NIESA Training Manual NEMATOLOGY TRAINING MANUAL FUNDED BY NIESA and UNIVERSITY OF NAIROBI, CROP PROTECTION DEPARTMENT CONTRIBUTORS: J. Kimenju, Z. Sibanda, H. Talwana and W. Wanjohi 1 NIESA Training Manual CHAPTER 1 TECHNIQUES FOR NEMATODE DIAGNOSIS AND HANDLING Herbert A. L. Talwana Department of Crop Science, Makerere University P. O. Box 7062, Kampala Uganda Section Objectives Going through this section will enrich you with skill to be able to: diagnose nematode problems in the field considering all aspects involved in sampling, extraction and counting of nematodes from soil and plant parts, make permanent mounts, set up and maintain nematode cultures, design experimental set-ups for tests with nematodes Section Content sampling and quantification of nematodes extraction methods for plant-parasitic nematodes, free-living nematodes from soil and plant parts mounting of nematodes, drawing and measuring of nematodes, preparation of nematode inoculum and culturing nematodes, set-up of tests for research with plant-parasitic nematodes, A. Nematode sampling Unlike some pests and diseases, nematodes cannot be monitored by observation in the field. Nematodes must be extracted for microscopic examination in the laboratory. Nematodes can be collected by sampling soil and plant materials. There is no problem in finding nematodes, but getting the species and numbers you want may be trickier. In general, natural and undisturbed habitats will yield greater diversity and more slow-growing nematode species, while temporary and/or disturbed habitats will yield fewer and fast- multiplying species. Sampling considerations Getting nematodes in a sample that truly represent the underlying population at a given time requires due attention to sample size and depth, time and pattern of sampling, and handling and storage of samples. Since plant parasitic nematodes feed on plant tissue, their distribution is influenced by the distribution of their hosts, soil types, nematode species involved and season. In most situations, nematodes are clustered or aggregated. When the host crop is present, depth and lateral distribution of nematodes in soil generally mirrors the 2 NIESA Training Manual degree of proliferation of the root system (particularly the fine roots on which nematodes feed). When the field is fallowed, the existing distribution will remain except that the bulk of the population may be slightly deeper in soil (largely due to desiccation of nematodes near the surface). Nematodes are rarely distributed evenly across a field. Some species are favoured by certain soils and their distribution may change with subtle changes in soil texture. Besides, nematode populations fluctuate over time; numbers increase in the presence of a host crop, the rate of increase being greatest when the environmental conditions are favourable. When the field is under fallow or a non-host crop is present, nematodes die of starvation and populations decline. The number of nematodes in a sample will therefore be affected by sampling time. Additionally, many plant parasitic nematodes have the capacity to survive periods of dryness through a behavioural adaptation in which their surface area is reduced by a process known as coiling. Since nematodes are very susceptible to mechanical damage in this desiccated state, the process of collecting samples from dry soils may damage nematodes. Nematode population densities are therefore likely to be underestimated in dry soil and it is preferable to wait until soil moisture is adequate before collecting samples. This uneven distribution makes collection of truly representative samples difficult as it introduces sampling errors. Other sources of sampling error are low population densities, latent infections, sub-sampling and counting of nematodes. Therefore, absence of nematodes in a sample may indicate their absence in the sampled field but may also indicate that the populations are too low to be detected by sampling. In order to increase the probability of detecting nematode infestations, roots of weeds and volunteer crops should be collected when soil is sampled. Sampling equipment For collecting soil samples to a depth of 20 cm or more, a soil sampling auger can be used. Garden trowels, narrow-bladed shovels or spades are also useful especially when sampling cropped areas or areas with rocky soil (Figure 1). Remember to clean all equipment and footwear of adhering soil before leaving a sampling site to reduce the risk of spreading nematodes to uninfected areas. Sample size The goal of a nematode sampling would be to capture information about the presence of nematode species occurring in one or more landscapes, ecosystems or geographic regions. The number of samples to be taken will depend on the breadth and depth of the sampling; where sampling breadth denotes the relative number of sites from which samples are collected, and sampling depth denotes the extent of taxonomic characterization per sampling site. Apportioning resources between sampling breadth and depth entails a choice between