Diagnosis of Plant Disease
DR. IR. HADIWIYONO, M.SI.
Study Program of Agrotechnology
Faculty of Agriculture
University of Sebelas Maret (UNS)
Surakarta
E-mail : hadi_hpt@yahoo.com.
Terminologi
Identification:

“the study of the characters of a thing or an
organism to determine its name” (APS, 1998)
Disease identification:
 Study on disease character to determine the
name of disease
Diagnosis


Diagnosis is identification of the nature or
cause of a disease or other condition
Diagnostic is a distinguishing characteristic
important for identification of disease or other
conditions (Glossary Plant Pathological Terms,
1998)
Why do diagnosis of disease

For control disease
 To plan the disease management in the field
 Certification of seed
 Plant quarantine to prevent disease spread inter area or
countries

For research
Principles of plant disease
diagnosis

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Simplicity-easy
Times spent-short
Reliability-accurate
Cost need-cheap
Applicability-practical
Standard operational prosedure in
identification and diagnosis of plant
diseases

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The first step is the determination whether the disease is
caused by pathogen or environmental factor
For some cases, the disease symptom and sign appear very
obviously or very specific, thus supported by the experience
and some available references prove the disease diagnosis to
be easy to do.
Most of cases however need detail observation and test to
the symptom of diseases.
Diseases caused by parasitic
higher plant

The existence of
the parasitic higher
plant on the host is
enough to
recognize the
causal agent
A. Benalu (Henslowia frutescens Champ.) on java apple
B. Tali putri (Cassytha filiformis L. or Cuscuta australis R. Br.) on an
ornamental plant.
Diseases caused by fungi and
bakteria
While miselia of fungi and spore or ooze of
bacterium exhibit in/on the part of diseased
plant thus two possibility should be
considered:
1. Fungi or bacterium is the causal disease
2. Fungi or bacterium is one of many
saprophytic fungi or bacterium living on
dead tissues caused by other causal agent
that is also belong to the group of fungi or
bacterium.

Fungi



Some disease have been reported that visual and
microscopic observation to structure of mycelia, fruit
bodies, and spores associating the symptom followed by
checking using relevant manual books or references is
enough to determine whether the fungi is the pathogen
or saprophyte
In most of cases however, the signs disappear in/on the
diseased plant thus identification of the fungi is
impossible to do.
Certain fungi must be isolated using selective medium
and promoting the sporulation in specific grow chamber.
Method for promoting formation
specific structure of fungi in/on
diseased tissues


Incubation in high humidity
Incubation under near ultraviolet
(NUV)
Method for promoting exhibition of
specific structure of certain fungi on
pure culture
1. Incubation under near ultraviolet (NUV)
Commonly, the radiation is able to
promote the fungi for sporulation
2. Specific treatment i.e. line cutting the
medium, enriching with specific
substrate, incubating in specific
temperature condition.
Isolation and characterization
of fungi on culture medium



Structure of colony
Specific structure fungi i.e. forming
sclerotium and chlamidospore
Coloring medium by extracellular compound
released by the fungi
Chemical and physiological
characterization of fungi


The method is less developed
Physiological character of fungi is limited in
grow response on the maximum and
minimal range of tolerance to salinity,
temperature, and pH of medium
Club root caused by Plasmodiophora
brassicae on cabbage
Sign and specific structure of
Rhizoctonia solani
The sign and specific structure
of Sclerotium rolsii
Symptom and structure to recognize
F.o. f.sp. melonis on melon
A
B
C
A. Discoloration in vascular systems of stem -seem browning
B. Wilting melons
C. Microconidia (a), makroconidia (b), clamidospore (c).
Symptom and sign on scallion
Alterneria porrii
A. Purple blotch on leaf of scallion; B. Conidia under stereo microscope; C. Conidia
under compound microscope
Specific symptom of wilt disease
of banana (F.o. fsp. cubense)
Bakteri


