International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 59-74
TJPRC Pvt. Ltd

ANTIOXIDATIVE ENZYMES AND BIOCHEMICAL RESPONSES OF MANGO

(MANGIFERA INDICA L.) GENOTYPES TO MANGO LEAFHOPPER INFESTATION
ANUSHA KARKERA1, KIRAN KAMALAKAR MIRAJKAR2, RENUKA SUDARSHAN PATIL3,
VASTRAD ANDANAIAHA. S4 & PRABHU VIRUPAKSHA5
1,2,3
4

Department of Biochemistry, University of Agricultural Sciences, Dharwad, India

Department of Agricultural Entomolgy, University of Agricultural Sciences, Dharwad, India

5

Department of Plant Pathology, University of Agricultural Sciences, Dharwad, India

ABSTRACT
The biochemical responses and the enzymatic antioxidant system of five mango genotypes Totapuri, Mulgoa,
Neelam, Alphonso and Pairi under leaf hopper infestation at two stages of leaf maturity (new flush and old leaves) were
investigated. Changes in different biochemical parameters in infested leaves were observed as compared to healthy ones.
Assay of phenols and antioxidant enzymes namely superoxide dismutase (SOD), catalase (CAT), peroxidase (POX),
glutathione reductase (GR), polyphenol oxidase (PPO) and ascorbate oxidase (AO) revealed markedly higher
constitutive and induced antioxidant activities in Totapuri than all other genotypes except for phenols, PPO and AO

respectively) and the maximum biochemical response was of phenols (104% increase) to leafhopper infestation
indicating their major role in defense against leafhopper infestation. Pairi, Alphonso and Neelam recorded loss of
activity (up to 50 % decrease) as evident from very low inherent and induced antioxidative activities indicating the
susceptibility to leaf hopper infestation. Due to leaf hopper infestation total chlorophyll and reducing sugar content was
decreased in all the genotypes but to a varying degree. The biotic stress response of new flush was higher than old leaves

Original Article

which were induced more in Mulgoa. PPO and POX exhibited highest induction (86.0% and 72.2% increase

in all the genotypes. Our study suggested that Totapuri and Mulgoa with higher antioxidant capacity rendered the
resistance to leafhopper infestation
KEYWORDS : Mango Genotypes, Totapuri, Pairi, Mango Leafhopper, Phenols, Antioxidative Enzymes, Resistance

Received: Jul 24, 2016; Accepted: Aug 20, 2016; Published: Aug 24, 2016; Paper Id.: IJASROCT20168

INTRODUCTION
Mango (Mangifera indica L.) is one of the most important fruit crops of tropical and subtropical regions
of the world. The 2014/2015 harvest season in India produced 18.4 million tons on 2.5 million hectares
(Ahuja, 2015). However, the productivity remains to be low at around 7.8 tons per hectare. The major reason for
the low productivity is the threat of insect pests of which leaf hoppers cause devastating damage to leaf and
inflorescence, ultimately affecting fruit setting, and leading to severe yield loss (Chowdhury, 2015). The loss of
yield caused by mango leaf hoppers Amritodus atkinsoni, Idioscopus niveosparsus and Ideoscopus clypealis was
estimated to range from 25-60%.
Considerable evidence support the role of antioxidant enzymes in the host plant resistance, which is
presumably due to the scavenging of the reactive oxygen intermediates (ROS). In general antioxidative enzymes
such as superoxide dismutase (SOD), Peroxidase (POX), catalase (CAT), Glutathione reductase (GR); other

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enzymes like polyphenol oxidases (PPO), ascorbate oxidase (AO),lipoxygenases (LOX) are induced in plants in response
to herbivory (Felton et al., 1994; Zhao et al., 2009) and secondary metabolites including phenols, condensed tannins etc.
play an important role in induced resistance. (Usha Rani and Jyothsna 2010). Activitiy increases in CAT, SOD, POX, PPO,
other peroxidases and the accumulation of secondary metabolites have been observed in various plants like groundnut,
grass species and eucalyptus etc., particularly for resistant genotypes under insect feeding. (War et al., 2013; Punithavalli et
al., 2013; Khattab, 2007).
In the present study, the general objective was to evaluate the activity changes in the enzymatic antioxidant system
of mango genotypes to leaf hopper infestation and understand the mechanisms of resistance to leaf hopper in mango
genotypes.

MATERIALS AND METHODS

Plant Materials
Healthy and leafhopper infested leaves were collected from five mango genotypes namely Totapuri, Mulgoa,
Neelam, Alphonso and Pairi from Silver Jubilee Orchard , UAS, Dharwad, at two stages of leaf maturity i.e.New flush
( 25-35 days ) and old leaves (120-130 days). Leaf samples were kept in ice box during sampling and processed
immediately for enzyme extraction and assay. All the processing steps were carried out at 0C-4C.
Enzyme Extraction and Protein Determination
One gram of fresh leaf tissue was directly harvested into liquid nitrogen, ground into fine powder and
homogenized in a prechilled mortar with 4 ml of ice cold buffer of specific composition, molarity and pH for each
antioxidant enzyme. Catalase (CAT) and Superoxide dismutase (SOD) were extracted in 0.05 M sodium phosphate buffer
of pH 7.0 and pH 7.8 respectively. Ascorbate oxidase (AO), Polyphenol oxidase (PPO) and Guaiacol peroxidase (POX)
extracts were prepared in 0.1M potassium phosphate buffers of pH 5.6, 6.8 and 7.0 respectively. Grinding buffer for
Glutathione reductase (GR) contained 0.1 M Tris HCl pH 7.8 and 2mM DTT (dithiothreitol). 1mM
ethylenediaminetetraacetic acid (EDTA) and 1.5% w/v insoluble polyvinylpolypyrrolidone were used in all extraction
buffers. The homogenate was centrifuged at 14, 000 rpm for 20 min at 4 C and the supernatant was used immediately as
an enzyme source for the assay. An aliquot of supernatant was stored at -20C for protein analysis. The protein content in
the extract was determined by Bradford method using bovine serum albumin as standard (Bradford, 1976).
Superoxide Dismutase Assay
The activity of superoxide dismutase (SOD, EC 1.15.1.1) was assayed spectrophotometrically at 560 nm
( Beauchamp and Fridovich, 1971 ). One superoxide dismutase unit is defined as the amount of enzyme required to inhibit
50% of the NBT photoreduction per minute and expressed as IU per mg protein.
Catalase Assay
The catalase (CAT, EC 1.11.1.6) activity was determined spectrophotometrically (Beers and Sizer, 1952). One unit
of catalase is defined as the one mole of H2O2 decomposed per minute at pH 7.0 at 25C and was expressed as mole min
-1

per mg protein.

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Peroxidase Assay
The peroxidase (POX, EC 1.11.1.7) activity was determined spectrophotometrically (Chance and Maehly, 1955).
One peroxidase unit is defined as the amount of enzyme which catalyses the formation of one micromole of oxidized
guaiacol per minute at 25C and expressed as mole min -1 per mg protein.
Glutathione Reductase Assay
Glutathione reductase (GR, EC 1.8.1.7) activity was spectrophotometrically determined (Mavis and Stellwagen,
1968). One Glutathione reductase unit is defined as the amount of enzyme that oxidises 1.0 mole of NADPH per minute
at pH 7.6 at 25C and expressed as mole min -1 per mg protein.
Ascorbate Oxidase Assay
Ascorbate oxidase (AO, EC 1.10.3.3.) activity was assayed spectrophotometrically (Oberbacher and Vines, 1963).
One unit of Ascorbate oxidase is defined as the amount of enzyme catalysing the oxidation of 1 micromole of ascorbate per
minute at 25C and expressed as mole min -1per mg protein.
Polyphenol Oxidase Assay
Polyphenol oxidase (PPO, EC 1.10.3.1) activity was spectrophotometrically determined (Benjamin and
Montgomery, 1973). One polyphenol oxidase unit is defined as the amount of enzyme that increased the absorbance by
0.001 per minute under the conditions of the assay and expressed as mole min -1per mg protein.
Determination of Total Phenols
One gram of dried leaf tissue was extracted with10 ml of hot 80% alcohol (Sadasivam and Manikam, 1992).
The colorimetric method (Bray and Thorpe, 1954.) was used for the determination of total phenols using the Folin
Ciocalteau reagent. The phenol content was expressed as mg per gram dry weight.
Determination of Reducing Sugars
For quantitative estimation of reducing sugars one gram dry weight of leaf material was plunged in hot 80%
ethanol for 5 min and then crushed in pestle and mortar. The slurry thus obtained was filtered and the residue was
re-extracted two or three times and the filtrate were made up to 10 ml with 80% ethanol. Reducing sugars were measured
by Nelsons modification of Somogis method (Norton Nelson, 1944). The reducing sugar was expressed as mg per gram
dry weight.
Determination of Total Chrolophyll
Chlorophyll content present in leaf samples was determined by Dimethyl sulphoxide (DMSO) method (Hiscox and
Tsraelstam, 1979). The values obtained were expressed as mg/g fresh weight.
Statistical Analysis
The data of the experiment was analyzed statistically following the procedure described by Gomez and Gomez
(1984). The experimental design followed a three factorial complete randomized design with three replicates.
The results were expressed as mean and standard error of mean of three replicates of the enzyme assay for each sample.
The level of significance used in the F and t test was p = 0.01.

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RESULTS
Superoxide Dismutase Activity
SOD activity of mango genotypes differed significantly (p 0.01) in new flush healthy and infested leaves. Biotic
stress due to leaf hopper infestation induced SOD activity in Totapuri and Mulgoa genotypes however in Pairi, Alphonso
and Neelum loss of activity was observed. The basal and the induced SOD activity in the Totapuri genotype was
significantly higher (792.38 IUmg-1 protein and 916.7 IUmg-1 protein respectively) compared to all other genotypes
whereas Pairi genotype recorded the lowest basal SOD activity (204.47 IUmg-1 protein). SOD activity in infested new
flush leaves increased by 15.7% in Totapuri and by 12.9% in Mulgoa cultivars whereas decrease in SOD activity was
observed in Alphanso, Neelum and Pairi ( 27.25%, 26.75% and 4.13% respectively). A similar trend was observed in old
healthy and infested leaves but the degree of response in both constitutive and induced activity was significantly lower
compared to new flush. (Figure 1 A and 1.B)

Figure 1: Specific Activity of Superoxide Dismutase (SOD) (mol Min-1 Mg-1 Protein) in the
Leaves of Five Cultivars, Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri, under Leafhopper
Infestation (A And B). the Values are the Means of Three Replicates SEM. NFH
Newflush Healthy. NFI Newflush Infested. OH Old Healthy. OI Old Infested
Catalase Activity
CAT activity was altered significantly (p0.01) in all the cultivars by the stress imposed by leaf hopper infestation
(Figure 2.A & 2.B); however the CAT activity increased in Totapuri and Mulgoa whereas it decreased in Alphanso, Pairi
and Neelum. In newflush infested leaves, Totapuri and Mulgoa had significantly greater CAT activity (48.8 and 41.7mol
min-1mg-1protein respectively) than in the newflush healthy leaves. Increase was similar in Totapuri and Mulgoa
(34.5% and 31.7%) though a stronger response was observed for Mulgoa (29.82%) than Totapuri (19.57%) in old leaves.
Following infestation Alphanso, Pairi and Neelum showed drastic reduction in CAT activity as compared to newflush
healthy leaves (17.0, 14.3, 20.6 mol min-1mg-1protein).The biochemical response of old healthy and old infested leaves
of all the genotypes was significantly lower than newflush leaves .

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Figure 2: Specific Activity of Catalase (CAT) (mol Min-1 Mg-1 Protein) in the Leaves of
Five Cultivars, Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri, Under Leafhopper
Infestation (A And B). the Values are the Means of Three Replicates SEM. NFH
Newflush Healthy. NFI Newflush Infested. OH Old Healthy. OI Old Infested
Peroxidase Activity
Infested leaves of Totapuri, Mulgoa and Alphanso showed a significantly (p 0.01) strong induction of POX
activity in new flush (0.62, 0.34 and 0.24 mol min-1mg-1protein respectively) compared with the healthy leaves
(0.36, 0.22 and 0.21mol min-1mg-1 protein respectively) whereas decreased activity was observed in Pairi and Neelum.
Similar response was observed in old leaves but to a lesser extent (Figure 3.A & 3.B).The greatest increase (72.2% in new
flush) in POX activity between infested and healthy leaves was observed in Totapuri. In contrast Pairi recorded the
maximum decrease (50%) in POX activity in infested leaves in both newflush and old leaves.

Figure 3: Specific Activity of Peroxidase (POX) (mol min-1 mg-1 protein) in the Leaves of Five Cultivars, Neelum,
Pairi, Alphanso, Mulgoa ana Totapuri, under Leafhopper infestation (A and B). the Values are the
Means of Three Replicates SEM. NFH Newflush healthy. NFI Newflush Infested.
Oh Old Healthy. OI Old Infested
Glutathione Reductase Activity
GR activity in leaf hopper infested leaves of Totapuri and Mulgoa was significantly (p 0.01) higher
(7.3, 5.5 mol min-1mg-1 protein respectively) than in healthy leaves (4.9 and 4.3 mol min-1mg-1 protein) of new flush.
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The greatest induced activity was observed in Totapuri in both new flush and old leaves (increase of 48.9% and 38.71 %
respectively) as given in (Figure 4.A & 4.B). Pairi, Alphanso and Neelum showed marked decrease in GR activity
(52.5%, 40.6 % and 30.6 % decrease respectively) in old infested leaves and similar trend was observed in new flush.
Constitutive levels of GR activity were lower in Pairi, and Alphanso however Neelum recorded highest constitutive activity
(3.98 mol min-1mg-1 protein) in old healthy leaves among all the genotypes.

Figure 4: Specific Activity of Glutathione Reductase (GR) (mol Min-1 Mg-1 Protein) in the Leaves of
Five Cultivars, Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri, Under Leafhopper
Infestation (A and B). the Values are the Means of Three Replicates SEM. NFH
Newflush Healthy. NFI Newflush Infested. Oh Old Healthy. OI Old Infested
Ascorbate Oxidase Activity
The AO activities were significantly (p 0.01) greater in the infested leaves of Totapuri and Mulgoa genotypes in
both the new flush and the old leaves than those of the healthy leaves whereas in contrast Pairi, Alphanso and Neelum
exhibited drastic reduction in AO activity in the infested leaves (Figure 5.A & 5.B). Across the genotypes Totapuri showed
a higher constitutive AO activity in the infested new flush and old leaves (3.95 and 2.00 mol min-1mg-1 protein
respectively) however the strong induction of AO activity was observed in Mulgoa in both the new flush and old leaves
(59.36% and 68.49%). Infested leaves of Pairi, Alphanso and Neelum had decreased AO activity in the range of 30%-48%
in both new flush and old leaves.

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Figure 5: Specific Activity Ascorbate Oxidase (AO) (mol Min-1 Mg-1 Protein) in the Leaves of
Five Cultivars, Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri, under Leafhopper
Infestation (A and B). the Values Are the Means of Three Replicates SEM. NFH
Newflush Healthy. NFI Newflush Infested. OH Old Healthy. OI Old Infested
Polyphenol Oxidase Activity
Leaf hopper infestation had profound effect on PPO activity. The cultivars Totapuri and Mulgoa showed the
maximum increase in PPO activity in infested leaves when compared to healthy leaves in both new flush and old leaves.
In the present study high phenol content had strong influence on induction of PPO activity. Maximum induction
(170% increase in PPO activity) was observed in Totapuri in old infested leaves whereas Mulgoa recorded maximum
induction (86%) in new flush infested leaves. However, in Pairi, Alphanso and Neelum cultivars the opposite effect
(decrease) in PPO activity was observed. Alphanso expressed very low constitutive PPO activity than all other cultivars.
(Figure 6.A&6.B).

Figure 6: Specific Activity Polyphenol Oxidase (PPO) (mol Min-1 Mg-1 Protein) In the Leaves of
Five Cultivars, Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri, under Leafhopper
Infestation (A And B). the Values Are the Means of Three Replicates SEM. NFH
Newflush Healthy. NFI Newflush Infested. OH Old Healthy. OI Old Infested
Total Phenols

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We observed that phenol content in the leaves of all mango genotypes, differed significantly (p0.01) under
healthy and infested condition caused by mango leafhopper. Accumulation of phenols in high concentration was observed
in Totapuri and Mulgoa (increased by 104% and 70% respectively) whereas decrease in phenols was observed in Alphanso,
Pairi and Neelum (decreased by 9%-18%) under infestation (Figure 7.A&7.B) in the new flush and a similar trend was
observed in old leaves.

Figure 7: Total Phenol Content (Mg. G-1 DW) in the Leaves of Five Cultivars, Neelum, Pairi,
Alphanso, Mulgoa Ana Totapuri, under Leafhopper Infestation (A And B). the
Values are the Means of Three Replicates SEM. NFH Newflush Healthy.
NFI Newflush Infested. OH Old Healthy. OI Old Infested
Reducing Sugar Content
In the present study total reducing sugar content was decreased significantly (p0.01) in infested leaves as
compared to healthy leaves in all the cultivars. Leaf hopper infestation caused higher decrease of reducing sugars in old
leaves (51%-70%) than in new flush (16%- 43%). Alphanso, Pairi and Neelum cultivars exhibited more decline in reducing
sugars under infestation than the Totapuri and Mulgoa cultivars. (Figure 8.A. &8.B).

Figure 8: Reducing Sugar (RS) (Mg. G-1 DW) in the Leaves of Neelum, Pairi, Alphanso, Mulgoa Ana Totapuri,
Under Leafhopper Infestation (C And D). the Values Are the Means of Three Replicates SEM.
NFH Newflush Healthy. NFINewflush Infested. OH Old Healthy. OI Old Infested

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Total Chlorophyll
Leaf chlorophyll content was significantly affected by leaf hopper infestation in all the cultivars. Infested leaves
had lower chlorophyll content than the healthy leaves (decreased by 15%-49%). The reduction in chlorophyll content was
least in Totapuri (15% -25%) while all the other cultivars showed marked decrease in chlorophyll content(30%-49%) in
both the new flush and old leaves(Figure 9.A&9.B)

Figure 9: Chlorophyll (A+B) Content (Mg. G-1 DW) in the Leaves of FiveCultivars, Neelum, Pairi, Alphanso,
Mulgoa Ana Totapuri, Under LeafhopperInfestation (A And B). the Values Are the Means of Three
Replicates SEM. NFHNewflush Healthy. NFI Newflush Infested. OH Old Healthy. OI Old Infested

