plant defense activators

1. “ Defence inducing molecules against plant pathogens
Lessons learned and way forward”
Indian Agricultural Research Institute
Division of Plant Pathology
Speaker - M. Ashajyothi, 10863
Ph.D first year
Seminar leader: G. Prakash
Chairman: Dr. A.Kumar
Credit seminar: Pl.Path 691

2. CONTENT
 INTRODUCTION
 DEFENSE INDUCING MOLECULES - BABA
- PROBENAZOLE
- SA ANALOGUES AND JASMONIC ACID
- CHITOSAN
- OLIGO GALACTURONOIDES
- HARPIN PROTEIN
- VITAMINS (3)
- AZELAIC ACID
- HEXANOIC ACID
 CASE STUDY I
 CASE STUDY II
 ADVANTAGES
 CHALLENGES
 WAY FORWARD
 CONCLUSION

3. A great loss to plant yield......
INTRODUCTION

4.  Their use at commercial level is uneconomical.
 Application is cumbersome.
 Some are proved to be carcinogenic.
 Environmental issues.
 Efforts have been accomplished to devise environmental-friendly strategies for the
check of plant diseases.
 Plants can activate separate defense pathways depending on the type of pathogen
encountered (Garcia-Brugger et al.,2006).

5.  Discovery of natural and synthetic compounds called elicitors that induce
similar defense responses in plants (Gómez-Vásquez et al.,2004).
 Term elicitor = phytoalexins now commonly used for compounds stimulating
any type of plant defense (J. Ebel.,1994).
Universal plant defense pathways

6.  Putative binding of elicitor and receptor a signal transduction cascade is
activated and lead to the activation of a variety of plant defense responses.
Types of elicitors

7.  Priming is a mechanism which leads to a physiological state that enables
plants to respond more rapidly and/or more robustly after exposure to biotic
or abiotic stress.
 This increased alertness correlates with no or minimal gene induction
(Slaughter et al., 2012).
 Priming evolved to compensate for the vulnerability of plant to pathogen before
defense responses trigger.
 It allow plants to sense environmental cues and to promote a state of readiness
to enable a quick, strong response upon pathogen attack (Frost et al., 2008).
Getting ready for battle

8. PLANT DEFENSE ACTIVATORS

9.  Alternatives to fungicides in plant protection have arisen with the discovery of
disease resistance inducers of biotic and abiotic origins.
 Depending on their efficacy, these compounds can be used in fields either
alone or in combination with fungicides.
 Many compounds have been commercially released in some countries as a
plant health promoter (P. Chen.,2006).
 Over the years, a range of chemical treatments has proven capable of
triggering IR, mostly through the priming mechanism.

10. β-Aminobutyric acid (BABA)
• An isomer of aminobutyric acid
• Chemical formula: C4H9NO2
• It has two isomers, α-aminobutyric acid (AABA)
γ-Aminobutyric acid(GABA)
• Kuc et al. were the first to notice in 1957 and 1959 that D phenylalanine, D-
alanine, and DL-tryptophan injected into apple leaves increased resistance against
scab without affecting the causal pathogen in vitro.
• 1958, Van Andel examined 50 amino acids for inducing resistance against
Cladosporium cucumerinum in cucumber
• In 1960, Oort and Van Andel first noted induced resistance to tomato late blight
following BABA treatment.
• In 1963, two groups reported on the activity of aminobutrates.
• Amino acid–mediated induced resistance was renewed about 30 years later - a
strong activity of BABA against disease in potato , tomato, and tobacco.
α
β
γ

11. Disease quantification
 Enhance disease defense against late blight of tomato, downy mildew of grape vine and
Phytophthora blight of pepper.
Phytophthora brassicae - Arabidopsis
Phytophthora infestans - Potato
Transformation with Vector P34gfn (nptII) + Reporter gene(gfp)
Quantification of pathogen growth in Planta by measuring gfp fluorescence
Transformants with high gfp expression and normal growth and virulence
Non distructive monitoring of infection process - To analyse the efficacy of chemical
inducers of disease resistance.
 Pre-treatment (300 μM) via soil drench applied 24 hpi protected susceptible
Arabidopsis - Landsberg erecta (Ler) from infection with P. brassicae.

