the rhizome rot disease caused by pythium aphanidermatum is the most destructive disease of turmeric plants in india , which reduces its economic and commercial value [ 1 , 2 ] . at present , effective fungicides are not available .
the possibility of stimulating internal plant defenses has become an interesting option for enhancing natural disease resistance .
higher plants have the ability to initiate various defense mechanisms when they are infected either by phytopathogens or after treatment with biotic and abiotic elicitors . among the elicitors known to date ,
chitosan , a polyvalent cation , has been shown to act as a potent oligosaccharide elicitor which can induce defense responses in plant tissue .
plant defense - related enzymes were known to participate in early defense mechanisms and to prevent pathogen infections [ 4 , 5 ] .
chitosan has attracted tremendous attention as a potentially important biological resource due to its biological properties including biocompatibility , nontoxicity , and biodegradability .
chitosan has been found to interfere with the growth of several plant pathogenic fungi [ 4 , 79 ] .
the potential of chitosan to protect fungal diseases of various horticultural plants has been studied in various investigations [ 4 , 911 ] .
the interest in the antimicrobial properties of chitosan was focused on its possible role in plant protection .
hence , the present study was carried out to induce resistance in susceptible turmeric plant ( erode local ) against rhizome rot disease by application of chitosan .
rhizomes of curcuma longa ( l. ) cultivar erode local ( susceptible ) were obtained from a farmer 's field at erode , tamilnadu , india .
the fungus p. aphanidermatum was obtained from itcc ( indian type culture collection ) , new delhi , india , and was maintained on potato dextrose agar . 1  g chitosan ( crab shell , sigma chem .
co. , usa ) was dissolved in 40  ml of distilled water containing 9  ml of 1  m acetic acid .
the ph was adjusted to 6.0 using sodium acetate . from this stock , 0.1% ( w / v )
rhizomes were thoroughly washed with running tap water thrice followed by glass distilled water ; surface was sterilized by immersion in a sodium hypochlorite 0.001% ( v / v ) solution for 15  min followed by several rinses of sterile distilled water . for the field experiment ,
rhizomes were sown in plots and each plot had 30 plants , respectively , at a farmer 's field , erode district of tamilnadu , india . for foliar spray ,
30-day - old plants were treated with 0.1% chitosan ( 10  ml / plant ) and the foliar spray was performed at a regular interval of 30 days up to 210 days .
turmeric leaves ( 1  g/2  ml ) were homogenized with potassium phosphate buffer ( 0.02  m , ph 7.6 ) and centrifuged .
one unit of chitinase was defined as the amount of enzyme that liberated 1 mol of n - acetyl glucosamine per minute under assay condition .
chitosanase activity was determined by the method of measuring the reducing sugars released from chitosan .
the reducing sugars were estimated by the nelson and somogyi [ 14 , 15 ] method using chitosamine hcl as standard .
one unit of chitosanase was defined as the amount of enzyme that liberated 1 mol of reducing sugar as chitosamine per minute under assay condition .
samples ( 50  g protein ) were separated on 10% sds - page . for chitinase localization ,
after electrophoresis , the gel was stained with calcofluor white m2r according to the procedure of trudel and asselin .
chitosanase localization was carried out according to the method of grenier and asselin   in which the separation gel contains 0.02% glycol chitosan .
control and chitosan treated plants ( 60-day - old ) were challenged with 5  ml spores of p. aphanidermatum ( 1  10 spores / ml ) .
all the data were subjected to one - way analysis of variance to determine the significance of individual differences in p < 0.01 and 0.05 levels .
an increase in protein content in leaves of chitosan ( 0.1% w / v ) treated plants was evident compared to control ( water treated ) plants ( figure 1 ) .
turmeric plants treated with chitosan showed higher chitinase activity than untreated plants ( figure 2 ) .
there was a significant increase in chitinase activity in leaves of chitosan treated plants after 2 months and maximum chitinase activity was observed on the 7th month . on sds - page , different new isoforms of chitinase were observed in treated plants ( figure 3 ) . in control plants , on the 1st month
there were four constitutive chitinase polypeptides observed with molecular mass 110 , 75 , 37 , and 19.3  kda .
in chitosan treated plants , in the 1st month the induced constitutive chitinase of molecular mass 110 , 75 , 37 , and 19.3  kda was observed and these isoforms remained up to the 7th month .
in addition to this , a new chitinase isoform of 35  kda was observed in the 4th month after treatment .
apart from this , another new isoform of molecular mass 32  kda was observed in 5th month , respectively .
a new constitutive chitinase isoform of 26  kda was observed in the 6th and 7th month , respectively ( figure 3 ) .
turmeric plants treated with chitosan showed an increase in chitosanase activity over a period of time and reached maximum on the 7th month .
nearly a threefold increase in chitosanase was observed in treated plants ( figure 4 ) .
chitosanase separated on sds - page showed new polypeptides in chitosan treated turmeric plants . in control plants , chitosanase polypeptide with molecular mass 19  kda
apart from this , 23  kda chitosanase polypeptide was observed in the 5th month of control plants . in chitosan
treated plants , apart from the constitutive chitosanase polypeptide ( 19  kda ) , a new isoform was observed after the 4th , 5th , 6th , and 7th month with molecular mass ranging from 2330  kda ( figure 5 ) .
chitosan treated plants challenged with p. aphanidermatum showed a significant decrease in disease severity when compared to control ( figure 6 ) .
inducing the plants own defense mechanisms by application of a biological inducer is thought to be a novel plant protection strategy .
chitosan can induce defense reactions in plants , including the induction of chitinase , chitosanase , and -1,3 glucanase isoforms .
chitosan and its derivatives offer a great potential as natural biodegradable substances which have antimicrobial and eliciting activities [ 5 , 20 ] .
chitinase and chitosanase have been identified as pr proteins which might be implicated in plant defense system against pathogenic fungi [ 21 , 22 ] . in the present study
chitosan stimulated chitinase production in cucumber plant and offered protection from root rot disease caused by pythium aphanidermatum .
celery , apium graveolens , treated with chitosan showed a 20-fold increase in chitinase activity compared to that of chitosan - untreated plants and exhibited a delay in symptom expression caused by f. oxysporum .
it has been reported that a constitutive high level expression of chitinases in transgenic plants can enhance resistance to a variety of pathogens .
our results could imply that the application of chitosan might sensitize the turmeric plant in protecting themselves from the phytopathogenic fungal invasion by elaboration of chitinase and chitosanase activity .
chitosan played an important role in the growth suppression of p. aphanidermatum infection in turmeric plants .
increase in chitinase and chitosanase activity may play a role in enhanced resistance in turmeric plants against p. aphanidermatum infection .
 chitosan was evaluated for its potential to induce antifungal hydrolases in susceptible turmeric plant ( curcuma longa l. ) . under field conditions , 
 the application of chitosan ( crab shell ) to turmeric plants by foliar spray method induces defense enzymes such as chitinases and chitosanases . 
 such an increase in enzyme activity was enhanced by spraying chitosan ( 0.1%  w / v ) on leaves of turmeric plants at regular intervals . 
 gel electrophoresis revealed new chitinase and chitosanase isoforms in leaves of turmeric plants treated with chitosan . 
 treated turmeric plants showed increased resistance towards rhizome rot disease caused by pythium aphanidermatum , whereas control plants expressed severe rhizome rot disease . increased activity of defense enzymes in leaves of chitosan treated turmeric plants may play a role in restricting the development of disease symptoms . 
 the eliciting properties of chitosan make chitosan a potential antifungal agent for the control of rhizome rot disease of turmeric . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion