Penicillium oxalicum directed systemic resistance in tomato against Alternaria alternata

ORIGINAL PAPER

Penicillium oxalicum directed systemic resistance in tomatoagainst Alternaria alternata

Aqeel Ahmad • Sobiya Shafique • Shazia Shafique •

Waheed Akram

Received: 3 December 2013 / Revised: 2 February 2014 / Accepted: 4 February 2014 / Published online: 19 February 2014

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract Enhanced yield of tomato has ever been an

important issue due to its nutritional value and various

dietary consumption forms. But, efficient yield increase can

never be achieved without using environmentally safe

means e.g. innate resistance. In present study, tomato innate

antifungal resistance has been boosted up using Penicillium

oxalicum, and then various aspects of resistance modulation

have been explored in details. Two tomato varieties of

differential antifungal resistance (Dinaar and Red Tara)

were treated with six P. oxalicum strains which screened the

best inducer strain (Pn 5); which remarkably controlled

disease incidence (DI) of Alternaria alternata. Inducer was

not only responsible for almost two times production of

phenolics, alkaloids and terpenoids in Red Tara, but it also

non-significantly triggered same biochemicals in Dinaar.

Hemicellulose showed only 40 % increase in variety of

least antifungal resistance. During quantification assays of

peroxidase (POD), phenyl ammonia lyase and polyphenol

oxidase, more or less the same doubling trend was recorded

in susceptible variety, while only POD had significant

enhancement in resistant variety under the influence of

fungal inducer. It was also recorded that inducer not only

modulated quantity of enzyme (glucanase), but its isozyme

package was also altered. Colorimetric quantifications of

lignin, cellulose and pectins proved that biotic inducer

strengthened the physical structure of plant cells by

increasing these contents from 30 to 120 %. The above

investigation collectively comes with the recommendation

of an efficient and environmentally safe inducer (P. oxali-

cum); which, can be used to control fungal pathogens.

Keywords Biological inducer � Enhancement of

defenses � Isozyme modulation � Colorimetric assay �Physical barriers

Introduction

Tomatoes occupy a significant position among dietary

elements of humans globally. Its consumption is both direct

and indirect through salad, curries, jams and ketchups etc.

due to its unique nutritional combination; it cannot be

replaced with any other food stuff because it provides

minerals and vitamins to human body (Hobson et al. 1979;

Baloch 1994). According to nutritional analysis there is

0.9 % protein, 94.1 % water, 0.1 % fat and 3.5 % carbo-

hydrates in tomato fruit.

Due to these features it is necessarily important to fulfill

tomato consumption requirement of world population. But,

unfortunately, this important agriculture crop has to face a

number of yield reducing factors including fungal diseases.

Fungi not only infect tomato crop in field but also in store

houses, and Alternaria alternata is an emblematic species

among fungal pathogens. A. alternata has a vast host

spectrum and growth conditions range as well (EL-Morsy

1999, 2000; Guo et al. 2004). Due to high yield losses of

Communicated by M. J. Reigosa.

A. Ahmad (&) � Sobiya Shafique � Shazia Shafique �W. Akram

Institute of Agricultural Sciences, University of the Punjab,

Lahore, Pakistan

e-mail: [email protected]

Sobiya Shafique

e-mail: [email protected]

Shazia Shafique

e-mail: [email protected]

W. Akram

e-mail: [email protected]

123

Acta Physiol Plant (2014) 36:1231–1240

DOI 10.1007/s11738-014-1500-5

tomato each year caused by A. alternata (Akhtar et al.

1994), it is imperative to control its infection on tomatoes.

Control of fungal pathogens using plant internal resis-

tance has been considered as an effective and inexpensive

measure and always preferred over other disease-control-

ling strategies. Moreover, plant systemic resistance may

also be stimulated by exposing the plant surface to bio-

logical agents (Lawrence and Novak 2004; El-Khallal

2007; Kurabachew and Wydra 2010). By keeping in view

the biocontrol agents, role of Penicillium oxalicum can

never be overemphasized as it has been successfully used

for resistance induction in previous researches (De-Cal

et al. 1995). Sempere and Santamarina (2010) had also

described the antagonistic behavior of P. oxalicum against

A. alternata. Thus P. oxalicum was selected to evaluate its

resistance induction potential in tomatoes.

