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??