“ Defence inducing molecules against plant pathogensLessons learned and way forward”

Indian Agricultural Research InstituteDivision of Plant Pathology

Speaker - M. Ashajyothi, 10863 Ph.D first year

Seminar leader: G. Prakash

Chairman: Dr. A.Kumar

Credit seminar: Pl.Path 691

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

A great loss to plant yield......

INTRODUCTION

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).

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

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

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

PLANT DEFENSE ACTIVATORS

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.

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

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.

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)

Stem

injection

Soil

drench

Foliar

spray

Protection of NahG tobacco against Peronospora tabacina.

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

Rice plantsFungus

Probenazole

30% Sequence Similarity to PR-10

PBZ1

Infection response defense - participating protein

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

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

Arabidopsis mutant npr1

avirulent pathogens

No enhanced SA levelsPR expression

Over expression NPR1 in transgenic plants

npr1

SA

XSAR

Stronger PR gene expressionEnhanced disease resistance

NPR1 seems to play a key role in the SA-independent induced systemic resistance response.

PR Proteins

X

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

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.

• 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

• 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)

HEXANOIC ACID PRIMING AGENT

Hexanoic acid (6Cmono carboxylic acid-Hx) ( C6H12O2 )

4 wk old Tomato roots

Callose deposition Increased caffeic acid levelsBotrytis cineraria

Hx-IR

Castle mart

X

Additional mechanisms 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.

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

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.

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

Compounds commercially released as plant health promoters

• 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

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- TomatoActivators: JA, Beta-aminobutryric acid (BABA)- nonprotein amino acidChallengers: spider mites, caterpillars and aphids, and against the necrotrophic fungal pathogen, Botrytis cinerea.

CASE STUDY I

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) caterpillarsSignificant reduction in populations of the green peach aphid (M. persicae)Reduced lesion area of necrotrophic fungal pathogen, B. cinerea.

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

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

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

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

• 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

CASE STUDY - II

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

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 : CRDChemicals : BABA, INA, 2-DDG, AASpray : After 3 – 4 months when new flush presentScoring : 0-5 scale , AUDPC qPCR

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

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

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

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

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.

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.

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.

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

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.

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.

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.

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