Methodology

Procurement of tomato germplasm and fungal isolates

Two representative tomato varieties (Dinaar and Red Tara)

were procured from ‘‘Fungal Biotechnology Lab, Institute

of Agricultural Sciences, University of the Punjab, Lahore,

Pakistan’’, which were the most resistant and the most

susceptible varieties against A. alternata.

The most virulent isolate of A. alternata and six strains

of biocontrol agent, P. oxalicum, were also procured from

the same biotechnology laboratory to evaluate their indi-

vidual resistance induction potential in tomato varieties

against fungal pathogen.

Experimental setup

Tomato seeds were grown in pots under greenhouse con-

ditions to minimize the dynamics of environmental factors.

At the age of 1 month, pots were divided into 13 sets of

each variety with three pots in one set. Then six sets of

each variety were treated with foliar pathogen inoculum

(3,000 spores/ml) and a single strain of biocontrol agent at

one time. Application of biocontrol agent was carried out

by adding 100 ml of P. oxalicum spore suspension (3,000

spores/ml) in pot soil, 10 days before the application of

pathogen. Six sets of pots were left as positive control

(biocontrol agent only), for which individual set was

treated with a single biocontrol strain only. Moreover, a

single pathogen control was arranged for comparison with

other treatments. After 15 days of incubation data were

collected with reference to disease incidence (Fig. 1;

Table 1) and statistically analyzed to evaluate the most

efficient biocontrol agent. Further downstream analyses

were made of all treatments of the most efficient biocontrol

strain (EBS) in comparison with pathogen control.

Comparison of physiological defenses

Representative samples of tomato plants from each set

were evaluated for their phenolics, alkaloids, terpenoids

and hemicellulose contents.

Method of Mujica et al. (2009) was adopted for the

estimation of phenolics, while, evaluation of alkaloid

contents in plant tissue was carried out by the method of

Arvind et al. (2007). So, plant material was extracted with

0.1 M aqueous HCl solution and then those acidic wash-

ings were freed from chlorophyll, tissue residues and

Fig. 1 Scale for visual

representation of disease

incidence on leaf tissue

Table 1 Scale for percentage DI with denoted resistance status

Percentage disease incidence (DI) Status

01–20 Highly resistant

21–40 Resistant

41–60 Moderately resistant

61–80 Susceptible

81–100 Highly susceptible

1232 Acta Physiol Plant (2014) 36:1231–1240

123

lipophilic substances. Then its pH was raised to precipitate

alkaloids from mixture.

Ethyl acetate was used for 15 min at 40 �C to extract

terpenoids from plant material. Then extracts were filtered

to remove tissue debris and treated with 300 ml, 5 % each

of KOH and HCl solution to remove acidic and basic

substances, respectively. Remaining fraction was concen-

trated and vacuum evaporated to isolate solid terpenoids.

Comparison of physical defenses

Strength of plant cell wall and its toughness is increased

with the increased quantity of cellulose, lignin and pectin.

The visual estimation of all these cell-wall components was

made by examining the stained sections of plant tissue,

microscopically.

For cytostaining of cellulose, a stain was prepared by

dissolving 0.2 g iodine in 2 % aqueous KI solution and

applied on 0.05-mm-thick plant tissue sections for 15 min

before the application of H2SO4. Lignin was stained with a

specific stain of phloroglucinol (0.1 %), prepared in 20 %

aqueous HCl solution. Pre-weighed plant material was

boiled in 0.5 %, H2SO4 and percentage weight loss on

drying was denoted as hemicellulosic contents. Cytostain-

ing of pectin in tomato plant tissues was carried out

through toluidine blue O (0.05 %) method. Furthermore,

all above-described stains were separately applied to plant

tissue sections for microscopic observations.

Micrographs of stained plant sections were captured by

keeping the settings EV = 0 and ISO = 100, and reference

graphs for individual physical barrier on the basis of

spectral variations were plotted (Fig. 2). Moreover, cap-

tured micrographs were analyzed by ‘‘COLORS’’ to

determine inter and intra-spectral variations among them

after vertically slicing each image into nine. The quantities

of physical barriers were determined by comparing their

spectra with reference graphs (Ahmad et al. 2013).

Comparison of defense-related enzyme activity

Expression of pathogenesis-related protein genes is a key

factor for defining the plant resistance towards diseases

(Van Loon 1997). So, the activity of defense-related

enzymes was studied in tomato plants to get a clear view of

induced resistance in tomato varieties. For this purpose pre-

weighed plant material was crushed in cold sodium phos-

phate buffer (7.2 pH) and EDTA. Then mixture was freed

from tissue, and activities of phenyl ammonia lyase (PAL),

Polyphenoloxidase (PPO) and peroxidase (POD) were

determined through methods of Burrell and Rees (1974),

Mayer et al. (1965) and Putter (1970), respectively.

