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Hindawi Publishing CorporationISRN BiotechnologyVolume 2013, Article ID 601303, 6 pageshttp://dx.doi.org/10.5402/2013/601303

Research ArticleInduction of Defense-Related Enzymes in Banana Plants:Eect of Live andDead Pathogenic Strain of Fusarium oxysporumf. sp. cubense

Janki N. Thakker,1 Samiksha Patel,1 and Pinakin C. Dhandhukia21 Department of Biotechnology, PD Patel Institute of Applied Sciences, Charotar University of Science and Technology,CARUSAT Campus, Gujarat, Changa 388421, India

2Department of Integrated Biotechnology, Ashok and Rita Patel Institute of Integrated Study and Research in Biotechnology andAllied Sciences, Gujarat, New Vallabh Vidyanagar 388 121, India

Correspondence should be addressed to Janki N. akker; [email protected]

Received 29 October 2012; Accepted 15 November 2012

Academic Editors: H. S. Garcia and A. Kouzmenko

Copyright 2013 Janki N. akker et al. is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

e aim of the present study was to scrutinize the response of banana (Grand Naine variety) plants when interacting with dead orlive pathogen, Fusarium oxysporum f.sp. cubense, a causative agent of Panama disease. Response of plants was evaluated in termsof induction of defense-related marker enzyme activity, namely, peroxidase (POX), polyphenol oxidase (PPO), -1,3 glucanase,chitinase, and phenolics. Plants interaction with live pathogen resulted in early induction of defense to restrain penetration as wellas antimicrobial productions. However, pathogen overcame the defense of plant and caused disease. Interactionwith dead pathogenresulted in escalating defense response in plants. Later on plants inoculated with dead pathogen showed resistance to even forcedinoculation of live pathogen. Results obtained in the present study suggest that dead pathogen was able to mount defense responsein plants and provide resistance to Panama disease upon subsequent exposure. erefore, preparation from dead pathogen couldbe a potential candidate as a biocontrol agent or plant vaccine to combat Panama disease.

1. Introduction

Musa acuminata (Banana) is one of the most important fruitcrops of world as well as of India. Banana could be consideredpoor mans apple, and it is the cheapest among all otherfruits in the country. Fusarium wilt caused by Fusariumoxysporum f.sp. cubense (Foc) is the most destructive diseaseof banana [1]. e pathogen is soil-borne and remains viableup to several years and cause 20%80% loss of banana.Several disease management strategies can be used such ascrop rotation, burning infected plants or plant parts, andapplication of carbendazim [2]. Methods mentioned havelimited success, and the application of synthetic fungicidesmay result in undesirable eects on the environment.

An alternative to above strategies for managing fusariumwilt is the use of biological control. Biocontrol agent can be a

benecial organism (live or dead) or its part such as cell wall,protein, and oligosaccharides [3]. While using live organismsas a biocontrol agent, appropriate conditions for maintainingit should be strictly followed. Nevertheless, if part of theorganism such as cell wall, protein, oligosaccharide, orattenuated/killed organism is used then strict conditions arenot required. Plants, humans, and animals give instantaneousresponse to the pathogen or its part. Animals and humansproduce antibodies against pathogen or vaccine, similarlyplants response to pathogen attack by producing PR-proteins,defense-related enzymes [4], plantibodies, and phytoalexins.

Study of PR-proteins or defense-related enzymes arekey to any plant disease resistance mechanism. Farmersin Gujarat, India purchase tissue culture plants every yearfor sowing in the elds with the expectations of gettinghigh production and high prot. Foc being soil borne may

2 ISRN Biotechnology

enter the plant and cause disease anytime aer sowing andaects fruit production. If plants are immunized, that is,accumulation of defense-related enzymes occurs before theattack of pathogen then the pathogen can be successfullywarded o, and loss can be minimized. Same concept ofvaccination is used here. Vaccines used for animals andhumans are derived from the same disease causing organism.But vaccines have inactive organism or attenuated organism.Elicitor used here is acting as a vaccine (derived from the deadfungus) to protect the plant. e aim of the present studywas to compare the interaction of dead and live pathogenwith banana plants (Grand Naine variety). Grand Naine isa large fruit yielding dwarf Cavendish variety with height of6.5 to 7.5 introduced to India from Israel remains choiceof farmers as the bunches of banana fruits can be harvestedwithin twelve to thirteen months from the date of planting.e plant response was documented using several markerenzymes to analyze whether the plant could dierentiate thesignals from dead and live pathogen. Another important aimwas to check if the response generated using dead pathogenwas sucient to ward o the forced inoculated live pathogen.

