The Basics of Plant The Basics of Plant DiagnosticsDiagnostics
USAID IPM WorkshopUSAID IPM Workshop
October 22 - 25October 22 - 25
The Clemson University The Clemson University Plant Problem ClinicPlant Problem Clinic
An Interdisciplinary ClinicAn Interdisciplinary Clinic Diagnosis of plant diseases and other problemsDiagnosis of plant diseases and other problems• Insect identifications – DPR EntomologistInsect identifications – DPR Entomologist• Plant/Weed identifications – Botanist in Dept. Plant/Weed identifications – Botanist in Dept.
of Biologyof Biology• Specialists across campus assist with Specialists across campus assist with
recommendations.recommendations.
WebsiteWebsite:: www.clemson.edu/plantclinic
Plant Problem DiagnosisPlant Problem Diagnosis
Diagnosis – process used to identify the Diagnosis – process used to identify the cause of problems.cause of problems.
Tools:Tools: Plant sciences, especially plant pathology, Plant sciences, especially plant pathology,
mycology and entomology.mycology and entomology. Arts of investigation and detective work.Arts of investigation and detective work.
Consider the Possible AgentsConsider the Possible AgentsAbiotic AgentsAbiotic Agents
Damage from Weather Chemicals Mechanical
Nutritional problemsCultural problems
Biotic AgentsBiotic Agents
Pathogens or Pests:FungiBacteriaViruses, viroidsPhytoplasmasNematodesInsects and mites
Patterns of Abiotic DamagePatterns of Abiotic Damage
Damage occurs suddenly.Damage occurs suddenly.
Many species of plants Many species of plants affected.affected.
Symptoms often Symptoms often distributed in a large distributed in a large area. area.
Damage is uniform even if Damage is uniform even if in small area.in small area. Cotton field with chemical damage
Laboratory testsLaboratory tests
Diagnostic tests for abiotic causesDiagnostic tests for abiotic causesSoil, waterSoil, water
pH testingpH testingNutrient analysisNutrient analysisSoluble salts analysis Soluble salts analysis Analysis for chemicalsAnalysis for chemicals
Plant tissue testsPlant tissue testsNutrient analysisNutrient analysisAnalysis for chemicalsAnalysis for chemicals
Patterns of Biotic DamagePatterns of Biotic Damage
Symptoms usually Symptoms usually localized or scattered.localized or scattered.
Pattern is random.Pattern is random. Symptoms develop Symptoms develop
gradually and get gradually and get worse over time.worse over time.
Damage spreads to Damage spreads to plants of same or plants of same or related species.related species.
R. Billings
Loblolly pine killed by southern pine beetle
Fusarium wilt of tomato
Consult literature resourcesConsult literature resources
APS Compendia seriesAPS Compendia series US ARS Systematic Mycology and US ARS Systematic Mycology and
Microbiology websiteMicrobiology website Pestnet Diagnostic Service: Pestnet Diagnostic Service:
www.pestnet.orgwww.pestnet.org Other Online resourcesOther Online resources
Identify symptoms and signsIdentify symptoms and signs
SymptomsSymptoms - alterations of a plant’s - alterations of a plant’s appearance due to a disease, pest or appearance due to a disease, pest or disorder.disorder.
SignsSigns - actual pathogen, pest, parts or - actual pathogen, pest, parts or by-products seen on a host plant.by-products seen on a host plant.
Localized vs. Systemic SymptomsLocalized vs. Systemic Symptoms
Localized SymptomsLocalized SymptomsLeaf spotsLeaf spotsStem lesionsStem lesionsFruit rotsFruit rotsBlights Blights GallsGalls
Systemic SymptomsSystemic SymptomsVirus infectionsVirus infectionsLeaf yellowingLeaf yellowingStunting, slow growthStunting, slow growthWiltingWilting
Systemic SymptomsSystemic Symptoms
Diagnosis is often more difficultDiagnosis is often more difficult
First pin point sourceFirst pin point source
Vascular diseaseVascular disease
Root RotsRoot Rots
Nematode infestationNematode infestation
Isolations or other techniques often needed to Isolations or other techniques often needed to determine causal agent.determine causal agent.
Identification of SignsIdentification of Signs
MyceliumMycelium Distinctive appearanceDistinctive appearanceallows id. of some fungi.allows id. of some fungi.
Advantageous if fungus Advantageous if fungus produces no spores.produces no spores.
Mycelium of Rhizoctonia sp.
Mycelium of Phytophthora sp.
Signs for Localized SymptomsSigns for Localized Symptoms
Fungal fruiting bodies Fungal fruiting bodies or sporesor spores
Downy mildewsDowny mildews Powdery mildewsPowdery mildews SclerotiaSclerotia Bacterial oozeBacterial ooze
Downy mildew of veronica, Peronospora sordida
Cercospora leaf spot of watermelon, Cercospoora citrulina
Southern stem rot,Sclerotium rolfsii
Laboratory testsLaboratory tests
Moist IncubationMoist Incubation Goal – to induce Goal – to induce
sporulation.sporulation. Important for obligate Important for obligate
fungal pathogens. fungal pathogens. Avoid overly moist Avoid overly moist
conditions.conditions.Moist chamber
Identification of SignsIdentification of Signs
Fungal signsFungal signsFruiting bodies Fruiting bodies
Morphology of fruiting Morphology of fruiting body aids in fungal body aids in fungal identification.identification.
Spore-bearingSpore-bearing myceliummycelium Morphology of spores Morphology of spores
provides fungal provides fungal identificationidentification
Leaf Spot DiagnosisLeaf Spot Diagnosis
Yellow spots on Yellow spots on leaves only.leaves only.