12. Curative protection of tobacco against Peronospora tabacina
Control of Fusarium wilt of: A, watermelon (Fusarium oxysporum f.
sp. niveum) and B, muskmelon (F. oxysporum f. sp. melonis)
Protection of NahG tobacco against Peronospora
tabacina.

13.  3-allyloxy-1,2-bezisothiazole-1,1-dioxide
 It was developed by Meiji Seika Kaisha Ltd. in Japan.
 The compound is marketed as Oryzemate® for rice blast control and has been
used by Japanese farmers in rice seedlings and paddy fields since 1975.
 Activities of enzymes in the phenylpropanoid pathway, such as:
Phenylalanine ammonia-lyase,
Peroxidase and polyphenol oxidase,
Probenazole
Rice plants
Blast fungus
C10H9NO3S

15. Rice plants
Fungus
Probenazole
30% Sequence Similarity to PR-10
PBZ1
Infection response defense - participating protein

16.  SA is rapidly conjugate to an O-glucoside
 Storage form
 Inactive form targeted for catabolism
 These conjugates lack the phloem mobility of free salicylate.
Salicylic acid was first prepared by the Italian chemist Raffaele Piria in 1838 from
salicylaldehyde.
Around 3000 BC, the ancient Egyptians used the bark of willow trees to reduce pains
and fevers.
Salicylic acid

17. Jasmonic acid
 Induces systemic resistance against many pathogens by strengthening the
defense mechanisms in plants.
 Octadecanoid pathway
Synthetic SA analogs
 2,6 dichloro iso nicotinic acid and its methyl
ester (INA)
 Benzo(1,2,3) thiadiazole-7-carbothioicacid S-
methylester (BTH)
(SAR is analogous to the innate immune system found in animals)
SAR
JA

18. Arabidopsis mutant npr1
avirulent pathogens
No enhanced SA levels
PR expression
Over expression NPR1 in
transgenic plants
npr1
SA
SAR
Stronger PR gene expression
Enhanced disease resistance
NPR1 seems to play a key role in the SA-independent induced systemic resistance
response.
PR Proteins

19.  Potential elicitor having antiviral, antibacterial, and antifungal properties.
 Mechanisms:
• Direct toxicity or chelation of nutrients and minerals from pathogens.
• It can form physical barriers around the penetration sites of pathogens,
preventing them from spreading to healthy tissues.
• Induce reactions locally and systemically that involve signaling cascades.
• Chitosan was also shown to alter many other H+ mediated processes.
• Oxygen-species scavenging and antioxidant activities, as well as octa
decanoid pathway activation
• Role of priming in the complex chitosan-plant interaction framework are still
scarce.
Chitosan

20.  Oligogalacturonides(OGs) – Plantcell wall pectin-derived oligo saccharides which
consist in linear chains of α-(1-4)-linked D-galacturonic acid.
 Endogenous elicitors, and the degree of methylation and acetylation has been
found to affect the activation of defense responses.
 Some evidence indicates the involvement of OGs signaling in the octadecanoid
pathway, whereby LOX activities are enhanced.
 Harpin Protein: A 44-kDa protein encoded by hrp (hypersensitive reaction and
pathogenecity) gene of Erwinia amylovora.
 It elicits protective response in plants and makes them resistant to a wide range
of diseases.

21. • Riboflavin is involved in antioxidation and peroxidation resulting in the
production of reactive oxygen intermediates (ROI) in oxidative burst and
consequently hypersensitive response.
• Thiamine can modulate the cellular redox status to protect Arabidopsis against
Sclerotinia sclerotiorum at early stages of infection(Zhou et al.,2013).
• Para-aminobenzoicacid(PABA): cyclic aminoacid vitamin-B group
• Field experiments have proven that it is capable of enhancing resistance against
Cucumber mosaic virus and Xanthomonas by inducing(Song etal.,2013).
PAL gene
Peroxidase (cprx1) gene
Sugarbeet
Rice
Rhizoctonia solani
Riboflavin
Vitamins

