White Paper: Plant Diagnostics Jane Trolinger, Ph.D Syngenta Flowers Technical Services January 2011

Transcription

1 Jane Trolinger, Ph.D Syngenta Flowers Technical Services Introduction Plant disease diagnosis is the process of biotic or abiotic disease identification (Mullen, 2008). Sometimes plant disease agents cause very distinctive symptoms and the diagnosis can be accurately accomplished by inspection based on the knowledge of prior association of the agent and the symptoms. Powdery mildew caused by the fungus, Sphaerotheca pannosa, is an example of a plant disease that is often diagnosed by visual symptoms and/or signs. Another example is chrysanthemum white rust caused by the fungus, Puccinia horiana, which has distinctive white pustules easily observed as visual signs on the undersurfaces of chrysanthemum leaves. More frequently, however, additional testing is required since many microorganisms as well as abiotic agents can cause similar symptoms in the same crop. In addition to visual examination, the use of plant disease indices and plant disease manuals, and determination of soil ph and total soluble salts can be useful in specifically determining the problem. The use of microscopy, culture techniques, molecular studies, plant tissue analysis, soil analysis, and/or pathogenicity studies are often required to accurately identify the causal agent. Plant Disease Diagnosis Summary Plant disease diagnosis is the process of plant disease identification and it is a necessary step in solving plant problems. Plants should be initially examined in the field, landscape, greenhouse, or garden setting, and the site, plant, and the problem history should be recorded. If the plant disease or other problem cannot be diagnosed at the site location, collect a plant sample and ship it to a diagnostics laboratory or plant disease clinic in fresh condition. Make sure the sample includes enough symptomatic tissue for examination and testing, and it must represent the problem. Diagnosis of plant diseases may be done in private, state supported, or land grant university supported clinics or laboratories. Laboratories use a variety of techniques to identify the problem. Many labs charge for services that consist of the diagnosis and disease control recommendations. Visual study, use of references, soil ph analysis, soluble salt analysis and microscopy are all techniques that may be used initially for a diagnosis. The use of additional specialized procedures including culture work, serology, and molecular testing depends upon the disease suspected, the values of the crop, and the client. A diagnosis is typically based upon more than one technique. Plant Disease Diagnostics White Paper - Page 1

2 Historically, the process of disease diagnosis has existed in some form since plant diseases were first recognized (Mullen, 2008). The Romans were aware of rust diseases of cereals as early as 310 BC. Later this awareness was evidenced by their annual Robigalia festival to appease two gods, Robigus and Robigo, whom they believed to be responsible for the rust (Alexopoulos, 1962). The actual cause of the rust diseases was not known and not even studied until after the development of the microscope around In the mid-1800s, Pasteur and other early scientists dispelled the belief of spontaneous generation and proved that microorganisms were present and responsible for many diseases of both plants and animals (Mullen, 2008). It was actually not until 1861 that DeBary scientifically proved that the fungus, Phytophthora infestans, caused the disease of the potato called late blight. This was the disease that caused the widespread famine in Ireland in After DeBary s first plant disease identification, disease diagnosis studies progressed rapidly. In 1876, Anton Koch developed a procedure used to confirm the existence of a new plant disease (Agrios, 1997). The procedure, known as Koch s Postulates or Proof of Pathogenicity, is still used today for diagnosing and describing a new disease. It was modified by Robert Smith to include the re-isolation of the pathogen from the inoculated symptomatic plant. Following is the four step process: 1. The pathogen must be found with all symptomatic plants. 2. The pathogen must be isolated and grown in pure culture. 3. The pathogen from pure culture must be inoculated on the same species. 4. The pathogen must be re-isolated into pure culture. Koch s Postulates are performed infrequently in most diagnostic labs today unless the disease agent is suspected to be new and previously unreported. Some pathogens, however, such as the fungus, Fusarium, and the bacterium, Pseudomonas, can be frequently isolated but are not actually causing the disease, so a proof of pathogenicity test is needed to actually know if the identified organism is causing the disease. Plant disease diagnosis is better understood when some basic terms are defined. A plant disease may be defined as any disturbance that prevents the normal development of a plant and reduces its economic or aesthetic value (Lucas et al., 1985). Diseases are caused by living agents as microorganisms and parasitic plants, and also by nonliving agents as environmental factors, unbalanced nutrition, and chemical substances. The living agents are called pathogens or biotic agents and the nonliving agents are called abiotic agents. Most of the biotic diseases are caused by fungi, bacteria, viruses, viroids, nematodes and parasitic plants. Abiotic diseases can be caused by temperature and moisture extremes, low or high soil ph, fertilizer excesses or deficiencies, pesticide damage, pollution effects, or weather/soil problems. Some diseases can have both pathogens and abiotic agents involved. A plant disease develops over a period of time, whereas an injury that may be caused by an insect feeding on a plant or damage from equipment or machinery occurs immediately or over a relatively short period of time. Usually a disease has been developing for several days before any symptoms on the plant are visible. Plant Disease Diagnostics White Paper - Page 2

