Salicylic acid (10 mM) sprayed on cowpea leaves inoculated with Meloidogyne incognita reduced nematode
infection and promoted plant growth. Salicylic acid did not kill nematodes in an in vitro test and induced expression
and accumulation of pathogenesis related-1 protein in the leaves of sprayed plants. The presence of Tween-20
enhanced the effect of salicylic acid on the accumulation of pathogenesis related-1 protein

Banerjee, N.

Das, P.

Nandi, B.

Sengupta, S.

Sinha Babu, S.P.

Sukul, N.C.

B. Nandi

N. Banerjee

N.C. Sukul

P. Das

S. Sengupta

S.P. Sinha Babu

Directory of Open Access Journals

39Phytopathol. Mediterr. (2002) 41, 39–44IntroductionPlants have a number of defense mechanismsagainst pathogens. In addition to constitutive re-sistance, plants can activate protective mecha-nisms upon infection with widely different patho-gens. This is termed induced or acquired resist-ance. Resistance could be expressed locally but alsosystemically in tissue remotely located from theinitial treatment (systemic acquired resistance,SAR) (Ross, 1961). This form of resistance protectsplants from a broad spectrum of pathogens andworks systemically in many cases (Klessig andMalamy, 1994; Schneider et al., 1996; Mauch-Maniand Metraux, 1998).  In the resistant tissue thereis the expression of a complete set of defense-re-Salicylic acid enhances resistancein cowpea against Meloidogyne incognitaBIDYUT NANDI1, NIRMAL CHANDRA SUKUL1, NIRMALYA BANERJEE2, SHADAK SENGUPTA3,PRADIP DAS3 and SANTI PRASAD SINHA BABU11Department of Zoology, and 2Department of Botany, Visva-Bharati University,Santiniketan 731 235 West Bengal, India3National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta 10, West Bengal, IndiaSummary. Salicylic acid (10 mM) sprayed on cowpea leaves inoculated with Meloidogyne incognita reduced nema-tode infection and promoted plant growth. Salicylic acid did not kill nematodes in an in vitro test and induced expres-sion and accumulation of pathogenesis related-1 protein in the leaves of sprayed plants. The presence of Tween-20enhanced the effect of salicylic acid on the accumulation of pathogenesis related-1 protein.Key words: root-knot nematode, systemic acquired resistance, Vigna catjang.Corresponding author: S.P. Sinha BabuFax: +91 034 6353268E-mail: santi_p@rediffmail.comlated genes whose products, for example in tobac-co leaves infected with tobacco mosaic virus (TMV),include five or more families of pathogenesis-re-lated (PR) proteins usually 10 to 40 KDa in size(Carr and Klessig, 1989).  The synthesis of theseproteins have been detected in a wide variety ofplants including, in addition to tobacco, tomato,cucumber, potato, cowpea, rice, soybean, sugarbeet,wheat, barley, maize, beans and carrot (Carr andKlessig, 1989; White et al., 1989; Nasser et al., 1990;Cordero et al., 1992; Klessig and Malamy, 1994).The exogenous application of salicylic acid (SA)could induce expression of the same set of defense-related genes that are activated by TMV infection(Ward et al., 1991; Durner et al., 1997). In recentliterature SA has been reported as an endogenoussignal for the activation of certain plant defenseresponses, including PR gene expression and en-hanced resistance to pathogens (Greenberg et al.,1994; Conrath et al., 1995), but for some virus in-fections several authors demonstrated that neither40 Phytopathologia MediterraneaB. Nandi et al.PR protein nor exogenous SA induced resistance(Pennazio et al., 1983; Roggero and Pennazio, 1988,1991).  The expression of PR genes and the induc-tion of SAR can, however, occur even without SAaccumulation by means of a synthetic compoundsuch as 2, 6-dichloroisonicotinic acid (Vernooij etal., 1995). In plant-nematode interactions, newlysynthesized proteins including PR proteins havebeen found in potato plants infected with the pota-to cyst nematodes Globodera pallida and G. rosto-chiensis (Hammond-Kosack et al., 1989; Rahimi etal., 1993, Rahimi et al., 1996), but studies are rath-er limited. We decided therefore to investigate theeffect of exogenous SA both on the synthesis of PRprotein and on resistance to Meloidogyne incogni-ta infection of cowpea (Vigna catjang Walp.).Materials and methodsNematode and plant materialSecond-stage juveniles (J2) of Meloidogyne in-cognita (Kofoid and White) Chitwood were allowedto hatch from egg masses obtained from a culturemaintained on cowpea in the Department of Zool-ogy at Santiniketan, West Bengal, India. ActiveJ2 obtained from egg masses were kept in cavityblocks containing sterile tap water, each block con-taining 100±15 juveniles. Cowpea plants (cv. PusaRuby) susceptible to M. incognita were used forthe experiments.Experimental planAseptically germinated seeds of cowpea cv. PusaRuby were sown in 32-cm-diameter pots (one seed/pot) containing an autoclaved mixture of clay soiland composted manure (2:1, v:v). The pots weredivided into 7 groups of 10 pots each. The