Biochemical alterations in grape infected with three

phytonematode species with emphasis on root-knot

nematode control.

Hosny H. KesbaZoology and Agricultural Nematology Department, Faculty of

Agriculture, Cairo University, Giza 12613, Egypt.

Abstract

Grape rootstocks, Flame seedless, Flame

seedless/Freedom and Freedom were reacted differently

to Meloidogyne incognita, Rotylenchylus reniformis and Tylenchulus

semipenetrans according to rootstock progenitor. Freedom

reduced significantly the nematode criteria and build

up. Humic and fulvic acids were tested at the rate of

2 and 4 ml/pot against the root-knot nematode. On

Freedom, all treatments reduced significantly

nematode build up and the higher dose was more

effective than the lower one. As a result of humic

and fulvic applications, the lipid peroxidation (MDA)

and H2O2 contents were significantly reduced after

treatments while the antioxidant compounds

glutathione (GSH) and ascorbic acid (AsA) contents

significantly increased when compared with check.

Antioxidant defense enzymes (ascorbate peroxidase

(APX), superoxide dismutase (SOD) and catalase (CAT))

showed significant increase in their activities.

Total phenol content and polyphenol oxidase (PPO)

were improved significantly in treated plants

compared to inoculated untreated check.

Keywords- Meloidogyne incognita, Rotylenchulus reniformis,

Tylenchulus semipenetrans, grape, humic acid, fulvic

acid, non enzymatic antioxidants, Antioxidant

enzymes.

Introduction

Plant resistance to nematode infection is one of

the most important agricultural measures in nematode

management strategies especially in perennial crops.

Under such circumstances, perennials are subjected to

invasion with progressive numbers of nematodes due to

the unbroken build up notably when environmental

conditions are in favor to nematode activities.

Accordingly, cultivation of resistant rootstocks or

inducing resistance in growing plants is a smart

choice to control nematode populations. On grapes,

the root-knot and the reniformis nematodes are highly

distructive to most grape varieties, however, they

are found to be poor hosts for the citrus nematode

(Kesba, 2003).

Resistant grape varieties or rootstocks to

different plant parasitic species had been studied by

many researchers (Walker et al., 1994; Walker, 1997;

Al-Sayed et al., 1999 & 2005; Anwar & McKenry, 2000 &

2001; McKenry et al., 2001).

Humic and fulvic acids are among the organic

acids that induce plant resistance to nematode

infection (Al-Sayed et al., 1988; Nandi et al., 2000;

Daneel et al., 2000; Zhang & Schmidt, 2000; Zaki et al.,

2004; Kesba & Al-Shalaby, 2008). They enable plants

to produce different compounds that improve the

physiological defense mechanism in infected plants e.g.

enzymatic and non-enzymatic compounds against

parasitic nematodes (Kesba & El-Beltagi, 2008), H2O2

(Waetzig et al., 1999; Peltzer et al., 2002), jasmonic

acids (Reymond & Farmer, 1998), reactive oxygen

(Neill et al., 2002; Montes et al., 2004; Murgia et al.,

2004), enzymatic and non-enzymatic ascorbate (Asada &

Takahashi, 1987; Asada, 1992; Jespersen et al., 1997;

Yoshimura et al., 2004), peroxidase and polyphenol

oxidase (Vaughn & Duke, 1984; Sarowar et al., 2005),

antioxidant (Halliwell, 1996; Reed, 1995; Decker,

1997), polyphenols (Buffle, 1988; Stackhouse &

Benson, 1988; Laughton et al., 1989; Metodiewa et al.,

1999).

Therefore, the objectives of this study were to:

(1) study the relationship between rootstock and

scion against three phytonematode genera and its

biochemical alterations. (2) evaluate the effect of

humic and fulvic acids on root-knot nematode, M.

incognita control and there role in enhancing plant

resistance against nematode infection.