DISCUSSIONS
Five genotypes of mango were used to study the biochemical response in terms of phenols, reducing sugar,
chlorophyll and enzyme antioxidant system and to evaluate the responses of genotypes to leaf hopper infestation.
Superoxide Dismutase Activity
In this study, high constitutive and induced SOD activity exhibited by Totapuri and Mulgoa under leaf hopper
infestation may explain the better performance of these cultivars in the total stress response exhibited as supported by
findings of Hubert (2014). The SOD processing is known to be substrate inducible (Tsang et al., 1991), an increment in the
SOD activity may be attributed to the increased production of the superoxide (O2 ) as substrate that lead to induced
expression of genes encoding SOD. The decreased SOD activity in Alphonso, Pairi and Neelum appeared to be linked to
the low constitutive activity which is unable to protect functional biomolecules from oxidative damage and loss of activity
was also observed in studies on pest infested rice plants by Usha rani and Jyothsna (2010). Leaf hopper infestation might
have influenced the differential expression and activation of SOD isozymes leading to enhanced activity observed in
Totapuri and Mulgoa cultivars conferring greater resistance against leaf hopper. The biological stress induced SOD
response of cultivars in both new flush and old leaves followed the same trend.
Catalase Activity
In our study among the five mango cultivars Totapuri and Mulgoa exhibited a prominent increase in CAT activity
to oxidative stress induced by leaf hopper infestation. Similar elevated CAT activities were reported in cotton
(Bi et al., 1997) under infestation. The increased activity of CAT might be due to the enhanced superoxide dismutase
activity and the increase in CAT activity might be useful in dismutating hydrogen peroxide. The other mango cultivars i.e,
Pairi, Alphonso and Neelum recorded decline in CAT activity which has also been documented in other crops
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(Khattab, 2007). Such contrasting differences in CAT activity between the mango cultivars may be the result of genetic
differences in their metabolic pathways to scavange ROS. In plants, hydrogen peroxide (H2O2) plays a major signaling
role in triggering both a defense response and cell death. In the present study the Totapuri and Malgoa cultivars with higher
CAT activity in concert with other antioxidant enzymes may quench leaf hopper induced free radical damage and thus
impart resistance whereas the cultivars with insufficient CAT activity cannot overcome oxidative stress and may lead to
increased levels of H2O2 triggering cell death. The biotic stress response in new flush and old leaves of all cultivars
followed the same trend except that Totapuri exhibited maximum increase in new flush and Mulgoa in old leaves which
might be related to multiple molecular forms or isozymes of catalase encoded by multiple genes that exist in different
species (Scandalios, 1968).
Peroxidase Activity
Present studies showed increased peroxidase activity in leaf hopper stressed mango cultivars of Totapuri, Mulgoa
and Alphonso whereas decreased activity was observed in Pairi and Neelum in both new flush and old leaves. It might be
possible that the increased peroxidase activity is triggered by the cellular damage caused by leaf hopper infestation.
Quantitative alterations in phenols observed in Totapuri and Mulgoa may elevate activities of peroxidases in response to
insect attack leading to enhanced oxidation of phenolics to produce quinones and other oxidative radicals that can directly
deter feeding by insect herbivores and/or produce toxins that reduce plant digestibility. Induction in POX activity has been
implicated as an immediate response of plants in response to biotic stresses including insect attack (Moloi and van der
Westhuizen 2006). Observed patterns of different peroxidase activities in different mango cultivars in the present study
could be due to the differences in levels of resistance against leaf hopper as reported for black gram (Gaurav Kumar et al.,
2012). Alphonso with lower induced activities of other antioxidative enzymes showed marked elevation in peroxidase
indicating its major role in defense against leafhopper infestation. The current study suggests that the enhanced activity of
peroxidase might be associated with the leaf hopper resistance in mango cultivars.
Glutathione Reductase Activity
According to our data, GR activity increased substantially in the Totapuri and Mulgoa under infestation in both
new flush and old leaves which indicates that higher antioxidant capacity of the leaves rendered the tolerance to oxidative
stress. Both the above cultivars had similar constitutive activity; however the greatest induced activity was observed in
Totapuri in both new flush and old leaves resulting in the increased tolerance. According to the findings of Foyer et al
overexpression of GR led to increase in antioxidant capacity. The major involvement of GR in conferring stress tolerance is
the recycling of GSH and the maintenance of GSH/GSSG ratio in plant cell. Increases in GR as a result of biotic stress
have been previously reported for Barley and wheat (Keles and Oncel, 2002), indicating that the observed alterations in
activity may be related to cultivar tolerance. Pairi, Alphonso and Neelum cultivars under infestation exhibited a lower GR
activity than the healthy in both the new flush and old leaves. This decrease in GR activity leads to a decrease in ascorbate
and glutathione pools which may shift the redox balance towards the oxidative state and may be partly related to
leafhopper induced promotion of the disease in the susceptible cultivars.
Ascorbate Oxidase Activity
Ascorbate oxidase is another component of oxidative defense against insect herbivores (Felton and Summers,
1993). Ascorbic acid is essential for both nutritive and antioxidant functions in phytophagous insects and its bioavailability

