Chitosan induces resistance components in Arachis hypogaea against leaf rust caused by Puccinia arachidis Speg.

ELSEVIER

Chitosan induces resistance components in Arachis hypogaea against leaf rust caused by Puccinia arachidis Speg. M. Sathiyabama and FL Balasubramanian* Centre for Advanced Studies in Botany, 600 025, lndia

University

of Madras,

Guindy

Campus,

Madras

Chitosan (1000 ppm) reduced germination of uredospores of Puccinia aruchidis, the incitant of groundnut leaf rust disease. Chitosan treatment of groundnut leaves before inoculation reduced the number of lesions, lesion diameter and sporulation of t? aruchidis. Chitosan-treated leaves showed an

increase in endogenous salicylic acid, intercellular chitinase and glucanase activity. These compounds had been previously associated with induced resistance responses. Enzyme activity staining showed new isoforms of chitinase and glucanase in treated groundnut leaves. Western blot analysis of treated groundnut leaves also showed new isoforms of chitinase and glucanase. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Arachis hypogaea; chitosan; Puccinia arachidis

Research on the phenomenon of induced disease resistance in plants against phytopathogenic fungi has gained momentum in recent years because of its beneficial attributes such as environmental safety, long lasting effects and efficacy (Kuc’, 1993). There have been studies on the induction of defense reactions in plants against pathogens by prior treatment with various biotic and abiotic elicitors (Kuc’, 1987; Yoshikawa et al., 1993; Zhang and Punja, 1994). Among the biotic elicitors, chitosan is unique in that it not only activates genes of defense responses in plants but it acts as an inhibitor of fungal growth (Hadwiger et al., 1986). The dual effect of chitosan has been used in integrated disease management. Chitosan applied to pea tissue in advance of Fusarium solani f. sp. pisi inoculum induced resistance to this pea pathogen (Kendra et al., 1989) by accumulating defense response proteins such as chitinases and p-1,3 glucanases. Those compounds also increased when pea pods were treated with chitosan (Nichols et al., 1980; Mauch et al., 1984). Chitosan treatment induced synthesis of proteinase inhibitors in tomato leaves (Walker-Simmons et al., 1983), and phytoalexin in pea pods (Kendra and Hadwiger, 1984). Treating peanut seeds with chitosan induces cinnamyl alcohol dehydrogenase, glutamate dehydrogenase, polyphenol oxidase and peroxidase (Fajardo et al., 1994b). Tomato plants enhanced protection against

*To whom correspondence

treated crown

with chitosan and root rot

should be addressed.

caused by E oqsporum f. sp. radicis-lycopersici (Benhamou and Theriault, 1992). Krebs and Grumet (1993) reported that celery (Apium graveolens) plants susceptible to infection with Fusarium oxysporum f. sp. apii treated with chitosan resulted in an increase in chitinases to 20 fold with a new acidic chitinase and an increase in constitutive p-1,3 glucanase activity. Treated plants showed a delay in disease symptom expression. Treatment of cucumber plants with chitosan protected against root rot disease caused by Pythium aphanidermatum and stimulated antifungal hydrolases such as chitinases and p-1,3 glucanases (Ghauoth et al., 1994). Recent evidence suggests that salicylic acid (SA), is a systemic signal responsible for the induction of systemic acquired resistance (SAR) in tobacco to tobacco mosaic virus (Malamy et al., 1990). Cucumber plants inoculated with tobacco necrosis virus, or the fungus Colletotrichum lagenarium, showed a rise in SA levels that was preceded by the development of SAR; thus SA plays a role in plant defense against pathogen attack (Metraux et al., 1990). Transgenic tobacco plants carrying a bacterial salicylate hydroxylase gene cannot accumulate SA, and do not develop SAR (Gaffney et al., 1993). It was shown that peanut seeds treated with chitosan enhanced the synthesis of phenolic acids (such as p-coumaric), ferulic acids and resistance against Aspergillus jlavus (Fajardo et al., 1994a). Groundnut rust caused by Puccinia arachidis Speg. is one of the major destructive diseases in groundnut growing areas. Reduction in pod yield due to this disease may be as high as 50-60s (Subrahmanyam et

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Chitosan induces resistance components in Arachis hypogaea: M. Sathiyabama and R. Balasubramanian al., 1989). The

application of fungicides to control groundnut rust resulted in varying degrees of success. Although fungicides are effective against rust disease, their mammalian toxicity and possible phytotoxic nature (Subrahmanyam and McDonald, 1983) may restrict their use to control groundnut rust. Treating plant tissue with chitosan intensifies the natural defense mechanisms and consequently helps the tissue in restricting fungal colonization (Ghauoth et al., 1994). The present work describes the efficacy of chitosan in reducing leaf rust attack on groundnut, and the changes in levels of antifungal hydrolases and salicylic acid which have been associated with induction of resistance after treatment with chitosan.

Materials and methods Seeds of Arachis hypogaea L. cv. TMV7 were obtained from the Oilseed Research Station, Tindivanum, TNAU, Tamil Nadu, India. Groundnut plants were raised as described earlier (Sathiyabama and Balasubramanian, 1991). Preparation of uredospore suspension, method of inoculation and maintenance of rust on detached/ intact leaves was done according to the method of Govindasamy and Balasubramanian (1989). Uredospores from sori of 16 day old inoculated plants were used for further multiplication and studies. Chitosan (from crab shell, Sigma Chemical Co., St Louis, USA) purified by the method of Benhamou and Theriault (1992) was used for treatment of 10 mg/ml groundnut leaves. The stock solution, (10,000 ppm), of chitosan was prepared by dissolving purified chitosan in 0.25 N HCI under continuous stirring. The pH was adjusted to 5.5-6.0 using 2 N sodium hydroxide, dialysed for 12 h against cold distilled water, and autoclaved. From this stock solution various dilutions (100, 500, 1000 ppm) of chitosan were prepared with sterile distilled water. The final pH of the aqueous solution was adjusted to 5.6. Leaves from 30 day old plants were sprayed with different concentrations of chitosan on the abaxial surface (0.1 ml/leaflet) with a painting brush (Camlin Ltd, Bombay, India). Leaves treated with sterile distilled water served as controls. Leaves were inoculated with a uredospore suspension (1 x lo5 spores/ml) of p arachidis 24 h before chitosan treatment, during treatment, and 24 h after treatment (100 ul/leaflet) using a paint brush. Inoculated plants were covered with polyethylene bags for 48 h. They were maintained in a greenhouse at 25 _+5°C. Fresh uredospores of Z? arachidis (1 x 10’ spores/ ml) were mixed with chitosan solution (100 ul spore suspension +lOO ul of chitosan) and placed on depression glass slides and incubated in a moist chamber in total darkness at 25 f. 2°C for 4 h. Uredospores incubated in water served as a control. A spore was considered to be germinated when the germ tube was one and a half times its breadth (Manners, 1966). Germinated spores were counted