groupswere: uninoculated unsprayed (UU); inoculatedunsprayed (IU); inoculated and sprayed with 0.5%Tween-20 (IT); uninoculated and sprayed with 10mM SA (USA); uninoculated and sprayed with 10mM SA containing 0.5% Tween-20 (USAT); inocu-lated and sprayed with 10 mM SA (ISA); and inoc-ulated and sprayed with 10 mM SA containing 0.5%Tween-20 (ISAT). When the plants were at the 6-leaf stage, the IU, IT, ISA and ISAT groups wereinoculated with 1,400±100 freshly hatched M. in-cognita J2 in 5 ml of water. The J2 were injectedinto the soil at a depth of 2 cm, halfway betweenthe plant and the side of the pot. SA was appliedas a foliar spray by an atomizer 24 h before inocu-lation. During spraying, the soil surface under-neath each plant was covered with polyethylenesheeting. Plants in the uninoculated unsprayed andinoculated unsprayed groups received a distilledwater spray. Spraying was repeated after 4 days.The plants were regularly watered and the exper-iment was conducted outdoors at ambient atmos-pheric temperature (28±3°C) and humidity(75±4%). To assess the effect of SA on M. incogni-ta, the sterile tap water  in the cavity blocks (seeNematode and plant material section) where ac-tive M. incognita J2 were kept, was pipetted outand immediately replaced by 1 ml of 10 mM SA.Two cavity blocks containing only sterile tap wa-ter served as negative controls.  Mortality of thenematodes at room temperature (27±2°C) was re-corded every hour for 6 hrs.At 45 days after inoculation, shoot length, shootweight, root length, root weight, root nodulenumber, root gall number and number of eggs perg of root were recorded. Nematode eggs were ex-tracted from the roots with the sodium hypochlo-rite method (Hussey and Barker, 1973). Three sam-ples of root pieces were taken at random from eachplant group and the total protein fraction in eachsample was determined by the Folin-phenol meth-od (Lowry et al., 1951).  The experiment was re-peated twice with similar results. The data fromthe first experiment are here presented. Data wereanalyzed by ANOVA and the means were separat-ed by the least significant difference (LSD) test(P=0.05).PR proteins extractionPR proteins were extracted from the leaves ofthe various groups 2 days after the second spray-ing. Three samples of fresh leaves per group ofplants were taken at random, mixed and choppedinto pieces. Two g of these fresh leaves from eachgroup were homogenized in 50 mM Tris-HCl, pH8.0, 1 mM EDTA, 12 mM 2-mercaptoethanol/phe-nylmethylsulfonyl fluoride (10 µg ml-1). After clar-ification by centrifugation at 12,000 g (20 min, 4°C),the supernatant was fractionated by 12% SDS/PAGE and the separated proteins were stainedwith Coomassie blue following the method ofLaemmli (Laemmli, 1970). For western blot anal-ysis the homogenate was fractionated by 12% SDS-PAGE, and the separated proteins were electropho-41Vol. 41, No. 1, April 2002Induced resistance in cowpearetically transferred to a nitrocellulose filter. Im-munoblot analysis was performed using mousemonoclonal antibody MAb 2-11G5 against tobaccoacidic PR-1 protein at a dilution of 1:1000 (Con-rath et al., 1995).ResultsNo mortality of nematodes was recorded at 10mM SA.The sprayed plants did not show any toxic ef-fect, in the form of wilting or yellowing of leaves,in the next 30 days after the second spraying.The pot test indicated that: (i) SA increasedgrowth of inoculated plants in terms of shootlength, shoot weight and root length as comparedwith inoculated unsprayed plants; (ii) root gallnumber and number of eggs in roots were signifi-cantly greater in inoculated untreated plants thanin inoculated treated plants; (iii) root protein con-tent increased significantly in all SA-treated groupscompared with unsprayed inoculated plants; (iv)SA containing Tween did not differ from SA alonein its effect on the bio-mass and root gall numberof the cowpea plants (Table 1). SA induced expres-sion of PR-1 protein (16 KDa) in both uninoculat-ed and inoculated SA-sprayed cowpea leaves (Fig.1 and 2). The presence of Tween-20, which mayincrease the uptake of SA into the leaves, enhancedthe effect of SA on the expression and accumula-tion of PR-1 protein (Fig. 1). Tween-20 alonesprayed on the leaf surface did not induce expres-sion of PR-1 protein in cowpea leaves. No PR-1protein accumulated in unsprayed plants, wheth-er inoculated or uninoculated (Fig. 1 and 2).DiscussionSA is known to be an endogenous regulator oflocalized and systemic acquired resistance. Whensome plants become infected with disease organ-isms, SA accumulates in them, and the exogenousapplication of SA in these plants without infectioncould induce expression of the same set of defense-related genes as when they were infected (Klessigand Malamy, 1994). Some of the genes activatedduring the induction of SAR by exogenous SA en-code a group of PR proteins (Ryals et al., 1994).The antimicrobial and enzyme activities of thesegene