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in herbivorous insects may be hindered via significant conversion of ascorbate to dehydro-L-ascorbic acid by ascorbate
oxidase. In the present study the constitutive activity of Ascorbate oxidase in Totapuri was markedly higher than all other
cultivars. However highest activity of AO as biochemical response to leaf hopper was seen in Mulgoa. Similar induction of
AO by herbivory or wounding was reported in some plants (Garcia-Pineda et al. 2004). Ascorbate oxidase may cause
indirect harm to the insect through deprivation of ascorbate and/or damage to dietary proteins, may facilitate lignin
biosynthesis and phenolic oxidation implicated in defense against pest invasion by neutralizing the inhibitory effects of
ascorbic acid on lignin forming enzymes and accelerate the hypersensitive reaction to pest attack in plants (Adam et al.,
1989). Pairi, Alphonso and Neelum showed a negative response with decreased activity under infestation in both new flush
and old leaves and it may be due to depletion in Ascorbate Glutathione pools owing to reduced GR activity. The different
responses of antioxidant enzymes may be one of the possible mechanisms of the differences in infestation sensitivities of
the mango cultivars.
Polyphenol Oxidase Activity
Plant polyphenoloxidases are widely distributed oxidative enzymes which act as an anti-nutritive defense against
leaf-eating insects (Felton et al., 1989). Our studies showed induction of polyphenol oxidase activity in infested leaves of
Totapuri and Mulgoa cultivars and it was maximum in comparison with induced levels of other antioxidant enzymes.
The induction of PPO has been shown for a taxonomically diverse group of plants (Constabel et al., 2000). Increased PPO
activity contributed to disease resistance due to its property to oxidize phenolic compounds to more reactive quinones
which directly reduce protein quality and redox cycling of quinones in insect gut produces oxidative stress which exerts
toxic effect on herbivores. Totapuri and Mulgoa with higher PPO activity substantiated the role of PPO in defense against
leaf eating insects. Alphonso, Pairi and Neelum showed decrease in activity when compared to healthy leaves indicating
greater susceptibility to leafhopper feeding which is similar to the findings of Mitra and Majumdar (1977). This suggests
that increase in PPO activity is not a general defensive mechanism in mango cultivars against leaf hopper infestation.
Bioprotection due to PPO may only occur in some resistant mango cultivars however other possible mechanisms like
activation of other related enzyme systems (lipoxygenase or arginase) or the influence of morphological traits of leaves
may also be part of the defense system of other cultivars to leaf hopper. The basal and induced activity of PPO in the
cultivars were higher in new flush when compared to old leaves suggesting higher phenolic content in new flush may
correlate with higher PPO activity. However Totapuri exhibited high induced activity in old leaves indicating upregulation
of PPO encoding genes and activation as an important defense related reponse in old leaves of Totapuri to leaf hopper.
Total Phenols
In the present study a significant increase in phenol content was observed in leafhopper infested leaves of Totapuri
and Mulgoa whereas a decrease was observed in Alphonso, Neelum and Pairi in comparison with the healthy leaves.
Synthesis of high amount of phenols by Totapuri and Mulgoa compared to healthy suggests their role in inducing
resistance against leafhopper in mango. It was observed that resistant plants contain more phenols or produce polyphenols
more rapidly than susceptible ones (Surekha et al., 2014). Alphonso, Neelum and Pairi with constitutively low phenolic
content was unable to resist insect feeding resulting in extensive damage to leaf tissue indicating the susceptibility to leaf
hopper. In addition, this study revealed higher constitutive and induced phenolic content in young leaves compared to old
leaves suggesting that the phenolic content of mango leaves was significantly affected by maturity as observed in other