Spores (sign) form on Spores (sign) form on leaf undersides.leaf undersides.
Microscopic mount Microscopic mount reveals reveals Fulvia fulva Fulvia fulva is is causal.causal.
Can now diagnose Can now diagnose visuallyvisually
Leaf Spot DiagnosisLeaf Spot Diagnosis
Leaves defoliating.Leaves defoliating. Dark, water-soaked Dark, water-soaked
spots, more obvious spots, more obvious when held up to lightwhen held up to light
Microscopic mount Microscopic mount shows bacterial flow.shows bacterial flow.
Doesn’t id. species, Doesn’t id. species, but control is same.but control is same.
Sally Miller
SignsSigns
Bacterial signsBacterial signs ““Streaming” from Streaming” from
freshly cut stemfreshly cut stem ““Stringing” from cut Stringing” from cut
stems pushed together, stems pushed together, then pulled apartthen pulled apart..
Bacterial oozeBacterial ooze Bacteria “flow” and Bacteria “flow” and
morphology seen on morphology seen on compound microscopecompound microscope..
Bacterial streaming from tomato stem infected by Ralstonia solanacearum.
Laboratory testsLaboratory tests
Bacterial isolationsBacterial isolations Suspend infected Suspend infected
tissue in sterile water.tissue in sterile water. Streak suspension on Streak suspension on
bacterial medium.bacterial medium. Can use selective Can use selective
media.media. Transfer single, Transfer single,
isolated colony.isolated colony.
Cultures of Ralstonia solanacearum
Isolate identificationIsolate identification
Bacterial identificationBacterial identification Biochemical tests.Biochemical tests. Carbohydrate utilization Carbohydrate utilization
(BIOLOG).(BIOLOG). Fatty acid methyl ester analysis Fatty acid methyl ester analysis
(FAME).(FAME). Polymerase chain reaction (PCR).Polymerase chain reaction (PCR). Serological tests, e.g. ELISA.Serological tests, e.g. ELISA.
Melon Vine Wilt DiagnosisMelon Vine Wilt Diagnosis
Wilt of vine - look for Wilt of vine - look for symptoms and signs. symptoms and signs.
GSB lesions have GSB lesions have gumming and gumming and pycnidia; if not present pycnidia; if not present may be Fusarium wilt.may be Fusarium wilt.
Fusarium wilt shows Fusarium wilt shows vascular discolorationvascular discoloration
Laboratory testsLaboratory tests
Fungal isolationFungal isolation Nutritive agar Nutritive agar
media, can be media, can be selective.selective.
Diagnostician can Diagnostician can often id. to genus often id. to genus from appearance of from appearance of culturecultureFungal culture.
Isolate identificationIsolate identification
Fungal identification – Other methodsFungal identification – Other methods Utilizing keys in literature.Utilizing keys in literature. Morphological comparison with Morphological comparison with
drawings in literature.drawings in literature. Literature sources for host diseases and Literature sources for host diseases and
disorders.disorders. Serological, molecular, other tests.Serological, molecular, other tests.
Investigate PathogenicityInvestigate Pathogenicity
If symptoms match, but not a known host, If symptoms match, but not a known host, Kochs Postulates may be next stepKochs Postulates may be next step
A lengthy process, rarely used in routine A lengthy process, rarely used in routine diagnoses.diagnoses.
Problem arises when organism cannot be Problem arises when organism cannot be cultured.cultured.
Koch’s PostulatesKoch’s Postulates
1.1. Note constant association of organism with Note constant association of organism with diseased plants and consistent, observable diseased plants and consistent, observable symptoms.symptoms.
2.2. Isolate and characterize organism in pure Isolate and characterize organism in pure culture.culture.
3.3. Inoculate healthy plant with organism, and Inoculate healthy plant with organism, and observe the same disease and symptoms.observe the same disease and symptoms.
4.4. Re-isolate same organism.Re-isolate same organism.
SymptomsSymptomsViral SymptomsViral SymptomsMosaicsMosaics Irregular patches of Irregular patches of
discolored tissue.discolored tissue. Often with distortionOften with distortionRingspots or line patternsRingspots or line patterns Distinct ring shaped Distinct ring shaped
lesions, various line lesions, various line patterns.patterns.
Very few can be Very few can be identified visually.identified visually.
Identification ofIdentification of non-culturable Pathogens non-culturable Pathogens
MethodsMethods Serological tests, e.g. ELISA .Serological tests, e.g. ELISA . Electron microscopy.Electron microscopy. Staining for virus-induced inclusion bodies.Staining for virus-induced inclusion bodies. Molecular tests, e.g. PCRMolecular tests, e.g. PCR DNA SequencingDNA Sequencing Host range testing.Host range testing.
Pathogen IdentificationPathogen IdentificationELISA : a very ELISA : a very
pathogen specific pathogen specific serological testserological test
ELISA uses animal ELISA uses animal antibodies to antibodies to detect pathogens.detect pathogens.
Multi-well test for multiple Multi-well test for multiple samplessamples
Individual test ELISA kitIndividual test ELISA kit
Plant Disease Diagnosis StepsPlant Disease Diagnosis Steps
8.8. Final DiagnosisFinal Diagnosis All information compiled and analyzed. All information compiled and analyzed.
Organism identified, found to be pathogen of Organism identified, found to be pathogen of host ORhost OR
Lab tests or other information reveal an Lab tests or other information reveal an abiotic abiotic cause.cause.
9. Develop control recommendations and 9. Develop control recommendations and present to grower.present to grower.
Questions??Questions??