To study isozymes of glucanase enzyme, blue native

PAGE of plant proteins was performed and obtained gel

was incubated at 35 �C for 2 h with glucanase antiserum.

Results

Disease incidence on tomato varieties varied greatly with

change in inducer strains. Among six P. oxalicum strains,

‘‘Pn 5’’ provided maximum control of fungal disease with

maximum DI of 26.43 % at the time of combine applica-

tion of inducer and pathogen on susceptible variety

(Fig. 3). ‘‘Pn 3’’ gave 100 % control in case of both vari-

eties when applied in the absence of lab-prepared inocu-

lum. If treated plants are combined with inducer (Pn 3) and

pathogen inoculum, then remarkable DI of 74.38 and

80.78 % was recorded in Dinaar and Red Tara,

respectively.

Disease-control behavior of three inducer strains ‘‘Pn

1’’, ‘‘Pn 4’’ and ‘‘Pn 6’’ was more or less identical as they

were unable to protect plants from infection in any treat-

ment. However, application of ‘‘Pn 2’’ was successful only

in one treatment of Dinaar, where, zero DI was recorded

after application of inducer’s inoculum (Fig. 3). The

Fig. 2 Reference spectral graphs for colorimetric quantification of biochemicals (Ahmad et al. 2013)

Acta Physiol Plant (2014) 36:1231–1240 1233

123

overall disease-controlling potential of ‘‘Pn 5’’ was found

to be the best among all strains used in this study. So, this

strain was selected for further downstream analysis.

Evaluation of biochemical defenses

Generally, higher quantities of all biochemicals were

quantified in ‘‘Pathogen ? Inducer’’ treatment of Dinaar

with non-significant differences from ‘‘Inducer’’ treatment

of same variety (Table 2). Hemicellulose was the only

biochemical which exhibited no quantity variation with

respect to treatments in Dinaar. Phenolics and alkaloids

were also recorded in non-significant lesser quantities in

negative control of Dinaar than the other two treatments.

Moreover, negative control of Dinaar had only terpenoids

in significantly lesser amounts in plant tissues than its

‘‘Pathogen ? Inducer’’ treatment (Table 2).

Quantities of all defensive phytochemicals (i.e. pheno-

lics, alkaloids, terpenoids and hemicellulose) dynamically

changed between all treatments of Red Tara. So, quantities

of phenolics, alkaloids and terpenoids were recorded at

about half in negative control of Red Tara than its other

two treatments; while, increase in hemicellulosic contents

of ‘‘Inducer’’ treatment of Red Tara was only 32 % greater

than its negative control. ‘‘Inducer’’ and ‘‘Patho-

gen ? Inducer’’ treatment of Red Tara had non-significant

differences in all biochemicals quantified with the general

trend of decreased quantity in later treatment. But, in case

of hemicellulose, non-significantly higher quantity was

calculated in ‘‘Pathogen ? Inducer’’ than ‘‘Inducer’’.

Analysis of defense-related enzymes

Results clearly revealed that ‘‘Pn 5’’ was involved in the

enhanced production of all the three defense-related

enzyme studied. Application of ‘‘Pn 5’’ significantly

increased the quantity of POD than its negative control in

Red Tara; whereas, its difference from ‘‘Patho-

gen ? Inducer’’ was non-significant. Significantly

increasing trend of PPO quantities was also found in Red

Fig. 3 Disease incidence of Alternaria tomato leaf spot on two

different tomato varieties (Dinaar and Red Tara) under different

treatment combinations. Two blank bars (without any filling pattern)

on the right side of the figure show normal disease index on tomato

varieties under favorable controlled conditions of pathogen

Table 2 Quantification of phytochemicals (g/kg) from tomato varieties

Biochemicals Dinaar Red Tara

Negative control Inducer Pathogen ? Inducer Negative control Inducer Pathogen ? Inducer

Phenolics 0.47 ± 0.04a 0.51 ± 0.06a 0.53 ± 0.06a 0.22 ± 0.04b 0.44 ± 0.03a 0.43 ± 0.03a

Alkaloids 0.49 ± 0.02a 0.49 ± 0.04a 0.50 ± 0.08a 0.26 ± 0.04b 0.49 ± 0.03a 0.44 ± 0.05a