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at 200 x under the compound microscope magnification. All plants were monitored for first visible symptom appearance and the number of pustules. The number of uredospores/sorus was counted. Disease severity was estimated by determining the leaf area occupied by lesions using an area meter (Delta-T Devices, Cambridge, UK). Twenty plants were used for each treatment. Experiments were repeated three times. Each experimental result was analysed statistically by the method of Denenberg (1976). Preparation of extract for the determination of salicylic acid (SA) was done by the procedure of Raskin et al. (1989). Control and chitosan-treated leaf samples were ground with an aqueous solution of 90% (v/v) methanol (1 g/2.5 ml) and centrifuged at 15,000g for 15 min. The pellet was reextracted with 100% (v/v) methanol and centrifuged at 15,000g for 15 min. The supernatants were combined and dried in air at 4°C. The residue was resuspended in 90% methanol and separated by preparative thin layer chromatography using silica gel G. Samples that co-migrated at the place of standard SA were eluted with 90% methanol, air dried and resuspended in methanol. SA levels in the samples were quantified by the procedure of Schneider-Muller et al. (1994). The intercellular washing fluid (IWF) was prepared from control and chitosan-treated leaves at 2 day intervals up to 16 days by the method of Rohringer et al. (1983). Leaves were washed thoroughly with distilled water, cut into 5 cm2 bits, immersed in pre-cooled distilled water, and infiltrated ‘in vacua’ for 15 min. The leaf bits were then blotted dry with tissue paper, rolled up and placed in a plastic syringe and centrifuged (3OOg, 3 min). The collected fluid was used after centrifugation. The protein content of the intercellular fluid was determined by the dye binding method of Bradford (1976) with bovine serum albumin fr (V) (Sigma Chem. Co., St Louis, USA) as a standard. Chitinase activity was determined by mixing 1 ml of intercellular fluid with 1 ml of colloidal chitin (O.l%, w/v in sodium acetate buffer, 0.05 M, pH 5.0). The amount of N-acetyl glucosamine (GlcNAc) produced after 2 h incubation at 37°C was determined by the method of Reissig ef al. (1955) using GlcNAc as the standard. One unit of chitinase activity is defined as the amount of enzyme which produces 1 pm of GlcNAc/ml per min under the assay conditions. p-1,3 glucanase activity was determined by mixing 0.1 ml intercellular fluid and 0.9 ml of laminarin (0.1% w/v in sodium acetate buffer, 0.05 M, pH 5.0). The amount of reducing sugars produced after 15 min at 37°C was determined by the method of Dygert et al. (1965) using glucose as the standard. One unit of p-1,3 glucanase activity is defined as the amount of enzyme which produces 1 urn of glucose/ ml per min under the assay conditions. Mannosidase, a typical intracellular enzyme, was tested as described by Matta and Bahl (1972) to monitor the integrity of the cells during IWF preparation. SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) was performed in

Chitosan induces resistance components in Arachis hypogaea: M. Sathiyabama and R. Balasubramanian Effect of chitosan on the progression of rust disease in groundnut leaves

polyacrylamide slab gel (11% w/v, separating and 5% w/v stacking gel) according to the method of Laemmli (1970). The gels were stained with silver nitrate as described by Blum et al. (1987). Standard proteins for molecular mass determination were a gift from New England Biolabs Beverly, USA. Apoplastic chitinases were detected on native PAGE (Davis, 1964) gels (10% w/v, separating gel) as described by Trudel and Asselin (1989). p-1,3 glucanases were detected after native PAGE (Reisfeld et al., 1962) gels (10% w/v) by the method of Pan et al. (1989). Immunoblots were performed according to the procedure of Towbin et al. (1979). The antiserum against IWF of rust-infected groundnut leaf chitinase was kindly provided by Dr V. Govindasamy, CAS in Botany, University of Madras and the antiserum against IWF of infected potato leaf p-1,3 glucanase was a gift from Dr E. Kombrink, Max-Planck Institute, Koln, Germany.

Chitosan at 1000 ppm (w/v) was the most effective dose in reducing the development of rust disease on leaves, symptoms groundnut leaves. In treated appeared 18 days after inoculation while symptoms in the controls took only 6 days. Furthermore, the numbers of uredosori and uredospores/sorus formed on treated groundnut leaves were reduced when compared with the control leaves (Figure 1). It is evident that chitosan treatment not only delayed the development of rust on groundnut leaves, but also reduced the sporulation of p arachidis. Effect of preventive and curative applications of chitosan on leaf rust development Chitosan delayed symptom development by 20 days on groundnut leaves when applied either preventively or curatively. However, even through there was no differences in symptom development among the treatments, the lowest numbers of uredosori and uredospores/sorus were recorded in leaves inoculated 24 h after chitosan treatment (Figure 2). The least effective treatment was when leaves were inoculated with rust uredospores first followed by chitosan application (Figure 2). Thus, prior treatment of leaves with chitosan did affect (slow down) the development of rust. This treatment was used for biochemical analysis.

Results Effect of chitosan on germination of uredospores of P. arachidis

Purified chitosan inhibited germination of uredospores of E arachidis. Chitosan treated uredospores did not germinate even after repeated distilled water washes (Table 1).