2. Materials andMethods2.1. Maintenance of Fungal Culture. Previously isolated cul-ture of Fusarium oxysporum f.sp. cubense (Foc) from fusarialwilt infected banana plants was maintained on potato dex-trose agar (PDA) at 27C [4]. For liquid culture of fungus, 8-mm agar plug of 3-4w k old culture was inoculated in potatodextrose broth (PDB) and incubated at 27C for 21 day onPDB. e media with mycelium was autoclaved at 121C for20 minutes, crushed intensely in grinder, and was used asdead fungi for treatment.

2.1.1. Plant Material. Twomonths old Banana (Grand Nainevariety) plantlets were procured fromGujarat State Fertilizersand Chemicals (GSFC), Baroda, Gujarat. Plants were plantedandmaintained in campus garden. Health of these plants wasobserved regularly by visual inspection.

2.2. Dead and Live Fungus Treatment. Plant roots of GrandNaine variety were exposed without damaging them by care-fully removing the surrounding soil. Dead and live fungussuspensions were prepared by mixing 1 g of dead fungus andlive fungus per liter of distilled water. Per plant, 1mL ofdead and live pathogen suspension was administered againstcontrol plants treated with 1mL of distilled water in exposedroot region. Aer treatment, changes in levels of defense-related enzymes in leaves were assayed aer each successiveday till seventh day [4, 5].

2.3. Experimental Design. Disease-free in vitro propagatedtwo months old plantlets were selected. Dead and livefungus treatments were given as mentioned earlier to plants.Distilled water treated plants were used as control. Leaveswere excised up to seven days at regular interval of 24 h forestimation of defense related enzymes, namely, POX, PPO,-1,3 glucanase, chitinase, and total phenolics from both

control and treated plants. Experiments were repeated twiceunder similar conditions with dierent sets of plants, andall the analyses were performed in triplicates. Fresh Bananaleaves were washed in running tap water and homogenizedin liquid nitrogen. e homogenized leaves were kept at 4Cuntil used for enzyme analyses. POX activity was measuredas described by Sadashivam and Manickam [6]. PPO, -1,3glucanase, chitinase, and total phenolics assays were done asdescribed by Meena et al. [7].

2.4. Forced Inoculation. To ensure the induction of resistance,plant roots treated with dead pathogen was exposed to sporesuspension of Foc (104 spores/mL) where plants treated withdistil water was used as control and exposed to same quantityof spore suspension as experimental. Plants were kept underobservation for the development of the symptoms.

3. Resultse aim of this study was to investigate interaction of deadand live pathogen, Fusarium oxysporum f.sp. cubenseacausative agent of panamadisease, with banana plants (GrandNaine). Interaction was determined using accumulation ofseveral defense related enzymes, namely, POX, PPO, -1,3 glucanase, chitinase, and total phenolics as markers forinduction of defense. Hypothesis was that plants could dier-entiate signals fromdead and live pathogen and in response tosignals from dead pathogen, it can induce defense enzymes,which could protect plants upon subsequent exposure topathogen.

3.1. Eect of Live and Dead Pathogen on Defense-RelatedEnzymes. POX activity in control plants remained constantthroughout seven days of study. POX activity induced earlierin dead and live pathogen treated plants and retained elevatedlevels compared to control plants. POX activity increased4- and 2.5-fold in dead and live fungus treated plants,respectively. Highest POX activity in plant treated withdead and live pathogen was observed on 6th and 7th day,respectively (Figure 1). Comprehensible dierence in PPOactivity was observed on treatment of dead and live pathogen.With live fungus interaction, plants showed to induce 3-foldPPO activity from rst day onward, reached highest level on4th day, and thereaer reached near basal. However, deadfungus treatment failed to mount signicant induction inPPO activity for initial ve days of interaction followed by2- and 3-fold induction in PPO activity on 6th and 7th days,respectively compared to control plants (Figure 2).

Interaction of dead and live fungus with banana plantsresulted in induction of -1,3 glucanase activities from 1stday onward. p to ve days, pattern of -1,3 glucanaseactivity was very similar in both treatments. From the 5thday onward, -1,3 glucanase activities stabilized at 2- to 2.5-fold higher levels in live fungus interaction as compared tocontrol. In case of dead fungus interaction, gradual increasein-1,3 glucanase activity was observed from5th day onwardwith highest increase 3.5-fold on 7th day as compared tocontrol (Figure 3).