Terpenoids 0.21 ± 0.02b 0.25 ± 0.04ab 0.27 ± 0.01a 0.10 ± 0.01b 0.20 ± 0.06a 0.19 ± 0.01a

Hemicellulose 0.34 ± 0.02a 0.34 ± 0.03a 0.34 ± 0.02a 0.25 ± 0.02b 0.33 ± 0.03a 0.35 ± 0.07a

Data were statistically analyzed through Duncan’s Multiple Range Test (DMRT) at p B 0.05 and significance of values has been mentioned by using alphabets (a, b)

1234 Acta Physiol Plant (2014) 36:1231–1240

123

Tara from ‘‘negative control’’ to ‘‘Pathogen ? Inducer’’

(Table 3). Moreover, quantities of PAL and POD were

more or less same in negative control of Red Tara. But

when subjected to ‘‘Inducer’’, quantity of PAL was

increased up to 29.34 g/kg in comparison with 23.8 g/kg of

POD. It revealed that the production of PAL is more ‘‘Pn

5’’ dependent than POD. It was also noted that the quantity

of PAL was significantly decreased when Red Tara was

subjected to ‘‘Pathogen ? Inducer’’ (Table 3).

Quantities of defense-related enzymes in Dinaar gener-

ally had non-significant differences among different treat-

ments. In this variety POD was the only enzyme which

varied significantly under the influence of different treat-

ments (Table 3). Two enzymes PAL and PPO increased

after the application of ‘‘Inducer’’ and ‘‘Patho-

gen ? Inducer’’ in a non-significant way (Table 3).

Three isozymes of glucanase were recorded in Dinaar in

comparison with two isozymes of Red Tara. Quantity of Glu 02

increased when Dinaar was treated with ‘‘Inducer’’, and its

production again reduced when ‘‘Pn 5’’ treatment was

accompanied with pathogenic inoculum (Fig. 4). Joint effect

of A. alternata and P. oxalicum under ‘‘Pathogen ? Inducer’’

enhanced the quantity of Glu 03, which was not affected by

‘‘Inducer’’ (Fig. 4). Glu 01 was neither modulated by

‘‘Inducer’’ nor by ‘‘Pathogen ? Inducer’’; hence, expressed in

similar amounts in all treatments of Dinaar (Fig. 3). Under

normal conditions Glu 02 was absent in Red Tara plants and it

was induced under the influence of ‘‘Inducer’’. Whereas,

behavior of Glu 01 production in Red Tara is completely

opposite with Glu 02, as Glu 01 was only detected in negative

control but not in other two treatments. Glu 03 was found to be

continuously produced in Red Tara with the identical behavior

as in Dinaar.

Evaluation of physical defenses

Careful microscopic observations were visible manifesta-

tions which also argued upon the strength of physical

barriers of plants after treating them with ‘‘Inducer’’ and

‘‘Pathogen ? Inducer’’. As specific stains for physical

barriers were used in this study, intensity of color obtained

was a symbol of defensive strength of plant against

pathogens (Fig. 5). Plant tissues with no treatment (nega-

tive control) exhibited lesser antifungal strength, showed

lighter color in figures as compared to their respective

treated plants which were darker in color (Fig. 5).

Spectral analysis of ‘‘Fig. 5’’ to quantify biochemicals in

plant cell wall revealed that cell walls of Dinaar were

Table 3 Quantification of defense-related enzymes (g/kg) in tomato varieties

Dinaar Red Tara

Negative control Inducer Pathogen ? Inducer Negative control Inducer Pathogen ? Inducer

PAL 29.54 ± 1.2a 30.15 ± 1.57a 30.88 ± 1.11a 13.24 ± 0.42c 29.34 ± 0.9a 27.32 ± 0.12b

PPO 6.8 ± 0.41a 6.8 ± 0.3a 6.92 ± 0.07a 3.9 ± 0.14c 6.4 ± 0.16b 6.79 ± 0.06a

POD 23.79 ± 1.7b 25.67 ± 0.98ab 26.13 ± 0.11a 13.46 ± 1.82b 23.8 ± 0.7a 23.47 ± 0.34a

Data were statistically analyzed through DMRT at p B 0.05 and significance was mentioned by using alphabets (a, b, c)

Fig. 4 Glucanases in different

treatments of representative

tomato varieties

Acta Physiol Plant (2014) 36:1231–1240 1235

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1236 Acta Physiol Plant (2014) 36:1231–1240

123

maximum lignified (0.2925 g/kg) when exposed to ‘‘Path-

ogen ? Inducer’’ (Fig. 6a). Similarly, second most ligni-

fied walls were also of Dinaar variety (0.276 g/kg) when it

was treated with ‘‘Inducer’’ (Fig. 6a). Whereas, least lig-

nification of walls was recorded in Red Tara negative

control (0.1465 g/kg).

