If you are recirculating irrigation water or drawing water from a pond, testing the biological water quality can be important. What might you test for? In addition to waterborne pathogens, microbial water issues result in algae on the growing substrate and floors, and clogged equipment from bacteria and biofilm. For growers producing edible crops, human pathogens, including strains of E. coli, are regulated.
Working with a testing lab
Most tests of biological water quality require samples to be analyzed by professional laboratory services, which are provided by water treatment companies, university extension plant pathology labs and private microbiology labs. Onsite detection methods for biological water quality are being developed, but tend to be useful as indicators rather than specific diagnostic tools.
For laboratory tests, contact the lab before you submit samples. Things you should discuss with the lab include:
- What to test for. The lab needs to understand whether you are interested in testing for a particular pathogen species or a class of organisms (such as aerobic bacteria) so it is ready to run the appropriate test when the samples arrive.
- How to collect samples. The samples should be representative of the irrigation water. Take the samples after the irrigation water has run for 5 minutes or long enough to ensure that you are not sampling water that has been sitting in the piping. For ponds, the usual procedure is to sample from the intake pipe depth.
- The sample volume and containers. Some laboratories provide sterile sampling containers. Typically 350 milliliters is enough. If sterile containers are not available, then purchase distilled water containers from a grocery store, empty the container and flush several times with the sample.
- Proper labeling and documenting samples including lab forms.
- Storing samples and packaging (usually in insulated containers with a cool pack).
- When to ship samples (usually early in the week with overnight shipping).
|Figure 1. Eucalyptus leaf discs for baiting Phytophthora are placed onto a selective agar culture medium to test for the presence of the pathogen (the thread-like rings around the leaf discs).|
Testing for pathogens
Plant pathology laboratories use either baiting (attracting pathogen spores to a leaf) or filtration to concentrate pathogens in the water sample. The sample is then plated out on agar substrates (Figure 1) that selectively grow different classes of organisms, to identify the presence or absence of pathogens and the number of colonies. This type of test for pathogens is typically to the genus level, with tests for Pythium and Phytophthora being the most widely available. For example, the University of Massachusetts provides this service for $50 per sample, with a one to two week turnaround to allow time to culture the sample (Table 1).
Pathogen testing could be used to determine if a water treatment technology is controlling Pythium. To test treatment efficacy, water samples could be collected from sources before and after the point of water treatment (with activated peroxygens, chlorine, chlorine dioxide, copper, ozone, UV radiation or another technology) and tested for living colonies of Pythium. Biological water quality changes very quickly. Therefore, repeat testing over the growing season is an advised monitoring strategy if waterborne pathogens are suspected.
Identification of pathogens to the species level requires a specialist, but many species of Pythium and Phytophthora can be identified by conventional or molecular techniques. Identification to species level is most important when there is there is a specific pathogen suspected, such as Phytophthora ramorum.
The presence of Pythium does not necessarily mean there will be root disease but Phytophthora is more aggressive and should always be considered a problem. Nevertheless, if a lab reports the presence of Pythium or Phytophthora at the genus level, it is prudent to assume that a pathogen is present and to treat the water and crops accordingly. Follow up with additional water tests after crop management treatments are carried out.
The University of Guelph can identify the pathogen species based on characteristics of their DNA (Table 1). This test can evaluate a number of species in a single analysis. However, the test will not differentiate between live and dead microbes.
Whether pathogens are viable is important if you are treating water with a sanitizing agent technology and want to know whether the pathogens are killed by the treatment. In this case, culture-plating may be more useful than DNA analysis.
Researchers are developing protocols to use ELISA diagnostic kits as a preliminary screening test for waterborne pathogens. Field diagnostic kits are available for Pythium and Phytophthora. The kits use monoclonal or polyclonal antibodies to detect the pathogens. This is the same technology used in home pregnancy test kits. Kits are available as dipsticks or as lateral flow devices (see Table 1 for suppliers).
Many growers already use these ELISA kits to identify pathogens on leaves, with instant results. Trialing at Oregon State University has found that some ELISA tests for Phytophthora cross-react with a few Pythium species, so that if the test is positive, it is important to follow up with laboratory tests. Lab testing is also advised to rule out the presence of Phytophthora ramorum from water that has tested positive in a Phytophthora test kit. Test kits detect both live and dead forms of these pathogens.
Total microbial load
Total colony count expressed as colony forming units (CFUs) of either bacteria or fungus is a non-specific measurement, meaning these tests estimate the amount of large groups of diverse organisms. Total CFUs of bacteria or fungi are not a good indicator of plant pathogens, because most microorganisms in water samples are likely to be beneficial or benign, rather than pathogenic.
|Figure 2. High microbial concentrations in the irrigation water can lead to clogging of irrigation equipment.|
The goal of treating irrigation water is not to eliminate bacteria or fungus, but to keep the CFUs managed so that they don't degrade water quality or limit control of pathogenic organisms. Greenhouses are not sterile environments, and attempting to completely sterilize water, all surfaces and the growing substrate would require high doses of chemicals that are likely to be phytotoxic to crops. Removing beneficial organisms can also increase the likelihood that pathogenic organisms will cause disease. Clogging of irrigation equipment is, however, a common problem resulting from high microbial density in water (Figure 2).