ISRN Biotechnology 3

00.0050.01

0.0150.02

0.0250.03

0.0350.04

0.0450.05

1 2 3 4 5 6 7

Days

Control

Dead Foc

Live Foc

PO

X a

ctiv

ity

(ch

ange

in

ab

sorb

ance

/g F

T)

F 1: O activity prole of banana for reinforcement ofphysical barrier as well as ROS generation in response to distilledwater (control), live fungus, and dead fungus interactions for sevendays.

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

1 2 3 4 5 6 7

Days

PO

X a

ctiv

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(ch

ange

in

ab

sorb

ance

/g F

T)

Control

Dead Foc

Live Foc

F 2: O activity prole of banana for generation of antimi-crobials in response to distilled water (control), live fungus, anddead fungus interactions for seven days.

Chitinase activity induced from 1st day in banana plantstreated with dead and live pathogenic fungus. Figure 4showed sti rise (10-fold) in chitinase activity on 2nd and 3rdday followed by rapid fall and retention of about 1.5 fold chiti-nase activity in banana treated with live fungus comparedto control plants. Whereas, in dead fungus treated plants,chitinase activity increased twofold on 1st day followed bygradual increase up to 4-fold on 7th day.

In dead fungus treated plants, total phenolics contentgradually increased and reached to maximum on 7th day (2-fold). Whereas in live fungus treated plants, total phenolicscontent increased on 3rd day followed by sharp decline toreach basal level as compared to control plants (Figure 5).

3.2. Forced Inoculation. Control plants and plants treatedwith dead pathogen were forced inoculated with the live fun-gal spores and observed for the development of symptoms.In control plants, the characteristic symptoms of fusarialwilt were observed in the rst week aer forced inoculation

0

0.02

0.04

0.06

0.08

0.1

0.12

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F 3: -1,3 glucanase activity prole of bananafor directantifungal activity in response to distilled water (control), livefungus, and dead fungus interactions for seven days.

0

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G (

nM

/min

/g F

T)

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Dead Foc

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F 4: Chitinase activity prole of banana for direct antifungalactivity in response to distilled water (control), live fungus, and deadfungus interactions for seven days.

followed by aggravation of disease condition aer 15 days.However, no symptoms of fusarium wilt were observed evenaer two months aer forced inoculation in plants treatedwith dead pathogen.

4. DiscussionIn the present study, analysis of plants response towards deadand live pathogenic strain was carried out using inductionof several key marker enzymes associated with plant defensemechanism. Development of eective, durable, economic,and environmentally sound strategies for the control of cropdiseases could be possible through an improved understand-ing of the interactions between plants and pathogenic agents.e ability of a pathogen to produce a disease in a hostplant is usually the exception, not the rule. is is becauseplants have an innate ability to recognize potential invadingpathogens and to set up successful defenses. On the otherhand, successful pathogens produce diseases because they


00.020.040.060.08

0.10.120.140.160.18

1 2 3 4 5 6 7

Days

Control

Dead Foc

Live Foc

F 5: otal phenolics activity prole of banana for reinforce-ment of physical barrier as well as antimicrobial activity in responseto distilledwater (control), live fungus, and dead fungus interactionsfor seven days.

are capable to evade detection or suppress host defensemechanisms, or both [8].

When plants are challenged by a pathogen, early localdefense reactions and delayed, systemic responses get acti-vated in order to counteract the pathogen attack. Amongthe early local responses, the hypersensitive response (HR)leads to a local programmed cell death in order to deprivethe pathogens of their nutrition base [9]. Later on, the plantcan develop systemic acquired resistance (SAR) leading toresistance throughout the whole plant in an unspecic man-ner towards a broad spectrum of pathogens. In case of SAR,signal is transmitted from infected tissue in the whole plantfor induction of overall defense gene expression.is demon-strates that signal perception in initial pathogen recog-nition and signal transduction to initiate further defenseresponses is essential for plants to counteract phytopathogens[10].

Some defenses are constitutive, such as various pre-formed antimicrobial compounds, whereas others activatedby pathogen recognition. Recognition process includes prod-uct of a dominant or semidominant resistance R gene presentin the plant and the corresponding dominant avirulence(Avr) factor encoded by or derived from the pathogen.Recognition of Avr factor by host plant starts one or moresignal transduction pathways that activate several of plantsdefenses, thus compromising ability of pathogen to colonizeplant [11, 12].