products suggest that they may play a role inthe induction and maintenance of SAR in plants(Ohashi and Ohshima, 1992). However, the func-tion of the PR-1 protein has not yet been clearlyunderstood. Changes in the gene expression of po-tato leaves after root infection with the cyst nema-tode Globodera rostochiensis induced the expres-Table 1. Increase in cowpea growth, decrease in Meloidogyne incognita infestation of cowpea and root protein con-tent following treatment by exogenous salicylic acid at a 10 mM concentration.Cowpea plants growth Meloidogyne incognita infestation Root proteincontent Treat-  menta Shoot length Shoot weight Root length Root weight Root nodule Root gall(cm) (g) (cm)  (g) No. No. Eggs g-1 root (mg g-1)UU 264.6±6.5 ab 229.3±5.6 a 43.0±3.9 a 15.0±1.4 f 313.0±12.2 a _ _ 7.5±0.67 aIU 182.6±5.0 de 165.0±4.2 cd 28.6±2.4 c 31.0±5.0 a 166.0±9.8 g 745.0±18.7 a 8400±348.2 a 4.2±0.65 bcIT 175.4±4.9 e 157.5±5.3 d 31.0±1.8 bc 29.3±3.1 a 167.3±7.5 fg 738.0±29.4 a 7900±141.4 a 4.0±0.50 cUSA 255.0±5.3 a 225.0±6.8 ab 37.0±3.2 ab 22.3±2.2 bcdef 251.0±7.7 e _ _ 6.2±0.57 aUSAT 261.5±6.0 a 218.0±5.3 ab 41.6±3.1 a 20.0±2.8 def 274.6±8.8 cd _ _ 6.6±0.68 aISA 242.0±3.7 bc 215.0±3.7 ab 36.0±5.3 abc 21.6±3.5 cdef 265.0±6.9 d 423.2±17.7 bc 3800±277.8 bc 6.4±0.43 aISAT 235.6±7.4 c 209.0±6.0 b 40.0±6.1 a 17.0±3.5 ef 285.0±6.4 bc 410.0±28.8 c 3500±179.4 c 7.1±0.56 aa Cowpea seedlings with six compound leaves were sprayed with salicylic acid at a 10 mM concentration. Each plant wasinoculated with 1400±100 second-stage juveniles of  M. incognita 24 h after the first treatment. Data are means of ten replicates.UU, uninoculated untreated; IU, inoculated untreated; IT, inoculated and treated with Tween; USA, uninoculated and treatedwith salicylic acid; USAT,  uninoculated and treated with salicylic acid containing Tween; ISA, inoculated and treated with sali-cylic acid;  ISAT, inoculated and treated with salicylic acid containing Tween.b Different small letters in a column indicate significant differences according to the least significant difference test (P=0.05).-, no root galls or eggs in this group.42 Phytopathologia MediterraneaB. Nandi et al.Fig. 2. Immunoblot analysis of proteins extracted from cowpea plants. Acidic PR proteins were extracted fromleaves on day 2 after the 2nd treatment. Proteins were fractionated by SDS-PAGE, and immunoblotted with mousemonoclonal antibody MAb 2-11G5 against tobacco acidic PR-1 protein. lane a, uninoculated and treated with salicyl-ic acid; lane b, uninoculated and treated with salicylic acid containing Tween; lane c, inoculated and treated withsalicylic acid  containing Tween; lane d, inoculated and treated with salicylic acid; lane e, inoculated and treatedwith Tween; lane f, inoculated untreated; lane g, uninoculated untreated; lane h, molecular weight marker.Fig. 1. SDS-PAGE of proteins extracted from cowpea plants. Acidic PR proteins were extracted from leaves on day 2after the 2nd treatment. Separated proteins were stained with Coomassie Blue. Lane a, inoculated and treated withsalicylic acid containing Tween; lane b, inoculated and treated with salicylic acid; lane c, uninoculated and treatedwith salicylic acid containing Tween; lane d, uninoculated and treated with salicylic acid; lane e, inoculated andtreated with Tween; lane f, inoculated untreated; lane g, uninoculated untreated; lane h, molecular weight marker.The induced band is indicated with a dot to the right of the respective lanes.a b c d e f g hKDa97664531211416 KDaa b c d e f g hKDa97664531211416 KDa43Vol. 41, No. 1, April 2002Induced resistance in cowpeasion of PR proteins (Hammond-Kosack et al., 1989),while infection of potato roots with Globodera pal-lida induced the expression of proteins with a mo-lecular weight of 70–82 KDa, larger than that ofcommon PR proteins (Rahimi et al., 1993). In to-mato root infected with root-knot nematodes, sev-eral homologous plant-defense genes (includingperoxidases, chitinase, lipoxygenase and protein-ase inhibitor protein) are expressed locally with-in 24 h of inoculation (Lambert, 1995; William-son and Hussey, 1996). In our experiments, spray-ing with SA, which induced the accumulation ofPR-1 protein in the sprayed leaves, significantlyincreased resistance to root-knot nematode infec-tion, reflected in a lack of nematode reproduction.It appeared that SA-spraying did not have anydirect influence on plant growth but improvedgrowth in the sprayed plants, perhaps as a resultof the reduction in root-knot disease it broughta-bout. The mechanisms governing SA-induced re-sistance to M. incognita in cowpea are under fur-ther investigation.AcknowledgementsThe work described here is part of a project fund-ed by the Council of Scientific and Industrial Re-search, New Delhi to SPSB. We thank Prof. Dan-iel F. Klessig, Waksman Institute and Departmentof Molecular Biology and Biochemistry, Rutgers,The State University