plant species (Nhuan and Hwang, 2014).
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Reducing Sugars
Reducing sugar content in healthy and infested leaves at both stages of leaf maturiy differed significantly among 5
cultivars (p<0.01). Alphanso and Pairi recorded highest reducing sugar content while Totapuri the least in healthy leaves.
High carbohydrate content correlates with high infections in plants (Horsfall and Dimond, 1957). Accordingly the present
study suggests that Alphonso and Pairi with leaves rich in sugars, providing good nutritional conditions to the leafhopper
for their growth and development resulting in higher rates of multiplication are the susceptible varieties whereas Totapuri
with lowest reducing sugar is the resistant cultivar. A significant decrease in reducing sugars was observed in infested
leaves of all cultivars (P0.01) compared with the healthy leaves. These results are in accordance with those of Lokeshwari
et al (2014). Decrease of total reducing sugars in a highly infected host tissue is due to the excess utilization of sugars by
the organisms for their growth and sustainability . In the present study the reducing sugar content decreased significantly in
old leaves when compared to new flush in both healthy and infested condition as supported by Khan et al (2013) who
reported the higher quantity of reducing sugar in young leaves of musk melon than in the matured leaves. Decreased
amount of reducing sugar in infested old leaves may be correlated to impaired phloem transport and inhibition of
photosynthesis.
Total Chlorophyll
Leaf pigment composition is sensitive to plant stress, with a range of abiotic and biotic factors responsible for
either loss of photosynthetic pigments (e.g. chlorophylls) or the production of photoprotective pigments (such as
zeaxanthin or -carotene. In this work, leaf hopper infestation reduced total chlorophyll content in the leaves of all mango
cultivars in both new flush and old leaves with Totapuri cultivar showing the least decline in chlorophyll content when
compared to all other cultivars and is in accordance with the reports of Nabil (2013) in mango.T he decrease in the
photosynthetic pigments may be due to the inhibition of pigment biosynthesis which may result from the alteration in
mineral nutrition or lack of assimilates which drain towards the insect or to the effect of reactive oxygen species on these
pigments. Wang et al (2004) interpreted the decrease in chlorophyll due to unbalanced chlorophyll biosynthesis and
degradation and may be due to loss in chlorophyll synthetase activity in response to insect infestation (Das et al., 1994).
Heavy infestation caused by most sap sucking insects such as adult leaf hoppers, aphids or thrips which drain sap from the
phloem sieve elements of the plants vascular tissue leads to chronic shortages of photosynthates and thus severely reduce
the photosynthetic potential of the plant (Kim and Jones, 2000). Besides the direct damage, leafhoppers excrete honeydew,
which supports the growth of black sooty mold organism (Capnodium mangiferae), thus adversely affecting the
chlorophyll content and photosynthetic activity of the plant.

CONCLUSIONS
The present study revealed that the higher antioxidant enzyme activity and phenolic content play an important role
in induced defense mechanism of mango genotypes against mango leafhopper infestation as observed in Totapuri and
Mulgoa. Strong induction of Polyphenol oxidase and Peroxidase enzymes substantiated the antinutritive role of these
enzymes in insect defence mechanism. Alphonso, Pairi and Neelum exhibited lower induction of plant defensive
compounds which might be due to greater tissue damage in the leaves and thus indicating greater susceptibility to
leafhopper.

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Antioxidative Enzymes and Biochemical Responses of Mango

(Mangifera Indica L.)Genotypes to Mango Leafhopper Infestation

71

ACKNOWLEDGEMENTS
We thank Directorate of Research, UAS, Dharwad, Karnataka,India for financial support and Dr. Ashalatha Bhat.
(Dept. of Agril. Statistics) for statistical analysis help.
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