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Bacterium is too tiny to observe visually or
using compound microscope
Specific symptom and sign of disease are
often reliable to identification of certain
disease in a short time to common diseases
being well known
In most of cases, selective medium and
specific diagnosis technique are needed
Specific symptom and sign of banana wilt
caused by blood disease bacterium (BDB)
Sign disease of BDB on
banana
Sign of bacterial disease
B
A
A. Sticky strand test on cut stem with bacterial streaming
(oozing) from xylem vessels of melon infected Ervinia
tracheiphila, causing bacterial wilt
B. Oozing, bacteria come out from leaf vascular system
Performing bacterium on
selective media
B
A
C
A. Identification of fluorescence bacteria on King’s B medium; B. BDB
on CPG, and C. BDB on TTC medium
Observation of phenotipic
colony

Observation the colony structure on specific
medium is often used to recognize bacterial
pathogen
Colony of Xanthomonas
campestris pv. campestris, the
cause of black rot of cabbage
with Yellow colony and forming
halo
Hypersensitive Reaction Test

Principle: all of bacterial pathogen exhibit
hypersensitive reaction on leaf of tobacco
Bacterial suspension 108 sel /mL are injected into the leaf of tobacco
(Nicotiana tabacum)
Microscopic observation


In severe diseased tissue, the
abundance of bacteria can reach 108
– 109 cell/gram of diseased tissue
Washed ooze from diseased tissue or
pure culture can observed at 4001000x
 motile bacteria will
more observed
Pathogenesity test of bacteria
 Pathogenisity test on susceptible host can be employed to
diagnose bacterial diseases.
 Various inoculation techniques may be applied i.e.
- Injection
- Spraying on abaxial foliages
- Soil drenching
- Dropping on infection site
- Seed germination on infested medium in plate
Biochemical and physiological test



Well developed and various
Gram reaction test, chemotropic test, capability of
carbon degradation, substrate oxidation, nitrate
reduction and so forth.
Spend along time and impractical diagnosis but it is
a conventional standard characterization in
complete identification
Reaction to bacteriophage

Susceptible bacteria to
bacteriophage may be
one of specific
character of certain
pathogenic bacteria
Structure of bacteriophage
Diagnosis bacterial disease Commercial
automated techniques: Biolog

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Spesies bahkan strain memiliki keragaman kemampuan dalam
memanfaatkan berbagai sumber karbon
569 taksa bakteri gram negatif dan 223 gram positif dapat
diidentifikasi dengan perangkat ini
Cawan mikrotiter terdiri dari 95 subtrat sumber karbon dan
satu kontrol
Inkubasi pada suhu 27-28 oC, selama 4-24 jam
Reaksi terlihat adanya prubahan pewarnaan oleh TTC
Hasil dianalisis dengan perangkat lunak Microlog
Diagnosis bakteri dengan Commercial
automated techniques: FAME

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FAME (Fatty Acid Methyl Ester Analysis)
Prinsip teknik
1. Bakteri dikulturkan pada kondisi standar
2. Melepaskan asam lemak dari permukaan sel bakteri
melalui saponisasi
3. Methylasi asam lemak untuk meningkatkan volatilitas
4. Analisis dengan gas kromatografi resolusi tinggi
5. Membandingkan profil asam lemak yang diperoleh dengan
profil mikrobia standar
Identification method based
serology (immunology)

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High sensitive, specific, accurate, fast, and simple to do
Antiserum monoclonal and polyclonal have been avalable
for some plant pathogenic bacteria
Sensitivity 104-106 sel/mL
Diagnosis through DNA finger
printing
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Many method of identification based
molecular techniques (Polymerase Chain
Reaction) have been developed
Specific primer derived from many plant
pathogenic bacteria heve been available
Sensitivities 102-103sel/mL
DNA finger printing based on PCR
1 C+ C- M
A
A.
B.
C.
1
B
2
3
C- M
M
1
2
3
4
C
Identification of BDB using primer specific 121F and 121R : 1. BDB, C+.
Control-positive, C-. Control negative
Identification Ervinia spp using PCR-RAPD with Universal Primer : 1. E.
stewartei, 2. E. carotovora, 3. P. syringe, C-. Kontrol negatif (Oliver, 1993
Identifikasi X.campestris dengan rep-PCR; 1-2 Xc pv carotae, 3-4 Xc pv
coriandri
Virus