Total colony counts within irrigation systems indicate the biological productivity of the water. If water treatment technology has been installed, you want to know whether waterborne pathogens are likely to be controlled if they arise in the source water. You can test the active ingredient dose of the sanitizing agent compared with published recommendations, and analyze indicator organisms such as total bacteria and fungus CFUs from samples both before and after the point of treatment in the irrigation line. The change in microbial density before and after treatment may be a useful bio-indicator of the general efficacy (or failure) of the treatment system to control microorganisms.
Sampling at points along an irrigation system can also identify where conditions favor growth of microorganisms. Excessive organic matter increases the number of microorganisms and the risk of clogging equipment. Changes in CFUs from the source water to the emitters can also help monitor the growth of microbes within the irrigation lines.
There is currently little standardization across private horticulture laboratories on how samples are processed for total density of bacteria and fungus. A threshold of 10,000 colony forming units (cfu) of aerobic bacteria per milliliter is generally recommended to reduce clogging of drip lines and micro-emitters.
One technology used (see sidebar) at the University of Florida to measure total density of bacteria and fungus is 3M Petrifilms (www.3m.com). Petrifilms are plastic cards coated in a dehydrated nutrient film with microbial indicators. A droplet of water placed on the Petrifilm can be stored for three to five days in the dark at room temperature, and each colony forming units makes a colored dot on the Petrifilm to allow counting. Different types of Petrifilm are available for aerobic bacteria, yeasts and molds (i.e., fungus and fungal-like organisms), or other pathogens of importance to human health. University of Florida researchers have trained growers with the Petrifilms, who have found the method easy to use.
In horticulture, algae are often thought of as the "green stuff" that workers slip on, causes blooms in ponds and coats the growing substrate, benches, floors and irrigation lines. In reality, algae are a complex of many unrelated species and groups (cyanobacteria, green and red algae) that range from unicellular bacteria-like organisms to complex aquatic plants.
The horticulture industry will probably need to become more educated about algal biology as control methods are evaluated. For example, pond chemicals vary in control of different types of algae (www.extension.purdue.edu/extmedia/HO/HO-247-W.pdf).
Samples can be sent to a testing laboratory for identification and quantification of algae and other aquatic weeds, which is most likely to benefit growers drawing water from catchment basins with algal bloom issues.
|Figure 3. Algae at different total chlorophyll concentrations on white filter discs after filtering pond water with a Lamotte testing kit.
|Click on TABLE 1 above to see full table.|
Growers can quantify algal concentration by sending a sample to a specialist laboratory that will measure total chlorophyll. A grower-friendly kit is available for extracting algae (Table 1, Figure 3), which involves filtration to concentrate the algae on a disc, and extraction of the chlorophyll. However, this testing method is qualitative. The color of the filter disc or extracted solution can be viewed and determined if it is "clear", "green" or "very green". In a laboratory setting, a spectrophotometer is used to quantify chlorophyll concentration in the water sample. Standards have not been defined for "acceptable" or "problem" levels for algae in greenhouse applications, although researchers at the University of Florida are starting to accumulate some data.
Human pathogen testing
Levels of human pathogens are regulated when using water for drinking supplies, irrigating edible fruits (especially with methods that wet foliage or fruit), and for post-harvest washing of produce. Some states have regulations for coliform levels in water used to irrigate non-food crops.
|Figure 4. Testing water quality for human pathogens is essential when producing edible crops.|
Outbreaks of human disease caused by contaminated produce emphasize that monitoring to ensure irrigation and wash water are free of human pathogens is a critical food safety issue (Figure 4). More information is available on water testing for human pathogens at http://edis.ifas.ufl.edu/ss482.
Why you should test
Tests for biological quality can help avoid crop losses from diseases, unsightly algae, clogging of equipment, and health issues from contaminated produce. Most organisms in water are too small to see with the human eye. Consider monitoring to catch biological water problems before they cost you money.
Paul Fisher is associate professor and extension specialist, Dustin Meador is graduate student, Environmental Horticulture Department, University of Florida, email@example.com. Robert Wick is professor of plant pathology, Department of Plant, Soil and Insect Sciences, firstname.lastname@example.org. Jennifer Parke is associate professor, Department of Crop and Soil Science, Oregon State University, email@example.com. William Argo is technical manager, Blackmore Co., firstname.lastname@example.org.
The authors thank sponsors of the Water Education Alliance for Horticulture (www.watereducationalliance.org) for supporting this series, including AquaPulse Systems, BioSafe Systems, Blackmore Co., Chem Fresh, Chlorinators Inc., DRAMMwater, Ellepot USA, Fafard, Greencare Fertilizers, Griffin Greenhouse and Nursery Supplies, Hanna Instruments, Phyton Corp., Pindstrup, Premier Horticulture, Pulse Instruments, Quality Analytical Laboratories, Sun Gro Horticulture and Trueleaf Technologies. The authors also thank the USDA-ARS Floriculture and Nursery Research Initiative and the Young Plant Research Center (www.floriculturealliance.org) for supporting the underlying research.