of New Jersey, USA, for pro-viding us with mouse monoclonal antibody MAb 2-11G5.Literature citedCarr J.P. and D.F. Klessig, 1989. The pathogenesis-relatedproteins of plants. In: Genetic Engineering: Principlesand Methods (J.K. Setlow, ed.) Plenum Press, New York,NY, USA, 65–109.Conrath U., Z.X. Chen, J.R. Ricigliano and D.F. Klessig,1995. Two inducers of plant defense responses, 2,6-dichloroisonicotinic acid and salicylic acid, inhibit cat-alase activity in tobacco. Proceedings of the NationalAcademy of Sciences, USA 92, 7143–7147.Cordero M.J., D. Raventos and B. San Segund, 1992. In-duction of PR proteins in germinating maize seeds in-fected with the fungus Fusarium moniliforme. Physio-logical and Molecular Plant Pathology 41, 189–200.Durner J., J. Shah and D.F. Klessig, 1997. Salicylic acidand disease resistance in plants. Trends in Plant Sci-ence 2, 266–274.Greenberg J.T., A.L. Guo, D.F. Klessig and F.M. Ausubel,1994. Programmed cell death in plants: a pathogen-trig-gered response activated coordinately with multipledefense functions. Cell 77, 551–563.Hammond-Kosack K.E., H.J. Atkinson and D.J. Bowles,1989. Systemic accumulation of novel proteins in theapoplast of the leaves of potato plants following rootinvasion by the cyst-nematode Globodera rostochiensis.Physiological and Molecular Plant Pathology 35, 495–506.Hussey R.S. and K.R. Barker, 1973. A comparison of meth-ods of collecting inocula of Meloidogyne spp., includ-ing a new technique. Plant Disease Reporter 57, 1025–1028.Klessig D.F. and J. Malamy, 1994. The salicylic acid signalin plants. Plant Molecular Biology 26, 1439–1458.Laemmli U.K., 1970. Cleavage of structural proteins dur-ing the assembly of the head of the bacteriophage T4.Nature 277, 680–685.Lambert K.N., 1995. Isolation of genes induced early in theresistance response to Meloidogyne javanica in Lyco-persicon esculentum. PhD Thesis, University of Cali-fornia-Davis, Davis, CA, USA.Lowry O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall,1951. Protein measurement with Folin phenol reagent.Journal of Biological Chemistry 193, 265–275.Mauch-Mani B. and J.P. Metraux, 1998. Salicylic acid andsystemic acquired resistance to pathogen attack. An-nals of Botany 82, 535–540.Nasser W., M. Tapia and G. Burkard, 1990. Maize patho-genesis–related proteins: Characterization and cellu-lar distribution of 1,3-β-glucanases and chitinases in-duced by brome mosaic virus infection or mercuric chlo-ride treatment. Physiological and Molecular Plant Pa-thology 36, 1–14.Ohashi Y. and M. Ohshima, 1992. Stress-induced expres-sion of genes for pathogenesis-related proteins in plants.Plant Cell Physiology 33, 819–826.Pennazio S., P. Roggero and R. Lenzi, 1983. Some charac-teristics of the hypersensitive reaction of  ‘White Bur-ley’ tobacco to tobacco necrosis virus. Physiological PlantPathology 22, 347–355.Rahimi S., R.N. Perry and D.J. Wright, 1993. Inductionand detection of pathogenesis-related proteins in leavesand roots of potato plants infected with pathotypes ofGlobodera pallida. Fundamental and Applied Nema-tology 16, 549–556.Rahimi S., R.N. Perry and D.J. Wright, 1996. Identifica-tion of pathogenesis-related proteins induced in leavesof potato plants infected with potato cyst nematodes,Globodera species. Physiological and Molecular PlantPathology 49, 49–59.Roggero P. and S. Pennazio, 1988. Effects of salicylate onsystemic invasion of tobacco plants by various viruses.Journal of Phytopathology 123, 207–216.Roggero P. and S. Pennazio, 1991. Salicylate does not in-duce resistance to plant viruses, or stimulate pathogen-esis-related protein production in soybean. Microbio-logica 14, 65–69.44 Phytopathologia MediterraneaB. Nandi et al.Ross A.F., 1961. Systemic acquired resistance induced bylocalized virus infections in plants. Virology 14, 340–358.Ryals J., S. Uknes and E. Ward, 1994. Systemic acquiredresistance. Plant Physiology 104, 1109–1112.Schneider M., P. Schweizer, P. Meuwly and J.P. Metraux,1996. Systemic acquired resistance in plants. Interna-tional Journal of Cytology 168, 303–340.Vernooij B., L. Friedrich, P. Ahl-Goy, T. Staub, H. Kessmannand J. Ryals, 1995. 2,6-Dichloroisonicotinic acid-inducedresistance to pathogens without the accumulation ofsalicylic acid. Molecular Plant-Microbe Interaction 8,228–234.Ward E.R., S.J. Uknes, S.C. Williams, S.S. Dincher, D.L.Wiederhold, D.C. Alexander, P. Ahl-Goy, J.P. Metrauxand J.A. Ryals, 1991. Coordinate gene activity in re-sponse to agents that induce systemic acquired resist-ance. Plant Cell 3, 1085–1094.White R.F., E.P. Rybicki, M.B. Von Wechmar, J.L. Dekkerand J.F. Antoniw, 1989. Detection of PR1-type proteinsin Amaranthaceae, Chenopodiaceae, Graminae, andSolanaceae by immuno-blotting. Journal of GeneralVirology 68, 2043–2048.Williamson V.M. and R.S. Hussey, 1996. Nematode patho-genesis and resistance in plants. The Plant Cell 8,1735–1745.Accepted for publication: February 4, 2002

