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Questions about the healthy plant
• What are the genus, species, and cultivar names of the plant in question?
• Is this particular plant suited to the production area? Is the cultivar resistant or especially suscepti- ble to diseases and other problems?
• Is the plant sensitive to certain environmental factors (salinity, excess or deficient soil moisture, etc.)?
• What are the characteristics, appearance, and growth habits of a healthy plant?
• How does the plant normally appear when grown under various conditions (greenhouse vs. outdoors, coastal vs. inland production locations, winter vs. summer) or at different stages of growth and devel- opment (seedling vs. transplant vs. mature plant)? • What is the normal growth rate?
Questions about the unhealthy plant
SYMPTOMS
• What are the symptoms of the affected plant? • Which plant parts are affected?
• Are symptoms restricted to external plant surfaces (spots and lesions) or are there also internal symptoms (vascular streaking, discolored pith or crown tissue)?
• Are symptoms present only on exposed plant surfaces or also on protected, covered tissues such as unexpanded inner leaves or unopened flowers? • What is the distribution of the symptoms on any
one particular plant (do symptoms occur on one side of the plant, only on older or newer leaves, on secondary roots but not on primary roots, etc.)? • What were the initial symptoms?
• How do early symptoms differ from more advanced symptoms?
• How rapidly do early symptoms change into advanced ones?
• How long have the symptoms been present?
GROWTHSTAGE
• What is the growth stage of the affected plant (seedling, new transplant, mature plant, flowering/fruiting plant, senescent plant)? • Is a particular growth stage associated with the
problem?
• What was the condition of the plant when first placed in the production area?
• How does the growth rate of the affected plant compare with that of a healthy plant?
SYMPTOMS ONOTHERPLANTS
• Are symptoms restricted to one species or one cultivar of a vegetable plant or are multiple cultivars and different species involved?
• Do the same symptoms occur in only one field or one greenhouse, while adjacent or nearby plantings of the same crop remain symptomless, or are many plantings of that particular vegetable affected? • Do adjacent plantings, weeds, or nearby crops
exhibit similar symptoms?
• If other plants are affected, do they belong to a common group or family of plants?
• Are symptomatic plants associated with a particu- lar set of transplants or lot of seed?
PATTERNS
• How are the symptoms distributed within the specific production area of concern?
• Are there patterns (repeating numbers of plants or plant rows) to the symptoms or are they completely random throughout the planting?
• Are symptomatic plants found in clustered groups? • Do the symptomatic plants occur in lines, streaks,
circles, or other discernable pattern?
• Are symptomatic plants found mostly along the edges of the planting?
• Are affected plants next to buildings, roads, ditches, weedy areas, other crops, or other production areas?
• Are symptoms associated with sub-sets of plants within the planting, indicating an association with plants from certain transplant trays, different sources of plant material, or other production factors?
33 • Are symptoms associated with physical features in
the field such as low or high spots of the field, places where water does not drain well, presence or absence of underlying gravel or clay, changes in soil types?
• Do such areas become flooded after rains or receive irrigation runoff? Take note of the irrigation system and possible patterns associated with each type. For example, in the field a plant growth pattern can occur in which linear stretches of plants grow poorly depending upon how the sprinkler lines were arranged.
TIMING
• What is the timing of symptom occurrence in relation to other factors?
• When did the symptoms first occur?
• Are there various stages of symptoms indicating new infections vs. older ones?
• Have symptom features or severity changed over time?
• Do symptoms appear to have developed gradually over a period of time or rapidly and all at once? • Have the same or similar symptoms occurred
before?
BIOTIC ORABIOTIC
• Do symptoms resemble those caused by biotic agents such as pathogens, nematodes, arthropods, or vertebrate pests, or are symptoms more sugges- tive of physiological or abiotic factors such as nutri- tional problems, physiological disorders, genetic mutations (chimeras), chemical damage, or envi- ronmental extremes?
• Do the symptoms provide evidence that more than one factor or pathogen is involved?
Questions about possible agents and factors
BIOTIC
• Which pathogens, nematodes, and arthropod pests are known to occur on the host?
• Which biotic agents occur in the geographic area of concern? Compile a list of common biotic agents that occur in the area.
ABIOTIC ANDPHYSIOLOGICAL
• Compile a list of physiological and abiotic factors known to cause problems for the plant in question (examples: tipburn in lettuce, blossom end rot of tomato).