3 Diagnosis on site or information needed from site: Diagnosis actually begins at the site of the diseased plants. Careful observation of symptoms, distribution, environmental conditions, and records of pesticide applications are all involved in the diagnosis process. Knowledge of the normal appearance of the plants in question and their growth requirements are very important. Site history, fertilization, lime and pesticide applications, and recent weather conditions add to the assessment of the problem. If the problem can be accurately diagnosed by observations on site, then lab confirmation may not be necessary. However, many problems do need the expertise of a plant pathologist diagnostician so then samples will need to be collected and submitted to a plant disease clinic or diagnostics lab. Collecting, packaging, and shipping samples: When diagnosis is not possible at the site location, then samples need to be collected and submitted to a lab. Important points to remember are: 1. Make sure the sample is representative of the problem. 2. Keep the sample fresh. If it cannot be collected the day it is shipped, then refrigerate until shipping. Overnight shipment is preferable since degradation in shipment can be an obstacle to accurate diagnosis. 3. Make sure the sample is adequate in size. If the problem is a leaf spot, then 20 leaves exhibiting the spots will be suitable (Mullen, 2008). This number of leaves is needed for the following procedures: microscopy, moist chambers, cultures, and possible virus testing. 4. Whole plants are needed for most problems. 5. Never collect dead samples. Also include some healthy plants with moderately and severely diseased plants. 6. Follow precisely the shipping instructions issued by the lab since they are designed to meet the requirements of the federal and state permits under which the lab operates. Overall operation of a plant disease diagnostics laboratory: Diagnostics labs may be private, state supported, or land-grant-university supported. Private diagnostic clinics usually receive samples from homeowners and commercial operations. The Syngenta Flowers Diagnostics Lab receives specimens from commercial operations through contact with the Syngenta Flower Technical Services Team or the Syngenta Flowers Grower Account Managers. State supported diagnostics labs usually accept samples only from state inspectors who examine commercial samples to certify plants free of specific diseases. Land grant universities in each state have a designated plant diagnostic laboratory/clinic and each of these clinics is a member of the National Plant Diagnostic Network (NPDN). The NPDN was created by the United States Department of Agriculture (USDA) and the Animal and Plant Health Inspection Service (APHIS) in conjunction with the Department of Homeland Security created after the terrorist attack on New York City in The NPDN strives to assist land grant Plant Disease Diagnostics White Paper - Page 3

4 university clinics by providing support for rapid and accurate diagnoses, a network communication system, a database for archiving diagnostic data on a national level and a protocol for reporting to responders and decision makers. In many diagnostic clinics, about 50 percent of the disease samples are diagnosed with biotic diseases, and the remaining samples are found to have problems caused by abiotic factors (Mullen, 2008). These percentages vary from year to year and from clinic to clinic depending on the weather conditions. In more typical years, about 80 percent of the biotic diseases seen in these clinics are caused by fungal pathogens. Bacteria and viruses together comprise about 15 percent or more. Plant parasitic nematode problems account for less than five percent of the plant problems submitted for diagnosis. The response to the client consists of the diagnosis and the control recommendation. The name of the disease and the name of the pathogen are provided. The name of the disease may or may not include the name of the pathogen so the scientific name of the pathogen is included for specific identification. Control recommendations vary with the disease, the disease severity, and the cropping situation. Certain states require those individuals who issue the pesticide recommendations to be licensed. Clinics, especially at land grant universities, were typically begun as free services and were totally supported by extension and/or university funding. This situation has changed in many states in the last 20 years (Mullen, 2008). Today many university-associated clinics charge for their services. Generally out of state samples have a higher cost. Most private labs charge for their services unless that service is offered freely to customers as a support for their business. Diagnostics methods used in clinics: Initial diagnostics procedures generally follow the order listed (Mullen, 2008): 1. Visual examination for symptoms and signs 2. Review of information sent with the sample 3. Consultation of plant disease compendia and/or host indices 4. Microscopic examination with stereoscope 5. Microscopic examination with compound light microscope 6. Possible further consultation with reference books or websites 7. Soil ph determination If visible symptoms are not diagnostic, microscopic structures are not observed, and soil ph does not account for the damage, then other diagnostics procedures are used. The exact procedures used and the order followed depends upon the symptoms and the possible agents that can cause these symptoms. Plant Disease Diagnostics White Paper - Page 4