Changes in salicylic acid levels Table 1. Effect of chitosan Puccinia arachidis Speg. Concentration of chitosan (ppm)

on

germination

of uredospores

Germination % after 4 h*

Germination % 10 h after wash

24+3 13*1 6kO 96il

28&l 1552 7*1

100 500 1000 Control (HzO)

Chitosan treatments induced an increase in endogenous SA which reached its peak on day 12. The SA level of control leaves remained unaltered

of

(Figure 3).

Changes in the protein

levels

The protein content in the intercellular washing fluid (IWF) of treated leaves increased after chitosan treatment. The highest protein content was detected

*Mean value of three experiments.

No. of uredosporeskorus

No. of lesions/cm 2 16

(x 1000) _r L3

14 20 12 IO

15

8

10

6 4

5 2 0

0

Days after inoculation -

Control

@zzJ Control

100ppm 100 ppm

~-+- 500ppm

-Pi IOOOppm

0

m

500 ppm

1000 ppm

Figure 1. Effect of chitosan on the progression of rust disease on groundnut leaves*. *Each value represents a mean of replicates experiments with + standard error. Bars without standard errors showed negligible value. Vertical bars indicate standard errors

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of two

309

Chitosan induces resistance components in Arachis hypogaea: M. Sathiyabama and R. Balasubramanian No. of lesions/cm

No. of uredosporeskorus

16

(x 1000)

14 12 10 8 6 4 2 0

Days after inoculation -o- uredospores inoculated 24 h after treatment 0

-

control m

+

uredospores inoculated during treatment

+

uredospores inoculated before treatment

m

Figure 2. Effect of inoculation time on the development of rust disease on groundnut leaves *. *Each value represents a mean of replicates of two experiments with *standard error. Bars without standard errors showed negligible value. Vertical bars indicate standard errors

on day 8 followed by a sharp fall on day 10 and subsequently it plateaued. Protein levels on the controls remained unchanged. Changes

in chitinase and p-1,3 glucanase activities

Chitinase activity of IWF of treated groundnut leaves increased over a period of time after treatment and peaked on day 10, followed by a fall in enzyme activity between days 12 and 16. The IWF of control leaves showed slight alteration in enzyme activity (Figure 4). activity of IWF of treated p-1,3 glucanase groundnut leaves increased over a period of time and peaked on day 10 followed by a fall in enzyme activity. The IWF of control leaves did not show alteration in enzyme activity (Figure 4).

ct-Mannosidase

activity in IWF of groundnut

leaves

Only a small amount of mannosidase activity was found in IWF. The low activity of mannosidase from IWF suggests that contamination by cytosolic protein was minimal. SDS-PAGE separation

of total proteins

The polypeptide profile of control leaves did not show any change over a period of time. A new 25 kDa polypeptide appeared on day 2 which was present until day 16 in treated leaves. Subsequently as many as 11 new polypeptides of molecular mass 27, 28, 30, 41, 47, 56, 62, 72, 83, 86 and 92 kDa appeared on day 8 in treated leaves. The induced polypeptides of molecular masses 56, 62, 72, 83, 86 and 92 kDa disappeared on day 10 after treatment.

SA @g/g f.wtI 1.6 -l

1

3.5

1.4 0.8

1.2

3

E .g

1

2.5

s 0.6 z

0.8

2

E

0.6

8 0.4 s! ‘Z= E 0 0.2

0.4 0.2

1.5 1

0

0.5

12

8

16 0

Days

10

12

14

16

Days

Control

m

Figure 3. Endogenous salicylic acid (vg/g fresh weight) and chitosan-treated leaves of Arachis hypogaea L.

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Treated

1998 Volume 17 Number 4

control (Chitinase)

+

u- control (s-1,3 glucanase)+ in control

treated (Chitinase) treated ( P- 13 @JcanW

Figure 4. Chitinase and b-1,3 glucanase activities in intercellular leaves of washing fluid (IWF) of control and chitosan-treated Arachis hypogaea L.