More or less similar trend of biochemical quantities was

prevailed in terms of cellulose and pectins (Fig. 6b, c). The

highest quantities of both of these defense elements

(0.41 g/kg = cellulose and 14.5 g/kg = pectins) were

recorded in Dinaar treated with ‘‘Pathogen ? Inducer’’.

However, slight trend variations were present at some

places but the least quantities of these biochemicals were

again found in Red Tara negative control (0.155 g/kg =

cellulose and 4.9 g/kg = pectins).

Discussion

Exposing plants to external stimuli induces resistance in

plants, which remarkably reduces disease incidence. Some

fungal species may also be good external stimuli in this

direction, and a lot of studies have also been reported in

this field (Harman et al. 2004; Ghosh et al. 2006; Djonovic

et al. 2007; Kempel et al. 2010). P. oxalicum induced

resistance in tomato against fungal pathogen which is a

redolent of the study of Ghosh et al. (2006) who also used

fungus to induce resistance in ginger for disease control.

This study is based upon the induction of horizontal

resistance in tomato plants and gives a control of a number

of pests on tomato crop.

Resistance induction potential of fungal bio-agents is

also modulated by the agent, which is applied jointly with

the inducer’s inoculum. This may increase or decrease the

disease severity in plants by affecting the inducer’s capa-

bilities (Abdel-Kader et al. 2012). This might be the reason

that DI of A. alternata varied between two inducer treat-

ments (Inducer and Pathogen ? Inducer). It can also be

simplified that inducer produced synergistic effects in the

presence of pathogenic inoculum. Moreover, efficiency of

resistance inducer is greatly dependent upon host genome

(Reglinski et al. 2007; Walters and Fountaine 2009).

Therefore, same level of resistance could not be achieved

using identical stimuli on two different tomato varieties. In

current investigation, Dinaar exhibited no DI in case of

three P. oxalicum strains. But same strains were unable to

give 100 % control of disease in Red Tara.

b Fig. 5 Deposition of different biochemicals, which play role in the

strengthening of physical barriers of plant cell wall against pathogen

invasions

Fig. 6 Colorimetric quantification of lignin (a), cellulose (b) and pectins (c) in two tomato varieties

Acta Physiol Plant (2014) 36:1231–1240 1237

123

Shoresh et al. (2010) explained that fungal inducers are

responsible for better growth and efficient nutrient uptake

of plants. They also govern photosynthesis and respiration

processes making them more synchronized. They not only

control pathogens but also enhance plant tolerance against

abiotic stresses by reprogramming plant gene expression

profile (Harman et al. 2004). By keeping in mind all these

studies, P. oxalicum treatment can be suggested to mini-

mize abiotic stresses on crop plants. These non-pathogenic

fungi can also be applied as a preventive measure prior to

pathogen infestations.

Basic mechanism behind induced resistance is the

enhanced production of defensive biochemicals to hinder

pathogen invasion. Phenolics are the secondary metabolites

of plants and are categorized into plant defense weapons

(Witzell and Martin 2008; Banerjee et al. 2012). Witzell

and Martin (2008) concluded in a study that oxidative

metabolism was responsible for high level of antifungal

resistance in plants. Meanwhile, Johansson et al. (2004)

reported increased quantities of phenolics behind enhanced

resistance of pine which was induced by white rot fungi.

Presently, a more quantitative enhancement of polyphenols

has been observed by invasion of a non-pathogenic fungus.

This phenomenon indicates significant resemblance

between infection processes of pathogenic and non-patho-

genic fungal species.

Alkaloids act as strong defense weapons of plants which

might be triggered by external stimuli (Mohammadi and

Kazemi 2002; Cherif et al. 2007; Vera et al. 2011a; Ashry

and Mohamed 2011). They have antimicrobial properties

and their increased biosynthesis ensures plant resistance

against pathogen. That was the reason behind resistance

modulation in tomato varieties through fungal inducer.