In the present study, interaction of banana plants withdead and live pathogen resulted in induction of POX activity.However, POX activity induced more with dead fungus. Asdead pathogen was capable to generate initial recognitionsignals; however, it cannot counter the plant response, whichleads to induction of POX activity more than live fungus.Peroxidases are a well-known class of PR proteins andinduced in host plant tissues by pathogen infection. eybelong to PR-protein 9 subfamily [13] and are expressed tolimit cellular spreading of infection through establishment ofstructural barriers or generation of highly toxic environments

by massively producing ROS and RNS [14]. Peroxidaseactivity or Peroxidase gene expression in higher plants is,indeed, induced by fungi [15], bacteria [16], viruses [17],and viroids [18]. Cross-linking of the phenolic monomersin oxidative coupling of lignin subunits has been associatedwith peroxidase using H2O2 as oxidant. Acidic and basic per-oxidases are capable of oxidizing p-coumaryl and coniferylalcohol. One signicant event in plant defense reactionsis oxidative burst, a common early response of host plantcells to pathogen attack and elicitor treatment. Our resultsshowed increase in POX activity in dead fungus treated aswell as fungus treated plants as compared to control. isresult indicates that elicitor slowly increases level of ligninformation, suberization, and hypersensitive response. Similarresults were reported in wheat heads [19].

Other enzymes, PPOs are a group of copper containingenzymes that catalyze oxidation of hydroxy phenols to theirquinone derivatives, which have antimicrobial activity [20].Because of its reaction products and wound inducibility,PPO plays a role in defense against plant pathogens [21].Plant immediately respond to pathogen so there is immediaterise in PPO indicating immediate synthesis of antimicrobialsto ward o pathogen. In elicitor treated plant PPO activityincreases slowly day by day indicating that plant has gotstimuli to increase PPO. In case of live pathogen interaction,there was immediate response by plants increasing PPO 5-fold on the rst day, which starts decreasing from the 6thday indicating multiplication of fungus in the plant system.However, dead pathogen completely fails to mount thisresponse, which strongly suggests that plants are capable todierentiate signals from live and dead pathogen. Increasein PPO activity was reported in banana roots treated withFoc-derived elicitors by akker et al. [4]. Marked increasein PPO activity was observed in banana roots treated withsuedomonas uorescens against fusarial wilt [22].

-1,3 glucan and chitin, polymer of N-acetylglucosamine(NAG) are major cell wall components of many fungi. Since-1,3 glucanase and chitinases have been shown to be capableof attacking cell wall of fungal pathogens, these enzymes havebeen proposed as direct defense enzymes of plants [23]. Weobserved an increase in chitinase and -1,3 glucanase activityindicating plants ready mechanism to ward o pathogen bydirectly degrading the pathogen cell wall and in turn pro-tecting the plant. In our previous study, we found antifusaricactivity of elicitor induced -1,3 glucanase that showedswelling of mycelia aer one hour incubation of pathogenwith puried elicitor [24]. ndochitinase puried frombarleywas capable of inhibiting the growth of Trichoderma rescei,Alternaria alternate and Neurospora crassa [25]. In addition,Mauch et al. [26] reported that in combination, chitinase and-1,3 glucanase act synergistically to inhibit fungal growth.

Phenolic acids are involved in phytoalexin accumulation,biosynthesis of lignin, and formation of structural barrier,and play a main role in the resistance against pathogen.Marked accumulation of phenolics was observed on the3rd third day in fungus treated plants indicating the plantssensitivity to pathogen, and it attempt to protect itselfby the formation of structural barriers. Further, from the4th day activity decreases showing successful multiplication

ISRN Biotechnology 5

and establishment of pathogen by overcoming structuralbarriers formed by plants. In dead pathogen-treated plants,we found slow increase in phenolics activity indicating abilityto recognize dead pathogen as some foreign body, but it isnot multiplying; therefore, it will not break structural barrier.Marked accumulation of phenols leading to suppression ofFusarial wilt was observed in tomato plants [27]. Similarresults were observed in banana plants in vitro condition[4]. When tomato plants were treated with catechol, markedaccumulation of phenols was observed and it resulted insuppression of Fusarium wilt of tomato [27]. Anna andDubery [28] investigated that subfraction of cell-wall boundphenolics, ester-boundphenolics, glycoside boundphenolics,and free phenolics increased 6.3-, 4.2-, 3.0-, and 2.3-folds,respectively, upon induction.

In plants treatedwith dead pathogen, a forced inoculationof live pathogen was performed to assess induced plantsability to ward o pathogen. Interestingly no symptoms offusarial wilt were observed in these plants even thoughpathogen was in close proximity. is supports the view thatelicited plants are less susceptible to infection.

5. Conclusione present study strongly supports the view of preparationof plant vaccines for combating devastating disease likeFusarium wilt of banana. Dead pathogen preparation wasnot only successful in mounting defense response but alsoin protecting plants upon subsequent infections. erefore,it could be potential candidate for plant vaccine preparationto combat panama disease.

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