22. • It has been suggested to be a phloem-mobile signal that primes SA-
induced defenses (Jung et al., 2009; Shah, 2009).
• The AA biosynthesis pathway is largely unknown.
• AA primes plants for more rapid SA accumulation by inducing glycerol-3-
phosphate (G3P) biosynthesis.
• G3P levels have been proposed to modulate primary and secondary
metabolic pathways, and to contribute to major physiological responses in
defense (Chanda et al., 2008).
• Both AA and G3P seem to be implicated with phytohormones SA and JA.
Signal transmitter - Azelaic acid (AA)

23. HEXANOIC ACID PRIMING AGENT
Hexanoic acid (6Cmono carboxylic acid-Hx) ( C6H12O2 )
4 wk old Tomato roots
Callose deposition
Increased caffeic acid levels
Hx-IR
Castle mart
Bio active signal - Jasmonoyl-isoleucine (JA-Ile)
Oxylipin12-oxo-phytodienoic acid (OPDA)
Significant increase in SA, in water treated plants but not in Hx-primed, post inoculation.

24. HEXANOIC ACID IS A BROAD-SPECTRUM NATURAL INDUCER
Arabidopsis (Hx – treated)Botrytis cineraria
 JA and ET defense-response marker gene PDF1.2
 Hevein-like protein gene PR4
 Specific JA-induciblemarkergeneVSP1
 The eds1-1 mutant (Zhou etal.,1998; Falketal.,1999) was unable to display Hx-IR.
 JA-impaired mutant jar1 and jin1-2 were unable to display Hx-IR
JAR1 JA with IsoleucineEnzyme (Staswick et al., 2002)
JAI1/JIN1 AtMYC2 Up regulate by JA content (Lorenzo et al., 2004)
Metabolic switch for
hexanoic acid

25. Hexanoic acid regulates and primes Botrytis-specific and non-specific genes
Botrytis cineraria
Gene expression studies
 Proteinase inhibitors
 Defense genes
 Transcriptional factors
 Signaling and hormone-related genes
 Oxylipins, ethylene & auxin related genes
 Redox relaated genes
24hpi
Hx preventively activates these genes, thus preparing plants for an
alarmed state, which would facilitate a quicker, better response against
pathogen attack.
Hx-treated plants
24hpi
All genes induced by Botrytis + set of genes
not induced by botrytis 24hpi
 These specific Hx early induced genes - as targets of new preventive defense
strategies.

26.  Many natural compounds have been claimed to be plant growth promoters,
plant activators or plant defense inducers :
• Oligosaccharides
• Glycosides
• Amides
• Carboxylic acids
• aromatic compounds
• Prohexadione – Ca
• Potassium phosphonate
• Fosetyl- Al

27. Compounds commercially released as plant health
promoters

29. • Composition
• Organic nitrogen at 1.0% by mass*
Phosphorus (P2O5) at 23.0% by mass*
Potassium (K2O) at 20.0% by mass*
Copper (Cu-EDTA) at 2.6% by mass

30. Aim: To check the effect of jasmonic acid (JA) and ⁄ or b-aminobutryric acid (BABA) treated
seeds on increased resistance of plant against insects and against the necrotrophic fungal
pathogen, powdery mildew.
Materials and methods:
Plant- Tomato
Activators: JA, Beta-aminobutryric acid (BABA)- nonprotein amino acid
Challengers: spider mites, caterpillars and aphids, and against the necrotrophic fungal
pathogen, Botrytis cinerea.
CASE STUDY I

31. Methods and results:
Seed treatment with JA enhances herbivore and disease resistance
Tomato seeds
Soaked in
3 mMsolution of JA
germinated
7- to 10-wk-old plants
After 9 days- populations and reproductive rate measured and found reduced
compared to control
Inoculation of Tetranychus urticae
Also observed:
 Reduced feeding of tobacco hornworm (Manduca sexta) caterpillars
Significant reduction in populations of the green peach aphid (M. persicae)
Reduced lesion area of necrotrophic fungal pathogen, B. cinerea.