Salicylic acid enhances resistance in cowpea against Meloidogyne incognita

Firenze University Press: E-Journals

39
Phytopathol. Mediterr. (2002) 41, 39–44
Introduction
Plants have a number of defense mechanisms
against pathogens. In addition to constitutive re-
sistance, plants can activate protective mecha-
nisms upon infection with widely different patho-
gens. This is termed induced or acquired resist-
ance. Resistance could be expressed locally but also
systemically in tissue remotely located from the
initial treatment (systemic acquired resistance,
SAR) (Ross, 1961). This form of resistance protects
plants from a broad spectrum of pathogens and
works systemically in many cases (Klessig and
Malamy, 1994; Schneider et al., 1996; Mauch-Mani
and Metraux, 1998).  In the resistant tissue there
is the expression of a complete set of defense-re-
Salicylic acid enhances resistance
in cowpea against Meloidogyne incognita
BIDYUT NANDI1, NIRMAL CHANDRA SUKUL1, NIRMALYA BANERJEE2, SHADAK SENGUPTA3,
PRADIP DAS3 and SANTI PRASAD SINHA BABU1
1Department of Zoology, and 2Department of Botany, Visva-Bharati University,
Santiniketan 731 235 West Bengal, India
3National Institute of Cholera and Enteric Diseases, Beliaghata, Calcutta 10, West Bengal, India
Summary. Salicylic acid (10 mM) sprayed on cowpea leaves inoculated with Meloidogyne incognita reduced nema-
tode infection and promoted plant growth. Salicylic acid did not kill nematodes in an in vitro test and induced expres-
sion and accumulation of pathogenesis related-1 protein in the leaves of sprayed plants. The presence of Tween-20
enhanced the effect of salicylic acid on the accumulation of pathogenesis related-1 protein.
Key words: root-knot nematode, systemic acquired resistance, Vigna catjang.
Corresponding author: S.P. Sinha Babu
Fax: +91 034 6353268
E-mail: santi_p@rediffmail.com
lated genes whose products, for example in tobac-
co leaves infected with tobacco mosaic virus (TMV),
include five or more families of pathogenesis-re-
lated (PR) proteins usually 10 to 40 KDa in size
(Carr and Klessig, 1989).  The synthesis of these
proteins have been detected in a wide variety of
plants including, in addition to tobacco, tomato,
cucumber, potato, cowpea, rice, soybean, sugarbeet,
wheat, barley, maize, beans and carrot (Carr and
Klessig, 1989; White et al., 1989; Nasser et al., 1990;
Cordero et al., 1992; Klessig and Malamy, 1994).
The exogenous application of salicylic acid (SA)
could induce expression of the same set of defense-
related genes that are activated by TMV infection
(Ward et al., 1991; Durner et al., 1997). In recent
literature SA has been reported as an endogenous
signal for the activation of certain plant defense
responses, including PR gene expression and en-
hanced resistance to pathogens (Greenberg et al.,
1994; Conrath et al., 1995), but for some virus in-
fections several authors demonstrated that neither
40 Phytopathologia Mediterranea
B. Nandi et al.
PR protein nor exogenous SA induced resistance
(Pennazio et al., 1983; Roggero and Pennazio, 1988,
1991).  The expression of PR genes and the induc-
tion of SAR can, however, occur even without SA
accumulation by means of a synthetic compound
such as 2, 6-dichloroisonicotinic acid (Vernooij et
al., 1995). In plant-nematode interactions, newly
synthesized proteins including PR proteins have
been found in potato plants infected with the pota-
to cyst nematodes Globodera pallida and G. rosto-
chiensis (Hammond-Kosack et al., 1989; Rahimi et
al., 1993, Rahimi et al., 1996), but studies are rath-
er limited. We decided therefore to investigate the
effect of exogenous SA both on the synthesis of PR
protein and on resistance to Meloidogyne incogni-
ta infection of cowpea (Vigna catjang Walp.).
Materials and methods
Nematode and plant material
Second-stage juveniles (J2) of Meloidogyne in-
cognita (Kofoid and White) Chitwood were allowed
to hatch from egg masses obtained from a culture
maintained on cowpea in the Department of Zool-
ogy at Santiniketan, West Bengal, India. Active
J2 obtained from egg masses were kept in cavity
blocks containing sterile tap water, each block con-
taining 100±15 juveniles. Cowpea plants (cv. Pusa
Ruby) susceptible to M. incognita were used for
the experiments.
Experimental plan
Aseptically germinated seeds of cowpea cv. Pusa
Ruby were sown in 32-cm-diameter pots (one seed/
pot) containing an autoclaved mixture of clay soil
and composted manure (2:1, v:v). The pots were
divided into 7 groups of 10 pots each. The groups
were: uninoculated unsprayed (UU); inoculated
unsprayed (IU); inoculated and sprayed with 0.5%
Tween-20 (IT); uninoculated and sprayed with 10
mM SA (USA); uninoculated and sprayed with 10
mM SA containing 0.5% Tween-20 (USAT); inocu-
lated and sprayed with 10 mM SA (ISA); and inoc-
ulated and sprayed with 10 mM SA containing 0.5%
Tween-20 (ISAT). When the plants were at the 6-
leaf stage, the IU, IT, ISA and ISAT groups were
