The Basics of Plant Diagnostics USAID IPM Workshop October 22 - 25

The Clemson University Plant Problem Clinic An Interdisciplinary Clinic Diagnosis of plant diseases and other problems Insect identifications – DPR Entomologist Plant/Weed identifications – Botanist in Dept. of Biology Specialists across campus assist with recommendations. Website: www.clemson.edu/plantclinic

Plant Problem Diagnosis Diagnosis – process used to identify the cause of problems. Tools: Plant sciences, especially plant pathology, mycology and entomology. Arts of investigation and detective work.

Consider the Possible Agents Biotic Agents Abiotic Agents Damage from Weather Chemicals Mechanical Nutritional problems Cultural problems Pathogens or Pests: Fungi Bacteria Viruses, viroids Phytoplasmas Nematodes Insects and mites

Patterns of Abiotic Damage Damage occurs suddenly. Many species of plants affected. Symptoms often distributed in a large area. Damage is uniform even if in small area. Cotton field with chemical damage

Laboratory tests Diagnostic tests for abiotic causes Soil, water pH testing Nutrient analysis Soluble salts analysis Analysis for chemicals Plant tissue tests

Patterns of Biotic Damage Symptoms usually localized or scattered. Pattern is random. Symptoms develop gradually and get worse over time. Damage spreads to plants of same or related species. Fusarium wilt of tomato R. Billings Loblolly pine killed by southern pine beetle

Consult literature resources APS Compendia series US ARS Systematic Mycology and Microbiology website Pestnet Diagnostic Service: www.pestnet.org Other Online resources

Identify symptoms and signs Symptoms - alterations of a plant’s appearance due to a disease, pest or disorder. Signs - actual pathogen, pest, parts or by-products seen on a host plant.

Localized vs. Systemic Symptoms Localized Symptoms Leaf spots Stem lesions Fruit rots Blights Galls Systemic Symptoms Virus infections Leaf yellowing Stunting, slow growth Wilting

Systemic Symptoms Diagnosis is often more difficult First pin point source Vascular disease Root Rots Nematode infestation Isolations or other techniques often needed to determine causal agent.

Identification of Signs Mycelium Distinctive appearance allows id. of some fungi. Advantageous if fungus produces no spores. Mycelium of Rhizoctonia sp. Mycelium of Phytophthora sp.

Signs for Localized Symptoms Fungal fruiting bodies or spores Downy mildews Powdery mildews Sclerotia Bacterial ooze Cercospora leaf spot of watermelon, Cercospoora citrulina Southern stem rot, Sclerotium rolfsii Downy mildew of veronica, Peronospora sordida

Laboratory tests Moist Incubation Goal – to induce sporulation. Important for obligate fungal pathogens. Avoid overly moist conditions. Moist chamber

Identification of Signs Fungal signs Fruiting bodies Morphology of fruiting body aids in fungal identification. Spore-bearing mycelium Morphology of spores provides fungal identification

Leaf Spot Diagnosis Yellow spots on leaves only. Spores (sign) form on leaf undersides. Microscopic mount reveals Fulvia fulva is causal. Can now diagnose visually

Leaf Spot Diagnosis Leaves defoliating. Dark, water-soaked spots, more obvious when held up to light Microscopic mount shows bacterial flow. Doesn’t id. species, but control is same. Sally Miller

Signs Bacterial signs “Streaming” from freshly cut stem “Stringing” from cut stems pushed together, then pulled apart. Bacterial ooze Bacteria “flow” and morphology seen on compound microscope. Bacterial streaming from tomato stem infected by Ralstonia solanacearum.

Laboratory tests Bacterial isolations Suspend infected tissue in sterile water. Streak suspension on bacterial medium. Can use selective media. Transfer single, isolated colony. Cultures of Ralstonia solanacearum

Isolate identification Bacterial identification Biochemical tests. Carbohydrate utilization (BIOLOG). Fatty acid methyl ester analysis (FAME). Polymerase chain reaction (PCR). Serological tests, e.g. ELISA.

Melon Vine Wilt Diagnosis Wilt of vine - look for symptoms and signs. GSB lesions have gumming and pycnidia; if not present may be Fusarium wilt. Fusarium wilt shows vascular discoloration

Laboratory tests Fungal isolation Nutritive agar media, can be selective. Diagnostician can often id. to genus from appearance of culture Fungal culture.

Isolate identification Fungal identification – Other methods Utilizing keys in literature. Morphological comparison with drawings in literature. Literature sources for host diseases and disorders. Serological, molecular, other tests.

Investigate Pathogenicity If symptoms match, but not a known host, Kochs Postulates may be next step A lengthy process, rarely used in routine diagnoses. Problem arises when organism cannot be cultured.

Koch’s Postulates Note constant association of organism with diseased plants and consistent, observable symptoms. Isolate and characterize organism in pure culture. Inoculate healthy plant with organism, and observe the same disease and symptoms. Re-isolate same organism.

Symptoms Viral Symptoms Mosaics Irregular patches of discolored tissue. Often with distortion Ringspots or line patterns Distinct ring shaped lesions, various line patterns. Very few can be identified visually.

Identification of non-culturable Pathogens Methods Serological tests, e.g. ELISA . Electron microscopy. Staining for virus-induced inclusion bodies. Molecular tests, e.g. PCR DNA Sequencing Host range testing.

Pathogen Identification ELISA : a very pathogen specific serological test ELISA uses animal antibodies to detect pathogens. Multi-well test for multiple samples Enzyme-linked immunosorbent assay; a sensitive immunoassay that uses an enzyme linked to an antibody or antigen as a marker for the detection of a specific protein, especially an antigen or antibody. Individual test ELISA kit

Plant Disease Diagnosis Steps 8. Final Diagnosis All information compiled and analyzed. Organism identified, found to be pathogen of host OR Lab tests or other information reveal an abiotic cause. 9. Develop control recommendations and present to grower.

Questions??