Chitosan induces resistance components in Arachis hypogaea: M. Sathiyabama and R. Balasubramanian

These bands appeared existing bands. Detection

of chitinase

again on day 12 along with the

isoforms on native PAGE

The chitinase isoforms native PAGE did show remained until day 12 IWF of control leaves chitinase profile.

of treated leaves separated on a new isoform on day 2 which in treated leaves whereas the did not show any change in

Detection of p-1,3 glucanase

isoforms on native

PAGE

The isoforms of p-1,3 glucanase separated PAGE showed 2-3 isoforms from day 2 to chitosan-treated groundnut leaves. The control leaves did not show any p-1,3 isoform. Western blot analysis

of apoplastic

on native day 14 in IWF of glucanase

chitinases

IWF of groundnut leaves treated with chitosan showed a new chitinase isoform of molecular mass 47 on day 4. This isoform of chitinase remained up to day 12 after treatment. In control leaves the apoplastic chitinase did not show any alteration over the entire experimental period. Western blot analysis

of apoplastic

o-1,3 glucanases

Immunoblot analysis of apoplastic glucanases of peanut leaves treated with chitosan showed alterations in their profiles. An increase in p-1,3 glucanase activity was evident from day 4 after treatment. A new glucanase isoenzyme (56 kDa) appeared on day 8 and remained up to day 10 in treated leaves. Subsequently this band disappeared on day 12. The constitutive glucanase band (32.5 kDa) continued to increase over the experimental period in treated leaves. In contrast, the constitutive glucanase of control leaves did not show any change in the experimental period. Discussion The components of resistance in groundnut leaves against the rust pathogen, Puccinia aruchidis, have been identified as: a longer incubation period; reductions in lesion diameter and number of pustules; and in number of uredospores/sorus (Mehan et al., 1994). Groundnut leaves treated with chitosan showed all the above mentioned components of resistance against the rust pathogen. The results presented in this study suggest that groundnut leaves could be protected from the rust disease by prior treatment with chitosan. Chitosan is a polymer of p-1,3 linked glucosamine and it is difficult to understand the entry of a polymer such as chitosan into the plant cell. It is possible that chitosan may enter through wounds on the leaf surface. Painting of the groundnut leaf surface with the chitosan solution might have dislodged trichomes on the surface of the leaf, resulting in microscopic

wounds through which chitosan could enter. After its entry the movement of chitosan inside the plant cell can be traced with radioactive chitosan. Using r4C chitosan, Hadwiger et al. (1981) localized it in the nucleus of pea leaves and suggested that chitosan actually interacts with the cellular DNA. Such an interaction of chitosan with the cellular DNA may lead to multiple, biochemical reactions in the plant (Hadwiger et al., 1986). The presence of salicylic acid in groundnut plant has been demonstrated (Raskin et al., 1990). In our work, treatment of groundnut leaves with chitosan enhanced SA levels. It has been shown that addition of fungal elicitors to carrot cell cultures induces rapid synthesis of SA and much of the SA is released into the medium (Schneider-Muller et al., 1994). Besides SA levels, enhanced activities of intercellular chitinases and glucanases in treated groundnut leaves suggest that SA may act as an endogenous signal in activating host resistance to the rust pathogen. Indeed, SA has been implicated as an endogenous signal in the resistance response of tobacco and cucumber in tobacco necrosis virus and Colletotlichum lagenarium (Metraux and Raskin, 1993). In cucumber the major extracellular pathogenesisrelated (PR) protein is an extracellular endochitinase which accumulates in leaves treated with SA (Metraux et al., 1989). One of the responses observed in groundnut leaves treated with chitosan was an increase in intercellular chitinases and p-1,3 glucanases. An increase in chitinase activity was evident from 48 h after treatment and this increase reached its peak on day 10 in treated leaves. During this time, there was no macroscopic rust symptoms on the groundnut leaves. A new, acidic isoform of chitinase was induced after chitosan treatment. Treatment of cucumber seedlings with chitosan did induce new isoforms of chitinase (Zhang and Punja, 1994). Besides chitinase, p-1,3 glucanase activity also showed qualitative and quantitative changes in the IWF of treated groundnut leaves. New isoforms of glucanase were detected in the intercellular washing fluid of treated leaves. These results indicate that application of chitosan solution may sensitize the plant to respond more rapidly to a pathogen attack by elaboration of chitinases and glucanases. A combination of isoforms of chitinases and glucanases may affect the growth of II uruchidis in the intercellular space. In a recent study, Benhamou and Theriault (1992) observed the chitin breakdown in fungal cell walls using WGA-ovomucoid gold complex and suggested that treatment induced the rapid production of tomato plant chitinases and accumulated extracellularly and interacted with fungal chitin. Localization of isoforms of chitinases and glucanases in the intercellular space of treated groundnut leaves by immunocytochemical techniques will substantiate this claim. It is well known that chitosan can inhibit germination and growth of several fungi such as Phytophthora cactorum, P megasperma, I? paroecandrum, Pythium debalyanum, Fusarium oxysporum, E culmorum (Allan