SIGNS
• A ‘sign’ is the visible presence of a biotic causal agent. Are such signs present? Examples of signs are fungal growth and spores, bacterial ooze, and insect bodies or frass (insect droppings).
• Are there multiple signs that indicate more than one factor may be involved?
• What is the distribution of signs on the affected plant (present on all or only on certain plant parts)? • Are signs present on all symptomatic plants? • Are signs present on adjacent plantings or non-
symptomatic plants?
• Are there signs on surrounding soil, irrigation pipe, or other inanimate objects in the area?
Questions about the surrounding environment, growing conditions, production practices
TIMING
• What time of year did the problem occur? • From year to year, does the problem recur during
the same month or time of year?
ENVIRONMENTALCONDITIONS ANDCONTEXT
• What are the current and past weather conditions? • Have there been any unusual weather patterns,
changes, or developments recently or in the past few weeks or months?
• Have there been any conditions that would hinder plant growth or favor pathogen and pest develop- ment?
• Is there evidence of abiotic stress factors (tempera- ture extremes, water stress or excess, salt buildup, mineral deficiencies and toxicities, pollution, wind or other mechanical damage, etc.)?
• What is the general location of the field or green- house (coastal vs. inland, next to other crops or production areas, next to roads, etc.)?
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ROOTINGMATERIALS ANDFIELDSOILS
• For greenhouse-produced transplants, what type of rooting medium is used?
• What is the condition of the mix (pH, porosity, salinity, nutrient level, etc.)?
• Has the rooting medium been subject to any envi- ronmental extremes (excess water or salts, water deficit conditions, high temperatures in the container, etc.)?
• What supplements have been added to or omitted from the rooting medium?
• Has the rooting medium been used previously, recycled, or exposed to contaminating factors? • What is the condition and cleanliness of the pots,
trays, or other containers used to hold the rooting medium?
• For field or greenhouse crops grown in the ground, what is the soil type (heavy clay, porous sand) and soil condition (compacted layers, change in soil type from affected to unaffected areas, pH, salinity levels, alkalinity, etc.)?
• Is there a history of this transplant crop grown at this site?
WATER
• What is the water source and quality? • How does the water drain off the site?
• Are there low spots where water collects or drains poorly?
• What type of irrigation system is used? • Does the system deliver water uniformly?
• What is the frequency and duration of irrigations? • How does the irrigation schedule correspond to
other production practices such as pesticide sprays, fertilizer applications, transplanting, and other pro- cedures?
GENERALPRODUCTIONPRACTICES
• What are the normal, typical production practices for the crop (propagation steps, planting arrange- ment, irrigating, fertilizing, pruning, pest manage- ment)?
• What is the condition of the facility, including struc- tures, fertilizer injection systems, irrigation systems, spray equipment, etc.?
• What experience do you have with this crop or par- ticular cultivar?
• What production steps were completed or omitted? • What was previously planted or placed in the area
of concern?
• What equipment, particularly spray equipment, was recently used in the area?
• Were any new production practices recently imple- mented?
• Did the onset of symptoms correspond with any cultural practice?
• Compile a complete record of production practices used on and around the symptomatic plants. • Which fertilizers, pesticides, or other chemicals
were applied to the plants, adjacent crops, green- house benches for transplants, and non-production areas in the vicinity? Compile information on all materials, formulations, rates, additives, and spray volumes used on symptomatic, non-symptomatic, and surrounding plants. Special attention should be placed on herbicide (selective, non-selective, pre- emergent, post-emergent) and pesticide tank mix applications.
• What weather patterns occurred before, during, and after the spray applications?
• What chemicals and additives were used previously in the spray equipment and how was the equipment cleaned afterwards? Chemical damage may be the subject of future litigation and careful records and photographs should be taken when such damage is suspected. Careful analysis will be required to determine if there is evidence that damage is associ- ated with spray patterns or if untreated plants lack such symptoms.
35 HISTORY
• What is the sequence of crops that was planted at this site in the past few years?
• Has the same crop been placed here frequently in the past few seasons?
• Have cover crops, composts, or other amendments and inputs been used here?
• What is the history of plant diseases that occur at this site?
• Are there field notes, laboratory reports, or other information that document the presence and problems due to soilborne or foliar diseases?