Terpenoids are plant defense icons and investigations

have confirmed their significance in plant survival tactics

(Martin et al. 2003; Miller et al. 2005). Increased biosyn-

thesis of terpenoids attracts predators of plant insect pests

and limits a variety of plant pathogens including viruses

(Mumm et al. 2008). Hence, triggering of terpenoids in

tomato plants treated with fungal inducer is a sign of

increased tomato resistance against vast categories of

pathogenic entities. Similar, defense phenomenon was

proved in terms of hemicellulose, which provided another

proof about the reliability of fungal-mediated resistance in

tomato because hemicellulose is also a defense icon in

plant cytological structures (Freeman and Beattie 2008).

Lignin is classified under plant physical defense barriers

and its biosynthesis normally involved the role of peroxi-

dase. Its presence in physical barriers provides rigidity to

cell wall and resistance to plant against pathogens as well

as abiotic stresses (Cosgrove 1997; Quiroga et al. 2000).

Therefore, increase in lignin recorded in this study pre-

cisely defines the efficiency of P. oxalicum as an inducer.

Freeman and Beattie (2008) classified cellulose in

structural defenses of plants. Meanwhile, there are some

investigators who explain role of cellulose as binding

domains of many proteins which directly or indirectly

inhibit pathogen attacks (Linder et al. 1995; Villalba-

Mateos et al. 1997). This finding of increased cellulose

contents in tomato plants is consistent with previous

studies as plants with high resistance also showed cellulose

augmentations in tissues. This might be happening to create

more space for integration of defense-related proteins

along walls of physical structures.

Pectin is a plant component which plays an important

role in the signaling pathways (Vorwerk et al. 2004).

Generally, it is assumed that plants with induced resistance

possess decreased amounts of pectins in their tissue

because of the activation of pectin degrading enzymes

(Ferrari et al. 2008). But pectin quantification in this study

did not agree with previous studies because pectin contents

were also recorded to be enhanced in plants with induced

resistance. This unusual increase in pectins may have

created complications in finding smooth trends of DI with

respect to given treatments. But in any case, it can easily be

concluded that there must be some metabolic changes and

signaling pathway alterations, triggered under the presence

of P. oxalicum, which finally resulted into altered quanti-

tation of biochemicals.

It is a frequently confirmed phenomenon that plants with

augmented resistance produce pathogenesis-related proteins

and enzymes when they are required; and this biosynthesis

raises protein level higher than usual (Castro and Fontes

2005). A research in same direction conducted by Veronese

et al. (2003) proved that higher glucanases confer microbial

resistance in plants which is in accordance with the findings

of current research. Resistant variety not only showed

increased number of glucanase isozymes, but the quantity of

each isozyme was also increased. Similarly, the quantity was

more enhanced after treating plants with biotic inducer.

Johansson et al. (2004) put responsibility of stopping

pathogen attacks in plants on peroxidase activity. It also

converts pectins into a form highly resistant against pec-

tinases; hence, making plant defenses more stable (Witt-

stock and Gershenzon 2002). Therefore, plant tissues with

enhanced resistance against pathogens should possess

enhanced quantities of peroxidases and this conclusion

falls in favor of present study.

Vera et al. (2011a) reported that during induction of

resistance, amounts of numerous antifungal proteins and

defense-related enzymes are significantly increased in

which phenyl ammonia lyase is a dominant enzyme.

Increased PAL is interlinked with accumulation of phe-

nylpropanoid compounds (Vera et al. 2011a), which may

explain increased quantities of phenolics and enhanced

resistance (Vera et al. 2011b).

1238 Acta Physiol Plant (2014) 36:1231–1240

123

One important phenomenon about PPO discovered in

this study is that its biosynthesis was not increased after the

inoculation of inducer. But, increase in its quantity was

recorded after inoculation of ‘‘Pathogen ? Inducer’’.

Those results were in agreement of Mohammadi and

Kazemi (2002). In a study conducted by Ashry and Mo-

hamed (2011), it was reported that a plant which is cate-

gorized as resistant against pathogen also exhibits higher

amounts of PPO in its tissues. These findings also support

current investigation.

Author contribution Aqeel Ahmad conceived the idea,

Aqeel Ahmad and Shazia Shafique realized experiments.

Aqeel Ahmad and Waheed Akram performed the research

work and recorded data. Sobiya Shafique technically

assisted the experimental work and data analysis. Aqeel

Ahmad and Shazia Shafique compiled and composed the

whole study.

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