5 Specialized analyses (Mullen, 2008) used to identify specific pathogens are: 1. Measurement of electrical conductivity as total soluble salts in a soil sample. This is very useful in determining if the damage was caused by excess fertilizer. 2. Moist chamber incubations of plant sample to help determine the presence of a fungal pathogen evidenced by sporulation. 3. Culture isolation techniques support the identification of fungal or bacterial pathogens. 4. ELISA (enzyme linked immunosorbent assay) or other serology method. Fungal, bacterial, and viral pathogens can be detected by these techniques. 5. Specific physiological tests depending on bacteria suspected of causing the disease. 6. Gas chromatography of bacterial fatty acids. 7. Gel electrophoresis for identification of pathogen species or strain by specific enzyme or protein identification. 8. DNA identification by various molecular methods to identify specific pathogens: methods include hybridization (dot blot using probe) and PCR (polymerase chain reaction). 9. Koch s Postulates or Proof of Pathogenicity when fungal or bacterial agents are suspected of being the pathogen. Please note that the Syngenta Flowers Diagnostics Lab will submit further testing to other specialized labs when the specific test needed is not available in our lab. For example, crown gall (Agrobacterium tumefaciens) confirmation by PCR will be submitted to the Oregon State Clinic. Specific bacterial identification after pathogenicity testing will, likewise, be submitted to the Oregon State Clinic for BIOLOG testing. BIOLOG is a combination of numerical taxonomy, molecular analysis, and the use of a computer software library program. Different carbon sources are used in the analysis. This system is considered to be fairly accurate for genus identification and sometimes accurate for species. Plant Disease Diagnostics White Paper - Page 5

6 Commonly used Diagnostics Procedures in Plant Diagnostic Clinics (From Mullen, 2008) Plant Pathogen Group Diagnostic Procedure Fungi Bacteria Viruses Nematodes Visual exam Microscopy + + (+) + Moist chamber Baiting Culture isolation ELISA Physiological tests Gas chromatography BIOLOG RT-PCR real time polymerized chain reaction (+) (+) (+) (+) Note: + = used routinely, (+) = used occasionally with a specific technique/situation, - = not used. Please note that the Syngenta Flowers lab is not equipped to handle nematodes or insects. Also, specialized tests such as PCR and BIOLOG will involve the assistance of other plant disease clinics. Plant Disease Diagnostics White Paper - Page 6

7 References Agrios, G. N Plant Pathology, 4 th ed. Academic Press, San Diego. Alexopoulos, C. J The Rusts. Pages in Introductory Mycology. 2 nd ed. John Wiley & Sons, Inc., New York. Eysinga, R. van and Smilde, K.W Nutritional Disorders in Chrysanthemums. Centre for Agricultural Publishing and Documentation, Wageningen. Lucas. G. B., Campbell, C. L., and Lucas, L.T Introduction to Plant Diseases. AVI Publishing Company, Westport, Connecticut. Mullen, J. M Plant Disease Diagnosis. Pages in Plant Pathology Concepts and Laboratory Exercises, 2 nd Ed. R.N Trigiano, M. T. Windham, A. S. Windham, eds. CRC Press, Boca Raton, FL. Pirone, P. P Diseases and Pests of Ornamental Plants. John Wiley & Sons, New York. Shurtleff, M. C. and Averre, C. W.III The Plant Disease Clinic and Field Diagnosis of Abiotic Diseases. APS Press, The American Phytopathological Society. Saint Paul, MN 2011 Syngenta Flowers, Inc., P.O. Box 1349, Gilroy, California The Syngenta logo is a registered trademark of a Syngenta Group Company. Plant Disease Diagnostics White Paper - Page 7