32. JA seed treatment has minimal impact on growth and development
 1–5 mM JA: Delay in germination by 1 day in JA treated plants but final germination
percentage was not significantly altered
10 mM:
 Significant alteration in germination
 Reduction in growth of the primary root
 On long term no differences in plant growth and development (plant height and fruit
dry weight
The priming agent β-aminobutyric acid influences plant pathogen responses when
applied as a seed treatment
 Seed treatments with BABA treatment caused no reduction in plant growth.
 Plants grown from treated seed were challenged, powdery mildew (O. neolycopersici) and
observed significantly lower degrees of colonization

33. Tradeoffs between different resistance mechanisms are minimized by seed
treatment-induced priming
A.
Plants raised by JA treated seeds
Challenge inoculation with
O. neolycopersici
Resistant as compared to control
A JA priming doesnot inhibit
SA mediated resistance
B.
Plants raised by β-aminobutyric acid treated seeds
Challenge inoculation with
B.cinerea
Susceptible compared to control
A BABA priming inhibit JA mediated
resistance
C.
Plants raised by b-aminobutyric acid and JA treated seeds
Challenge inoculation with B.cinerea
Resistant compared to control
A BABA cannot inhibit JA mediated
resistance when JA is primed

34. JA-induced priming of Botrytis resistance depends on JA and ethylene
signalling, and is associated with increased JA-dependent gene expression
A.
JL5 (def1)- defecient in JA
biosynthesis
B.
Never ripe plants- defecient in ethylene perception
No increased resistance to Botrytis

35. Quantitative real-time PCR (qPCR) was used to monitor expression of a
number of well-known defence-related genes from tomato
Observations:
• Early transcriptional activation of the JA biosynthetic gene ALLENE OXIDE SYNTHASE 2
(AOS2),
• Mid-phase activation of a JA-responsive defence gene PROTEINASE INHIBITOR II (PinII)
• Late activation of the pathogenesis-related gene PR1b1
Botrytis inoculated leaves were sampled over a 24-h
(qPCR)
JA treated seeds
Found no consistent difference between the timing or peak expression levels between
control and JA seed-treated plants, but in the case of the JA-dependent defence gene,
PinII, we observed higher expression in JA seed-treated plants

36. • Priming of defences, on the other hand, minimizes these costs whilst
improving future resistance to attack consistent with the effects of the
seed treatments
• Expression of the genes assayed was similar in control and treated plants
before biotic stress but increased expression once pathogen attacks
• JA priming cannot inhibit resistance against biotrophs but BABA priming
induces susceptibility to nectrotrophs. However BABA priming donot
affect susceptiliblity in JA+BABA treated plants
SUM UP

37. CASE STUDY - II
Aim: To evaluate the effects of various chemical inducer treatments on HLB
progression and fruit yield under field conditions.

38. Materials and methods
Experiment Treatments Replications Cultivar Age (years)
I 11 5 Mid sweet
orange
7
II 16 9 Mid sweet
orange
7
III 11 10 Murcott
mandarin
10
IV 11 10 Valentia sweet
orange
4
Design : CRD
Chemicals : BABA, INA, 2-DDG, AA
Spray : After 3 – 4 months when new flush present
Scoring : 0-5 scale , AUDPC
qPCR

39. Effect of plant defense inducer treatments on HLB disease development
Disease severity of huanglongbing as sAUDPS with
Midsweet orange at Mid Florida over time
HLB disease severity in the AA (60 µM),
BABA (15 µM), and BABA (150 µM) treated
groups reduced by 21.3, 28.6, and 21.4%,
respectively
Las bacterial titers in leaves of trees under these three treatments were also
significantly lower
Experiment: I

40. Experiment : II
Disease severity of huanglongbing as sAUDPS
with Midsweet orange at Mid Florida over time
Treatments AA (60 µM), BABA (0.2 to 1.0 mM),
BTH (1.0 mM), INA (0.1 mM), 2-DDG (100 µM),
BABA (1.0 mM) plus BTH (1.0 mM), BTH (1.0
mM) plus AA (600 µM), and BTH (1.0 mM) plus
2-DDG (100 µM) reduced HLB disease severity
by 15 to 25%
Treatments also relatively suppressed the growth of Las bacterial populations in citrus
leaves compared with the negative control