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Virus is obligate parasite, true biotrophic-
unculturable
Too small-visualized in light microscope except x
bodies belong to some viruses
Identification/diagnosis: specific symptom,
pathogenisity test, observation of specific structure
in infected cell, specify host test, molecular
techniques
Diagnosis of plant diseases
caused by virus
For some cases, the symptom of viral diseases are
specific.
 So using relevant manual book diagnosis is easy to
do.
 For some other cases, virus diseases cause specific
symptom
 For other cases, pathogen cause the symptom
being similar to other virus infection, tiny pests,
toxicity, deficiency, herbicide toxicity and so forth.

pathogenisity test of virus

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Some virus can be inoculated mechanically.
Nicotiana spp. and Chenopodium spp. are
often used as plant indicator
Type of symptom: local lesion and systemic
Viral diseases with specific
symptom
A
B
C
A. The symptom of cucumber mozaic virus on lettuce, B. Beet yellow stunt virus on
crisp head lettuce (Davis et al. 2002), C. Zucchini yellow mozaic virus onSummer
squash (Zitter et al.,1998)
Ex. symptom of pest are similar
caused by pathogenic diseases
A. Leaf symptom spider mites, B. Spider mites, Tetranychus sp.
Ex. of symptom of herbicide toxicity
being similar to virus infection.
A. Leaf of squash caused by 2,4-D, B. Glyphosate
Transmitting test

Some plant viruses are insect born
A. Aphid, Myzus persicae
(dewasa)
B. Whitefly, Bemissia sp.
(dewasa)
A
B
C. Aphid, Aphis gossypii
(dewasa)
D. Squash bug, Anasa
tristis (nimfa)
C
D
Observation of specific structure of virus
in infected cell

Potyviridae produce
‘pinwheel’ inclusions
Diagnosis virus using molecular
technique: serological techniques
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Virus is enveloped by protein called coat protein
Viral protein (antigen) will act to specific antibody
(antiserum) produced by mammalian injected with
the antigen
Enzyme-Linked Immunosorbent Assay (ELISA) is
the most popular technique
Many antiserum are available in market
Diagnosis virus using molecular
technique: based on PCR
Important information addition
for disease diagnosis in the fields
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Farmer’s name and address
Plant variety
Stage of plant
Pattern of the disease incidence
Insect or other organism
Land history or the previous plantings
Plant environmental condition
Around planting condition
Cultivation practices
Diagnosis of diseases haven’t
been well known yet


Certain diseases are well known so the diagnosis
are easy to do through observing the symptom and
pathogen by then comparing to relevant available
manual books or references.
In contrary, the others pathogens may be poorly
understood and haven’t been identified yet. For the
cases, Koch’s Postulate application is needed.
Koch’s Postulate
1. Disease agent must be associated with the symptom
2. The agent must be able to isolate in pure culture
3. the pure culture inoculated on the susceptible healthy host
must be able to induce the symptom on host where the
disease agent is isolated
4. The agent must be able to re-isolate from the inoculated
host plant
Standard equipment for diagnosis of
plant diseases
1.
2.
3.
4.
5.
6.
7.
8.
Lens or hand Loop
Digital camera with enough resolution
Stereo microscope and compound microscope
Grow or incubation chamber installed lamp and NUVgrow chamber
Isolation room, fungi and bacteria must be separated
Manual book for identification: ex. Compendium,
identification guides manuals
Computer with internet on line
KIT for isolation, culture, and microscopic isolation
Conclusions


Disease diagnosis is basic activity being important in plant
disease control
Diagnosis of pant disease can be done through:
1. Observation of the symptom and sign
2. Isolation and inoculation on susceptible plant indicator
3. Observation the biochemical and physiological
characters of the pathogen
4. Microscopic observation
5. Serological techniques
6. Molecular techniques based on nucleic acid .
An important statement

The plant disease identification is a scientific
art that is enhanced with experience and
constant study (Shurtleff & Averre, 1999)