Conducting the examination
Gather appropriate equipment for use in examining the production site. Equipment includes the following: notebook, hand lens, knife, pruning shears, shovel, plastic bags for plant and soil samples, bottles for water samples, labels and marking pens, soil sampling tube, flags for tagging plants for future observations, camera, ice chest/cooler, disinfectant for tools.
Examine all relevant propagation and production areas in an attempt to answer the pertinent informa- tion-gathering questions. Tour off-site areas such as adjoining roads, fields, and landscaped areas.
Thoroughly examine symptomatic plants. Carefully examine all roots, above-ground parts of the plants, and internal tissues of stems. Examine and dissect multiple examples of affected plants. Conduct a similar exami- nation of healthy or asymptomatic plants to make a comparison.
Record all observations. Draw maps to indicate patterns of symptoms and locations of plantings having the problem. Take photographs to document symptoms and distribution of patterns. Photograph asymptomatic plants for comparison.
Collect and label representative plant samples for possible laboratory analysis. Samples should include various stages of affected plants (initial and more advanced symptoms) taken from multiple sites. In many cases and if feasible, the entire plant should be collected and not just a few leaves and stems; foliar symptoms may be caused by root and crown problems, so laboratory personnel need to see the entire specimen. Include a healthy plant for comparison. Keep samples cool and deliver them in a timely manner to the lab. If the plant being examined is a sample brought in from the field or greenhouse, what is the condition of the sample? Samples that are in poor condition, incom- plete, or of limited size and number may not yield useful information. Is the sample representative of the problem? Have unaffected plants also been included in the sample for comparison? If warranted, collect appro- priate soil and water samples for various analyses.
Incorporating laboratory tests
Because diagnosis based solely on symptoms is risky and may lead to inaccurate conclusions, laboratory analysis is usually highly recommended. If pathogens are possibly involved, send good quality, representative samples to a plant pathology laboratory. Pathology labs use various tests and techniques to identify biotic causes of plant problems. Direct examination of plant tissues is used to search for signs such as fungal structures and bacterial ooze. Culturing techniques using microbio- logical media are designed to isolate and recover fungal or bacterial pathogens from symptomatic tissues.
Various extraction methods can recover nematodes from plant tissues and soils. Serological methods, such as the enzyme-linked immunosorbent assay (ELISA), employ antisera that will identify specific pathogens from either plant tissues or from cultures. Molecular identification techniques for plant pathogens are con- stantly being developed and improved. In some cases these molecular tests can be used directly on sympto- matic plant tissues to confirm the presence of the target pathogen. Note, however, that the detection or recovery of a pathogen does not necessarily mean that this agent caused the symptoms of the disease. Interpretation of lab results using any method often requires the expertise of diagnosticians and plant pathologists.
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For many fungi, fungal-like organisms such as oomycetes, and bacteria, isolating suspect pathogens from symptomatic plant tissue is a critical step in the diagnosing of plant pathogens. Different strategies are adopted for this purpose. The plant sample is first usually washed to remove debris and soil. Typically the sample is then surface sterilized to reduce interference from secondary decay organisms and non-pathogenic microflora that are present on the plant surfaces. Small pieces of diseased tissue, taken from the edges of the infection, are placed in or on solid agar media. For isolating fungi and oomycetes, general purpose micro- biological media are usually recommended: potato dextrose agar, corn meal agar, water agar. These media can be acidified if secondary decay bacteria are prob- lematic and might interfere with recovery of the pathogen. In some cases the use of semi-selective media can help recover suspect pathogens. Semi-selective media are used to inhibit the growth of non-target organisms while facilitating growth of the target pathogen. Examples of semi-selective media are PARP for Phytophthora, Sorensen’s NP-10 for Verticillium, and Komada’s medium for Fusarium.
Isolating plant pathogenic bacteria follows a similar strategy. Samples are prepared in similar ways as for fungal diseases. Small pieces of symptomatic tissue are taken from the edges of the infection and are macerated in a drop of water. A few microliters of this water are streaked onto general purpose media such as nutrient agar, yeast extract dextrose calcium carbonate agar, or sucrose peptone agar. At times a semi-selective medium is useful if a particular pathogen is suspected. Examples of semi-selective media for bacteria are SX agar for
Xanthomonas campestris pv. campestris and KBC
medium for several Pseudomonas syringae pathovars. Many book and journal references provide specific details on the appropriate media to consider for isolating and growing plant pathogenic fungi and bacteria. Several books are particularly useful in this regard and are listed in the references below.