41. Experiment: III
Disease severity of huanglongbing as sAUDPS
with Murcott mandarin
Treatments BABA (1.0 mM), BTH (1.0 mM), INA
(0.5 mM), and 2-DDG (100 µM) reduced HLB
disease severity by 15 to 20%
Suppressed the growth of Las bacterial populations in citrus leaves compared with the
negative control

42. Experiment: IV
Disease severity of huanglongbing as Saudps with
Valencia sweet orange
Treatments AA (600 µM), BABA (0.2 to 1.0
mM), BTH (1.0 mM), INA (0.1 to 0.5 mM), and
2-DDG (100 µM) were relatively more effective
in suppressing HLB disease development than
in experiment III
Suppressed the growth of Las bacterial
populations in citrus leaves compared with the
negative control

43. Effect of plant defense inducer treatments on fruit yield and quality
 Treatments AA, BABA, and INA exhibited a higher fruit yield in 2013 compared
with the negative control.
 There were no apparent differences among treatments in fruit yield (kg of
fruit/tree) in 2013.
 But in 2014, the treatments AA, BABA, BTH, 2-DDG, and INA exhibited a higher
fruit yield than the negative control.

44. Expression of plant defense-related genes
Relative expression of the β-1,3-glucanase gene (PR-2) in Midsweet orange leaves after a
single application of different plant defense inducer compounds.

45. SUM UP
 The treatments AA, BABA, BTH, 2-DDG, and INA have positive control effect on suppressing
Las population in plants and sustain fruit productivity to a certain extent compared with
the negative control.
 It is reasonable to speculate that the reduction of Las populations in citrus could also
impact the pathogen acquisition and spread by psyllids.
 Induction of plant defense showed relatively more effective to young trees with mild HLB
than to old trees with serious HLB.

46.  Reduced damage from insects, fungi, pests, and herbivores.
 Priming – A plant’s memory
 Reduced environmental hazards as elicitors affect directly the crop plant, and
their acute toxicity to other organisms is lower than that of pesticides.
 As protective agrochemicals, elicitors can be applied with the current spraying
technology.
 Elicitor treatments could be an alternative to genetically modified (GM) plants
for better attraction of natural enemies of pest organisms on cultivated plants
(I. F. Kappers.,2005)
 Elicitor-treated plants bear lower ecological risks than GM plants (G. M. Poppy
and M. J. Wilkinson.,2005).
Advantages of using Plant activators

47. Challenges to plant defense activators
Their effect is only transient and lasts only for a few days.
They are not curative and cannot eliminate an already established infection.
Phytotoxicity.
Plant activators would never be able to provide complete protection.
The mode of action of priming agents is eventually determined by hosts and
the stress challenging them.
This makes it difficult to decipher the molecular bases underlying the
priming mechanism.
Dosage for application must be optimized for various diseases.
Methods of application are need to be standardize.
They usually show antimicrobial activities at higher concentrations than
those required for priming.

49. The use of plant actvators in crop protection and pest management is still in the
very early stages of use as a new control method.
Defense activators represent an active area of research in pest and disease
management.
Studies on optimization of dose and method of application must be done.
Because of their versatility, their ability to prime JA-dependent defense and
their general low toxicity, which allows better crop tolerance.
They could be more suited as a component of integrated disease management.
Must be available at cheaper cost in the developing countries.
Awareness must be created among the farmers by the scientific community.

50. Conclusion
 Plant activators do not have any pesticidal or antibiotic activity, their
adverse effects on human health and environment are minimal.
 Since they do not interact directly with the pathogens, it is unlikely that
plant pathogens will develop resistance to these chemicals.
 The success of defense inducers for plant disease control depends on our
ability to manage their phytotoxicity either by chemical modification of
the compound or by modifying their formulation.

51. “Those who contemplate the beauty of the earth find reserves
of strength that will endure as long as life lasts”
- Rachael Carson