Materials and Methods

Nematode species stock cultures

Pure cultures of the root-knot nematode, M.

incognita, the reniform nematode, R. reniformis and the

citrus nematode, T. semipenetrans were obtained from

isolates belonging to the Nematology Research Center,

Faculty of Agriculture, Cairo University. Nematode

species have been propagated separately, (M. incognita on

eggplant cv. Classic), (R. reniformis on pigeon pea) and

(T. semipenetrans on sour orange) grown in 20 cm diameter

clay pots filled with sterilized loamy soil. To avoid

contamination, cultures of each species were arranged

separately, examined and periodically renewed in

order to ensure continuous supplies of inocula for

the experimental work.

Glasshouse experiments

One year old seedlings of grape rootstocks, Flame

seedless, Flame seedless/Freedom and Freedom with

uniform size were obtained from Grape Department,

Horticulture Research Institute, Agriculture Research

Center and cultivated singly in 20 cm diameter clay

pots filled with steam sterilized sandy loam soil

(1:1, v/v). One month after, 5 seedlings of each

rootstock were inoculated separately with 5000

infective stages of M. incognita, R. reniformis or T.

semipenetrans by pipetting the nematode water suspension

into 4 holes around the root system and immediately

covered with soil. Pots were labeled and arranged

randomly on a glasshouse clean bench, receiving

similar horticulture treatments. Seedlings were left

out for 4 months after inoculation. Soil populations

were extracted by means of (Hooper et al., 2005) and

counted. The nematode embedded stages were also

counted.

For testing the effect of humic and fulvic acids

(extracting and preparation at By-product Research

Department, Agricultural Research Center, Ministry of

Agriculture) on the root-knot nematode development

and reproduction, another 5 seedlings of each

rootstock were inoculated with 5000 J2 of M.

incognita/pot. Two weeks after inoculation, two

commercial products of humic and fulvic acids were

applied at the rate of (2, 4 ml/plant) as soil

drench. All treatments were arranged in a fully

randomized design on a clean bench in the glasshouse

at 32 ±5 oC receiving similar horticultural

treatments. After 4 months, nematode soil populations

were extracted and counted using a Hawksley counting

slide, under a binocular microscope. A subsample (5

g) of roots from each plant was stained and gall

numbers, embedded stages (developmental stages +

eggmasses) per root were calculated, final population

(embedded stages + nematodes in soil), nematode build

up (Pf/Pi), average of eggs/eggmass were estimated.

Plant chemical analysis

Subsamples of fresh root of each treatment were

chemically analyzed at Central Chemistry Lab.,

Faculty of Agriculture Research Park (FARP), Faculty

of Agriculture, Cairo University as follows:

Determination of non-enzematic antioxidants

(oxidative burst)

Assay of lipid peroxidation (MDA) according to

Hodges et al., (1999), hydrogen peroxide (H2O2) by the

method described by Capaldi and Taylor (1983), total

glutathione (GSH) according to De Vos et al., (1992),

total ascorbic acid (TAA) according to Abdulnabi et

al., (1997), total phenols by the method of Zieslin

and Ben-Zaken (1993).

Determination of antioxidant defense enzymes activity

Assay of superoxide dismutase (SOD) using the

method of Beauchamp and Fridovich (1971), ascorbate

peroxidase (APX) using the method of (Nakano & Asada,

1981), catalase (CAT) using the method of Dhindsa et

al. (1981), polyphenol oxidase (PPO) according to

Coseteng and Lee (1987).

Statistical analysis

Data were compared by Duncan's Multiple Range Test

(DMRT) at the 5% level of probability using MSTAT

version 4 (1987).

Results

Development and reproduction of the root-knot, the

reniform and the citrus nematodes on grape rootstocks

Data in (Table 1) revealed that both the root-

knot nematode, M. incognita and the reniform nematode, R.

reniformis reproduced normally on the three rootstocks

with significant differences in their susceptibility.