inoculated with 1,400±100 freshly hatched M. in-
cognita J2 in 5 ml of water. The J2 were injected
into the soil at a depth of 2 cm, halfway between
the plant and the side of the pot. SA was applied
as a foliar spray by an atomizer 24 h before inocu-
lation. During spraying, the soil surface under-
neath each plant was covered with polyethylene
sheeting. Plants in the uninoculated unsprayed and
inoculated unsprayed groups received a distilled
water spray. Spraying was repeated after 4 days.
The plants were regularly watered and the exper-
iment was conducted outdoors at ambient atmos-
pheric temperature (28±3°C) and humidity
(75±4%). To assess the effect of SA on M. incogni-
ta, the sterile tap water  in the cavity blocks (see
Nematode and plant material section) where ac-
tive M. incognita J2 were kept, was pipetted out
and immediately replaced by 1 ml of 10 mM SA.
Two cavity blocks containing only sterile tap wa-
ter served as negative controls.  Mortality of the
nematodes at room temperature (27±2°C) was re-
corded every hour for 6 hrs.
At 45 days after inoculation, shoot length, shoot
weight, root length, root weight, root nodule
number, root gall number and number of eggs per
g of root were recorded. Nematode eggs were ex-
tracted from the roots with the sodium hypochlo-
rite method (Hussey and Barker, 1973). Three sam-
ples of root pieces were taken at random from each
plant group and the total protein fraction in each
sample was determined by the Folin-phenol meth-
od (Lowry et al., 1951).  The experiment was re-
peated twice with similar results. The data from
the first experiment are here presented. Data were
analyzed by ANOVA and the means were separat-
ed by the least significant difference (LSD) test
(P=0.05).
PR proteins extraction
PR proteins were extracted from the leaves of
the various groups 2 days after the second spray-
ing. Three samples of fresh leaves per group of
plants were taken at random, mixed and chopped
into pieces. Two g of these fresh leaves from each
group were homogenized in 50 mM Tris-HCl, pH
8.0, 1 mM EDTA, 12 mM 2-mercaptoethanol/phe-
nylmethylsulfonyl fluoride (10 µg ml-1). After clar-
ification by centrifugation at 12,000 g (20 min, 4°C),
the supernatant was fractionated by 12% SDS/
PAGE and the separated proteins were stained
with Coomassie blue following the method of
Laemmli (Laemmli, 1970). For western blot anal-
ysis the homogenate was fractionated by 12% SDS-
PAGE, and the separated proteins were electropho-
41Vol. 41, No. 1, April 2002
Induced resistance in cowpea
retically transferred to a nitrocellulose filter. Im-
munoblot analysis was performed using mouse
monoclonal antibody MAb 2-11G5 against tobacco
acidic PR-1 protein at a dilution of 1:1000 (Con-
rath et al., 1995).
Results
No mortality of nematodes was recorded at 10
mM SA.
The sprayed plants did not show any toxic ef-
fect, in the form of wilting or yellowing of leaves,
in the next 30 days after the second spraying.
The pot test indicated that: (i) SA increased
growth of inoculated plants in terms of shoot
length, shoot weight and root length as compared
with inoculated unsprayed plants; (ii) root gall
number and number of eggs in roots were signifi-
cantly greater in inoculated untreated plants than
in inoculated treated plants; (iii) root protein con-
tent increased significantly in all SA-treated groups
compared with unsprayed inoculated plants; (iv)
SA containing Tween did not differ from SA alone
in its effect on the bio-mass and root gall number
of the cowpea plants (Table 1). SA induced expres-
sion of PR-1 protein (16 KDa) in both uninoculat-
ed and inoculated SA-sprayed cowpea leaves (Fig.
1 and 2). The presence of Tween-20, which may
increase the uptake of SA into the leaves, enhanced
the effect of SA on the expression and accumula-
tion of PR-1 protein (Fig. 1). Tween-20 alone
sprayed on the leaf surface did not induce expres-
sion of PR-1 protein in cowpea leaves. No PR-1
protein accumulated in unsprayed plants, wheth-
er inoculated or uninoculated (Fig. 1 and 2).
Discussion
SA is known to be an endogenous regulator of
localized and systemic acquired resistance. When
some plants become infected with disease organ-
isms, SA accumulates in them, and the exogenous
application of SA in these plants without infection
could induce expression of the same set of defense-
related genes as when they were infected (Klessig
and Malamy, 1994). Some of the genes activated
during the induction of SAR by exogenous SA en-
code a group of PR proteins (Ryals et al., 1994).
The antimicrobial and enzyme activities of these
gene products suggest that they may play a role in
the induction and maintenance of SAR in plants
(Ohashi and Ohshima, 1992). However, the func-
tion of the PR-1 protein has not yet been clearly
understood. Changes in the gene expression of po-
tato leaves after root infection with the cyst nema-
tode Globodera rostochiensis induced the expres-