extensive or intensive nematode sampling. An extensive sampling maximizes the number of sampling sites and characterizes a limited number of taxa per site. 3 NIESA Training Manual . Figure 1. Examples of tools for soil sampling to diagnose nematode infestations A B C D Figure 2. Recommended soil sampling patterns; A. and B: Patterns for perennial plants (When sampling tree crops, collect sub-samples from around the drip-line, towards and on alternate sides of the trunk) C: Pattern for annual crop or fallow field; D: Sampling pattern 4 NIESA Training Manual for diagnosing nematode problems (Take soil cores from the margin of affected areas and only from the roots of affected plants that are still alive). The size of the sampling unit will vary from crop to crop and is largely determined by the value of the crop. Thus, sampling can be more intensive on high value horticultural and ornamental crops than on low value field crops. A sample should consist of 10 or more sub-samples representative of the area being sampled. Any number of sub-samples can be combined to form a composite sample. If an entire composite sample can not be processed, sub-sample by mixing the soil or plant tissue sample very gently but thoroughly by hand, avoiding excessive handling to prevent mechanical damage to the nematodes. The size of the sub sample depends on the extraction procedure used but should be at least 100ml of soil and 50g of plant material. Sampling pattern The sampling pattern will depend on the purpose of sampling: sampling to predict a problem or sampling to diagnose a problem. When sampling for predictive nematode assays (Figure 2), take samples before the desired crop is in the ground. For annual crops, this is usually soon after (or just before) harvest of the existing crop and several months before planting the next crop. Sampling close to harvest ensures that nematode populations are at their peak and that the assay will be a good indicator of any potential problems An intensive sampling limits the total number of sampled sites but maximizes the number of characterizations per site. The rationale for sampling will determine whether an intensive or extensive approach is more appropriate. For perennial crops, plan to collect samples well before planting so that there will be time to treat the fields if necessary. It is very difficult to manage nematodes on an established crop. In high-value established landscapes like golf courses, however, it can be prudent to sample for nematodes on a regular basis so that management can be scheduled for off-peak seasons. Additional information required for predictive sampling includes: Current crop and cultivar (or the crop and cultivar to be planted in the case of pre-plant samples) Details of the site (e.g. area, soil type, variability of soil, previous cropping history). Standard of crop management, particularly with regard to irrigation and nutrition. Likely importance of other pests and pathogens of roots. Details of previous nematicide use and the responses that were obtained. When sampling to diagnose a problem, use a combination of agronomic tests when trying to diagnose whether an existing plant growth problem might be due to nematodes. Collect a soil sample with roots for nematode problem diagnosis as well as a comparison sample from a nearby area where growth is more nearly normal. Because most problems have more than one cause, submit samples for soil nutrient and plant tissue analyses as well. Package each different kind of sample separately. You can send matching samples from the same area for soil testing or plant analysis. You can collect samples for nematode problem 5 NIESA Training Manual diagnosis any time plants are actively growing and the soil is in good working condition. Take soil from the root zone of plants that are affected but still alive. Never sample beneath dead plants. Diagnoses are improved if adequate background information is provided. The following details are particularly important: Crop and cultivar Previous crop Area involved Description of symptoms and their distribution Soil texture, soil depth and variability of soil Frequency of irrigation and/or rainfall Previous nematode control methods used and details of responses obtained For larger cropped areas, divide the field into 1 hectare units and make a grid pattern that covers the entire 1 ha unit. The length of intervals between sampling points on the grid will depend on the sampling precision that you require. Very close intervals, e.g. 2m x 2m will reflect the nematode distribution more precisely than, for example, 10m x 10m. Collect a sub-sample from at least 20 points throughout the fields. Collect separate samples for areas with different soil types, different cropping histories, or different management objectives. Sampling depth The depth of sampling has to consider the depth of rooting of the crop being sampled. For most crops, a sampling depth of 20 cm is adequate; for deep rooted perennial crops, different depths can be sampled, for example, 15, 30, 60, 100 cm.