Diagnosing virus diseases
For any pathogen group (fungi, bacteria, viruses), the symptoms may vary due to any number of factors. However, for viruses, the incidence and expression of disease symptoms can fluctuate and vary a great deal depending on the strain and virulence of the agent, the particular crop and cultivar host, age of host when infected, mode of infection (mechanical abrasion, seedborne inoculum, or arthropod vector), factors involving the biology of the vector (type, strain, and population), and environmental conditions. With some exceptions, symptoms caused by different viruses often resemble each other, thereby making field diagnosis difficult and ill advised. Virus disease diagnosis is further complicated when more than one viral agent infects the host plant. Clinical tests are required to pos- itively identify viral agents in plants.
Digitally assisted diagnosis
Mention should be made of new technology involving digitally assisted diagnosis (DAD). DAD is the process of acquiring digital images of plant problems and sending these images to researchers, diagnosticians, and other experts for viewing and diagnosing. This diag- nostic approach therefore relies almost exclusively on digital photographs. In some cases, such images may be very helpful in reducing the time required to identify the cause of the problem. Digital images can also be appro- priate when distinctive symptoms and signs character- ize the problem (such as powdery mildews, downy mildews, clubroot of crucifers, and others). However, care should be taken not to rely on such images alone. DAD can be a useful supplement to the diagnostic process, but will not be able to completely replace field visits and hands-on examination of plant samples.
Compiling information and drawing a conclusion
Compile all notes, observations, maps, laboratory results, photographs, and other information. Consult with and record information from printed references and books, on-line website resources, and university and other experienced professionals. This compilation will be the information base for the present diagnosis and can also be a useful resource for future diagnostic cases. After considering all the information, render an informed diagnosis.
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References on disease diagnosis
Barnes, L. W. 1994. The role of plant clinics in disease diagnosis and education: A North American perspective. Annual
Review of Phytopathology 32:601–609.
Fox, R. T. V. 1993. Principles of Diagnostic Techniques in Plant
Pathology. CAB International.
Green, J. L., Maloy, O., and Capizzi, J. 1990. A systematic approach to diagnosing plant damage. Plant Diagnostics
Quarterly 11(3):139–165.
Grogan, R. G. 1981. The science and art of plant disease diagnosis. Annual Review of Phytopathology 19:333–351. Hansen, M. A. and Wick, R. L. 1993. Plant disease diagnosis:
present status and future prospects. Advances in Plant
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Henson, J. M. and French, R. 1993. The polymerase chain reaction and plant disease diagnosis. Annual Review of
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Holmes, G. J., Brown, E. A., and Ruhl, G. 2000. What’s a picture worth? The use of modern telecommunications in diagnosing plant diseases. Plant Disease 84:1256–1265.
Horne, C. W. 1989. Groundwork for decision: Developing recommendations for plant disease control. Plant Disease 73:943–948.
Kabashima, J. N., MacDonald, J. D., Dreistadt, S. H., and Ullman, D. E. 1997. Easy on-site tests for fungi and viruses in
nurseries and greenhouses. UC DANR Publication no. 8002.
Kim, S. H. 1988. Technological advances in plant disease diagnosis. Plant Disease 72:802.
Marshall, G. 1996. Diagnostics in Crop Protection. Symposium Proceedings No. 65. British Crop Protection Council. Major Print Ltd.
Newenhouse, A. C. 1991. How to recognize wind damage on leaves of fruit crops. HortTechnology 1:88–90.
Putnam, M. L. 1995. Evaluation of selected methods of plant disease diagnosis. Crop Protection 14:517–525.
Schubert, T. S. and Breman, L. L. 1988. Basic concepts of plant
disease and how to collect a sample for disease diagnosis.
Plant Pathology Circular No. 307. Florida Dept. Agric. & Consumer Services.
Shurtleff, M. C. and Averre, C. W. 1997. The Plant Disease
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Stowell, L. 1999. Digital disaster and the ethics of virtual plant pathology. Phytopathology News 33:62.
Thomas, M. B., Crane, J. H., Ferguson, J. J., Beck, H. W., and Noling, J. W. 1997. Two computer-based diagnostic systems for diseases, insect pests, and physiological disorders of citrus and selected tropical fruit crops. HortTechnology 7:293–298. Walker, S. E. and Schubert, T. S. 1997. Assessing plant problems
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