Table 1. Increase in cowpea growth, decrease in Meloidogyne incognita infestation of cowpea and root protein con-
tent following treatment by exogenous salicylic acid at a 10 mM concentration.
Cowpea plants growth Meloidogyne incognita infestation Root proteincontent Treat-
  menta Shoot length Shoot weight Root length Root weight Root nodule Root gall
(cm) (g) (cm)  (g) No. No. Eggs g
-1 root (mg g-1)
UU 264.6±6.5 ab 229.3±5.6 a 43.0±3.9 a 15.0±1.4 f 313.0±12.2 a _ _ 7.5±0.67 a
IU 182.6±5.0 de 165.0±4.2 cd 28.6±2.4 c 31.0±5.0 a 166.0±9.8 g 745.0±18.7 a 8400±348.2 a 4.2±0.65 bc
IT 175.4±4.9 e 157.5±5.3 d 31.0±1.8 bc 29.3±3.1 a 167.3±7.5 fg 738.0±29.4 a 7900±141.4 a 4.0±0.50 c
USA 255.0±5.3 a 225.0±6.8 ab 37.0±3.2 ab 22.3±2.2 bcdef 251.0±7.7 e _ _ 6.2±0.57 a
USAT 261.5±6.0 a 218.0±5.3 ab 41.6±3.1 a 20.0±2.8 def 274.6±8.8 cd _ _ 6.6±0.68 a
ISA 242.0±3.7 bc 215.0±3.7 ab 36.0±5.3 abc 21.6±3.5 cdef 265.0±6.9 d 423.2±17.7 bc 3800±277.8 bc 6.4±0.43 a
ISAT 235.6±7.4 c 209.0±6.0 b 40.0±6.1 a 17.0±3.5 ef 285.0±6.4 bc 410.0±28.8 c 3500±179.4 c 7.1±0.56 a
a Cowpea seedlings with six compound leaves were sprayed with salicylic acid at a 10 mM concentration. Each plant was
inoculated with 1400±100 second-stage juveniles of  M. incognita 24 h after the first treatment. Data are means of ten replicates.
UU, uninoculated untreated; IU, inoculated untreated; IT, inoculated and treated with Tween; USA, uninoculated and treated
with salicylic acid; USAT,  uninoculated and treated with salicylic acid containing Tween; ISA, inoculated and treated with sali-
cylic acid;  ISAT, inoculated and treated with salicylic acid containing Tween.
b Different small letters in a column indicate significant differences according to the least significant difference test (P=0.05).
-, no root galls or eggs in this group.
42 Phytopathologia Mediterranea
B. Nandi et al.
Fig. 2. Immunoblot analysis of proteins extracted from cowpea plants. Acidic PR proteins were extracted from
leaves on day 2 after the 2nd treatment. Proteins were fractionated by SDS-PAGE, and immunoblotted with mouse
monoclonal antibody MAb 2-11G5 against tobacco acidic PR-1 protein. lane a, uninoculated and treated with salicyl-
ic acid; lane b, uninoculated and treated with salicylic acid containing Tween; lane c, inoculated and treated with
salicylic acid  containing Tween; lane d, inoculated and treated with salicylic acid; lane e, inoculated and treated
with Tween; lane f, inoculated untreated; lane g, uninoculated untreated; lane h, molecular weight marker.
Fig. 1. SDS-PAGE of proteins extracted from cowpea plants. Acidic PR proteins were extracted from leaves on day 2
after the 2nd treatment. Separated proteins were stained with Coomassie Blue. Lane a, inoculated and treated with
salicylic acid containing Tween; lane b, inoculated and treated with salicylic acid; lane c, uninoculated and treated
with salicylic acid containing Tween; lane d, uninoculated and treated with salicylic acid; lane e, inoculated and
treated with Tween; lane f, inoculated untreated; lane g, uninoculated untreated; lane h, molecular weight marker.
The induced band is indicated with a dot to the right of the respective lanes.
a b c d e f g h
KDa
97
66
45
31
21
1416 KDa
a b c d e f g h
KDa
97
66
45
31
21
1416 KDa
43Vol. 41, No. 1, April 2002
Induced resistance in cowpea
sion of PR proteins (Hammond-Kosack et al., 1989),
while infection of potato roots with Globodera pal-
lida induced the expression of proteins with a mo-
lecular weight of 70–82 KDa, larger than that of
common PR proteins (Rahimi et al., 1993). In to-
mato root infected with root-knot nematodes, sev-
eral homologous plant-defense genes (including
peroxidases, chitinase, lipoxygenase and protein-
ase inhibitor protein) are expressed locally with-
in 24 h of inoculation (Lambert, 1995; William-
son and Hussey, 1996). In our experiments, spray-
ing with SA, which induced the accumulation of
PR-1 protein in the sprayed leaves, significantly
increased resistance to root-knot nematode infec-
tion, reflected in a lack of nematode reproduction.
It appeared that SA-spraying did not have any
direct influence on plant growth but improved
growth in the sprayed plants, perhaps as a result
of the reduction in root-knot disease it broughta-
bout. The mechanisms governing SA-induced re-
sistance to M. incognita in cowpea are under fur-
ther investigation.
Acknowledgements
The work described here is part of a project fund-
ed by the Council of Scientific and Industrial Re-
search, New Delhi to SPSB. We thank Prof. Dan-
iel F. Klessig, Waksman Institute and Department
of Molecular Biology and Biochemistry, Rutgers,
The State University of New Jersey, USA, for pro-
viding us with mouse monoclonal antibody MAb 2-
11G5.
Literature cited
Carr J.P. and D.F. Klessig, 1989. The pathogenesis-related
proteins of plants. In: Genetic Engineering: Principles
and Methods (J.K. Setlow, ed.) Plenum Press, New York,