and Hadwiger,

1979; Stossel and Leuba,

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Arachis hypogaea: M. Sathiyabama and R. Balasubramanian

Quere et al., 1995). This inhibitory action of chitosan on the rust pathogen is another example. Chitosan reduced germination of uredospores of I? aruchidis even at 100 ppm concentration. Chitosan, a polycationic polysaccharide, completely inhibits all the RNA synthesis in some fungi (Hadwiger et al., 1986). Thus, the effects of chitosan such as elevated levels of SA, induction of chitinases and glucanases in groundnut leaves and inhibition of germination of uredospores of p aruchidis, suggest its potential as a protectant against the rust pathogen. Treatment of groundnut leaves with chitosan may reduce the input of fungicides against rust disease.

suppression of Pythium aphanidetmatum reactions. Phytopathology 84, 313-320

and inducation of defense

Govindasamy, V. and Balasubramanian, R. (1989) Biological control of groundnut rust, Puccinia arachidis, by Trichoderma hanianum.

J. Plant Dis. Protection 96, 337-345

Hadwiger, L. A., Beckman, J. M. and Adams, M. J. (1981) Localization of fungal components in the pea-Fusatium interaction detected immunochemically with anti-chitosan and antifungal cell wall antisera. Plant Physiol. 67, 170-175 Hadwiger, L. A., Kendra, D. F., Fristensky, B. W. and Wagoner, N. (1986) Chitosan both activates genes in plants and inhibits RNA synthesis in fungi. In Chitin in Nature and Technology. eds R. A. Muzzarelli, C. Jeuniaux and G. W. Gooday, pp. 209-214. Plenum Press, New York Kendra, D. F. and Hadwiger, L. A. (1984) Characterization of the smallest chitosan chitosan oligomer that is maximally antifungal to Fusa-ium solani and elicits pisatin formation in Pisum sativum.

Acknowledgements

Exp. Mycol. 8, 276-281

The authors thank Prof. A. Mahadevan, Director, Centre for Advanced Studies in Botany, for providing facilities. M. S. thanks the CSIR, New Delhi, for the award of a fellowship, during which the present investigation was conducted. We thank Prof. Y. Ohashi, Japan for providing salicylic acid, New England Biolabs, USA for a gift sample of molecular weight markers and to Dr E. Kombrink, Max-Planck Institute, Germany for providing p-1,3 glucanase antibody.

Kendra, D. F., Christian, D. and Hadwiger, L. A. (1989) Chitosan oligomers from Fusarium solaniipea interactions, chitinase@-glucanase digestion of sporelings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. Physiol. Mol. Plant Pathol. 35,215-230

Krebs, S. L. and Grumet, R. (1993) Characterization of celery hydrolytic enzymes induced in response to infection by Fusarium oqspomm.

Physiol. Mol. Plant Pathol. 43, 193-208

Kuc’, J. (1987) Plant immunization and its applicability for disease control. In Innovative Methods for Disease Control, ed. I. Chet, pp. 303-318. Wiley, New York Kuc’, J. (1993) Induced resistance technology for the control of plant disease. In New Frontiers in Rice Research, eds K. Muralidharan and E. A. Siddiq, pp. 213-220. ICAR, India

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Received 6 February 1997 Revised 2 February 1998 Accepted 5 February 1998

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