SOIL AND PLANT SAMPLING TECHNIQUES
x,^> Soilborne fungal pathogens and the diseases they cause are inherently difficult to study because of the environment in which they are found. The infected root is not readily visible, and aerial disease symptoms are often similar to those caused by other factors such as flooding, drought, nutrient deficiency, soil compaction, insects, and herbicide misapplication. Therefore, before a given symptom can be positively attributed to a soilborne fungal pathogen, soil and/or root samples must be obtained. The following sections include general information that should be considered when sampling. More detailed information can be found in the chapters by W. L. Bland and K. B. Johnson in Section III.
Plant sampling- Two basic questions to consider when sampling are: "When did the plant become infected?" and "What part of the plant should be collected?" To answer these questions, it is important to have some understanding of both the pathogen and the disease of interest. Pathogens causing seedling diseases frequently infect their host(s) soon after seed germination. These same fungi may be essentially nonpathogenic to older plants. Likewise, some pathogens only infect mature plants and not seedlings. This phenomenon may represent a true difference in host-tissue susceptibility over time, or merely illustrate the importance of specific environmental conditions required for host infection and disease development. Regardless of plant age, if the purpose of plant sampling is to isolate a suspected pathogen, one should sample as soon as disease symptoms appear or even before. It is usually difficult to isolate pathogens from plant tissue in advanced stages of decay. If the purpose of sampling is to determine when infection occurs, it should be initiated long before any disease symptoms appear. Many soilborne fungi infect plants soon after seedling emergence, but disease symptoms are not expressed until the plant experiences some type of environmental stress such as heat or drought.
The second question (i.e., what part of the plant should be collected) also depends on the specific purpose of the sampling. Again, it is helpful to have some preliminary information on the pathogen and disease. Soilborne fungal pathogens are often localized or restricted to a particular part of the root such as the vascular system, cortex, or root tips. Attempts to detect or isolate a specific pathogen can be frustrating if the wrong plant tissue is sampled. For instance, Polymyxa betae Keskin, the vector of beet necrotic yellow vein virus, is restricted to cortical tissue and will not be found in older lateral roots or in the sugar beet tap root. Observation of these tissues could lead to the erroneous conclusion that Polymyxa was not present in a root sample. Such a wrong conclusion or diagnosis could have serious consequences.
When sampling to isolate a root-infecting fungal pathogen, isolation from root samples is not always the best choice. Even though the fungus may be isolated from roots, it may be easier to obtain pure cultures from other portions of the plant. An example of this is Bipolaris sorokiniana (Sacc.) Shoemaker, the cause of common root rot of wheat, which can be isolated very easily from the subcrown internode. Aphanomyces cochlioides Drechs. is best isolated from the hypocotyl of diseased sugar beet seedlings. Several vascular wilt pathogens
can be isolated from aboveground plant parts such as stems and leaf petioles. In this regard, it is important to be aware that different fungal species or anastomosis groups (AGs) of Rhizoctonia solani Kuhn, may predominate on a particular portion of the plant. On sugar beet, AG-4 is the predominant seedling pathogen, but older beets are mainly infected by AG2-2. Similarly, in wheat, AG-8, the cause of bare patch, mainly infects young roots and root tips, whereas AG-4 infects the crown and lower stem and leaf sheaths.
One should be open-minded when first sampling plants for purposes of pathogen isolation and identification. Producers are often acutely aware of disease problems, but frequently group them all together under the generic designation of "rot." There is always a possibility that unidentified pathogens are present and responsible for disease symptoms thought to be caused by another pathogen. If an individual begins sampling with a preconceived idea of the cause of the problem, the true causal agent might be overlooked. When possible, entire plants should be sampled with as much of the root system intact as is reasonably possible. In addition, apparently healthy plants also should be included when sampling, for comparison purposes.
Soil sampling- Soil sampling is usually done to determine the presence, inoculum density, or distribution of a particular pathogen. As with root sampling, timing is an important factor to consider. Pathogen populations are dynamic, both quantitatively and qualitatively. For instance, many pathogens, such as Phymatotrichwn omnivorum (Shear) Dugger, Sclerotium rolfsii Sacc., and R. solani, overwinter as sclerotia or some other dormant form in relatively low numbers. However, during the growing season when a susceptible crop is in the field, these fungi can achieve high populations as mycelium growing through the soil and colonizing host tissue or crop residues. Because fungi occur in various forms in the soil, inoculum or fungal density is often expressed as colony forming units per weight or volume of soil (i.e., CFU/g or CFU/10 mm3), as opposed to a more specific designation of fungal structure such as oospores, conidia, or chlamydospores. The fact that various forms do predominate at different times is important, however, because they can differ in pathogenicity (i.e. inoculum potential) and in their ability to survive storage.
Once sampling time has been determined, the actual process, while possibly labor intensive, is usually simple and straightforward. Numerous tools and devices have been developed for soil sampling, but the best one to use depends on the depth at which the sample must be taken, the volume of soil required per sample, and the total number of samples needed. Perhaps the most difficult part of soil sampling is determining how many samples must be taken, and preventing cross contamination between samples. This issue is especially important when sampling soil from different depths or across a large field with potentially different soil types. The following section includes information on statistical considerations when sampling.
Sampling strategy- If an investigator wishes to determine the amount of inoculum in a field, it is necessary to sample the soil. Although the mechanics are straightforward, one is immediately faced with determining the appropriate number of samples and their arrangement in the field, since fungal inoculum is not uniformly distributed throughout a field (Campbell and Noe, 1985). Several recent studies have focused on the problems associated with sampling soil in order
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to estimate the populations of soilbome fungi (Lin et al, 1979; Mihail, 1987). These studies have compared various methods of arranging sampling sites within a field. The simplest is to walk along a diagonal path from one corner of the field to the opposite corner and take 10-20 samples equally spaced along the path. However, this approach does not distribute the sampling sites well over the field (Lin et al, 1979; Mihail, 1987). A more effective approach is to traverse a "diamond" or "W" shaped path through the field, collecting 20-30 samples equally spaced along the path (Lin et al, 1979; Mihail, 1987). Once samples are collected, they may be bulked prior to assay, however, this does not permit the investigator to estimate the variability in pathogen population occurring within the field. If the samples are assayed individually, a mean and variance may be estimated for the pathogen population. However, these two measures do not provide information concerning the spatial pattern of the inoculum. If information of this type is desired, the reader should consult the review article of Campbell and Noe (1989) or studies of spatial pattern with specific soilborne fungi. The consideration of sampling strategies as they relate to crop loss assessment is treated in the
chapter by K. B. Johnson
Communicate in the upcoming series
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