NY, USA, 65–109.
Conrath U., Z.X. Chen, J.R. Ricigliano and D.F. Klessig,
1995. Two inducers of plant defense responses, 2,6-
dichloroisonicotinic acid and salicylic acid, inhibit cat-
alase activity in tobacco. Proceedings of the National
Academy of Sciences, USA 92, 7143–7147.
Cordero M.J., D. Raventos and B. San Segund, 1992. In-
duction of PR proteins in germinating maize seeds in-
fected with the fungus Fusarium moniliforme. Physio-
logical and Molecular Plant Pathology 41, 189–200.
Durner J., J. Shah and D.F. Klessig, 1997. Salicylic acid
and disease resistance in plants. Trends in Plant Sci-
ence 2, 266–274.
Greenberg J.T., A.L. Guo, D.F. Klessig and F.M. Ausubel,
1994. Programmed cell death in plants: a pathogen-trig-
gered response activated coordinately with multiple
defense functions. Cell 77, 551–563.
Hammond-Kosack K.E., H.J. Atkinson and D.J. Bowles,
1989. Systemic accumulation of novel proteins in the
apoplast of the leaves of potato plants following root
invasion by the cyst-nematode Globodera rostochiensis.
Physiological and Molecular Plant Pathology 35, 495–
506.
Hussey R.S. and K.R. Barker, 1973. A comparison of meth-
ods of collecting inocula of Meloidogyne spp., includ-
ing a new technique. Plant Disease Reporter 57, 1025–
1028.
Klessig D.F. and J. Malamy, 1994. The salicylic acid signal
in plants. Plant Molecular Biology 26, 1439–1458.
Laemmli U.K., 1970. Cleavage of structural proteins dur-
ing the assembly of the head of the bacteriophage T4.
Nature 277, 680–685.
Lambert K.N., 1995. Isolation of genes induced early in the
resistance response to Meloidogyne javanica in Lyco-
persicon esculentum. PhD Thesis, University of Cali-
fornia-Davis, Davis, CA, USA.
Lowry O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall,
1951. Protein measurement with Folin phenol reagent.
Journal of Biological Chemistry 193, 265–275.
Mauch-Mani B. and J.P. Metraux, 1998. Salicylic acid and
systemic acquired resistance to pathogen attack. An-
nals of Botany 82, 535–540.
Nasser W., M. Tapia and G. Burkard, 1990. Maize patho-
genesis–related proteins: Characterization and cellu-
lar distribution of 1,3-β-glucanases and chitinases in-
duced by brome mosaic virus infection or mercuric chlo-
ride treatment. Physiological and Molecular Plant Pa-
thology 36, 1–14.
Ohashi Y. and M. Ohshima, 1992. Stress-induced expres-
sion of genes for pathogenesis-related proteins in plants.
Plant Cell Physiology 33, 819–826.
Pennazio S., P. Roggero and R. Lenzi, 1983. Some charac-
teristics of the hypersensitive reaction of  ‘White Bur-
ley’ tobacco to tobacco necrosis virus. Physiological Plant
Pathology 22, 347–355.
Rahimi S., R.N. Perry and D.J. Wright, 1993. Induction
and detection of pathogenesis-related proteins in leaves
and roots of potato plants infected with pathotypes of
Globodera pallida. Fundamental and Applied Nema-
tology 16, 549–556.
Rahimi S., R.N. Perry and D.J. Wright, 1996. Identifica-
tion of pathogenesis-related proteins induced in leaves
of potato plants infected with potato cyst nematodes,
Globodera species. Physiological and Molecular Plant
Pathology 49, 49–59.
Roggero P. and S. Pennazio, 1988. Effects of salicylate on
systemic invasion of tobacco plants by various viruses.
Journal of Phytopathology 123, 207–216.
Roggero P. and S. Pennazio, 1991. Salicylate does not in-
duce resistance to plant viruses, or stimulate pathogen-
esis-related protein production in soybean. Microbio-
logica 14, 65–69.
44 Phytopathologia Mediterranea
B. Nandi et al.
Ross A.F., 1961. Systemic acquired resistance induced by
localized virus infections in plants. Virology 14, 340–
358.
Ryals J., S. Uknes and E. Ward, 1994. Systemic acquired
resistance. Plant Physiology 104, 1109–1112.
Schneider M., P. Schweizer, P. Meuwly and J.P. Metraux,
1996. Systemic acquired resistance in plants. Interna-
tional Journal of Cytology 168, 303–340.
Vernooij B., L. Friedrich, P. Ahl-Goy, T. Staub, H. Kessmann
and J. Ryals, 1995. 2,6-Dichloroisonicotinic acid-induced
resistance to pathogens without the accumulation of
salicylic acid. Molecular Plant-Microbe Interaction 8,
228–234.
Ward E.R., S.J. Uknes, S.C. Williams, S.S. Dincher, D.L.
Wiederhold, D.C. Alexander, P. Ahl-Goy, J.P. Metraux
and J.A. Ryals, 1991. Coordinate gene activity in re-
sponse to agents that induce systemic acquired resist-
ance. Plant Cell 3, 1085–1094.
White R.F., E.P. Rybicki, M.B. Von Wechmar, J.L. Dekker
and J.F. Antoniw, 1989. Detection of PR1-type proteins
in Amaranthaceae, Chenopodiaceae, Graminae, and
Solanaceae by immuno-blotting. Journal of General
Virology 68, 2043–2048.
Williamson V.M. and R.S. Hussey, 1996. Nematode patho-
genesis and resistance in plants. The Plant Cell 8,
1735–1745.
Accepted for publication: February 4, 2002


Salicylic acid enhances resistance in cowpea against Meloidogyne incognita

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

Firenze University Press: E-Journals