the crop management in coordination with the research team.