Flame seedless was the most susceptible, followed by

Flame seedless/Freedom but Freedom showed some

resistance to both nematodes as measured by numbers

of galls, embedded stages, final population and the

rates of build up and egg deposition. The three

rootstocks are not good hosts to the citrus nematode,

T. semipenetrans on which the nematode folded hardly. It

is noticeable that nematode population declined on

Freedom. In all cases, grafting Flame seedless on

Freedom rootstock resulted in reducing the final

population of the root-knot nematode by 20%, the

reniform nematode by 57% and the citrus nematode by

31% as compared to Flame seedless.

Effect of humic and fulvic acids on the development

and reproduction of M. incognita on grape

On Flame seedless, the susceptible rootstock,

both acids at the two concentrations significantly

reduced the number of galls and the embedded stages

on the infected roots as compared with the check. The

higher dose (4ml/pot) was significantly efficient

than the lower one. Similar results were observed

with the final population and the rate of nematode

build up as well as the number of eggs/eggmass.

Fulvic acid was significantly better than humic acid

in suppressing the root-knot nematode reproduction on

the susceptible rootstock (Table 2).

On Freedom, the less susceptible rootstock, an

opposite results were obtained whereas the

application of organic acids significantly increased

root galling and the number of embedded stages.

However, they reduced the final population, the rates

of build up and egg deposition. Also, significant

differences were observed between lower and higher

doses.

On Flame seedless/Freedom rootstock, the

increases in numbers of galls and the embedded stages

varied significantly but not drastically with the

check. Also, the reductions in the final population

and the rates of build up and egg deposition were not

satisfactory. No significant variations could be

observed between concentrations in the majority of

cases.

Results in (Table 3) manifested that single

infection of grape rootstocks with the root-knot, the

reniform and citrus nematodes resulted in increasing

root contents of both lipid peroxidation (MDA) and

H2O2 with significant differences within nematode

genera and between them and the uninoculated healthy

roots. The highest rates of increment were

implemented by M. incognita in all rootstocks and by all

nematode genera on Freedom.

Nematode infection also boosted the contents of

enzymatic antioxidant, total glutathione (GSH), total

ascorbic acid (TAA) and total phenols. It is clear

that the highest rates of enhancement of enzymatic

antioxidant were observed in roots infected with the

reniform nematode followed by those infected with the

citrus nematode, however, the rates of increase in

the roots infected with the root-knot nematode ranked

statistically in the third group. The utmost increase

rates were observed in roots of Freedom.

Concerning the influence of organic acids on the

contents of roots of lipid peroxidation, results

showed that both humic and fulvic acids significantly

reduced MDA and H2O2 in grape roots infected with M.

incognita as compared to non treated infected roots

(Table 4). With higher concentration, the greater

reduction in both MDA and H2O2 were observed with

humic acid which is efficient than fulvic acid with

Flame seedless and Flame seedless/Freedom rootstocks

and vice versa with Freedom. Both acids succeeded to

reduce the contents of lipid peroxidation on Freedom

to be almost near that in healthy plants.

All treatments significantly improved the levels

of non-enzymatic antioxidant molecules in the roots

of infected rootstocks especially at the higher

concentrations. Humic acid also achieved the best

results in the majority of cases. Also, all

treatments increased the levels of antioxidant

enzymes. Higher levels of the antioxidant enzymes

were recorded in roots of plants treated with the

higher dose. The highest significant increase in APX

was observed in roots of Flame seedless/Freedom

treated with humic acid at the high dose. With SOD

the highest significant increase was achieved by the

high dose of humic acid in roots of Freedom. However,

the highest significant increase in CAT and PPO was

accomplished by fulvic acid (the high dose) in roots

of Flame seedless and Flame seedless/Freedom

rootstocks, respectively.

Discussion

The present results proved that the nematode

species M. incognita, R. reniformis and T. semipenetrans

developed and reproduced variably on the tested

rootstocks. Flame seedless was the most susceptible

followed by Flame seedless/Freedom but Freedom showed

some resistance. Variability in the development and

reproduction of different nematode species on

different grape rootstocks had been documented by

many researcher workers (Saur, 1977; Walker et al.,

1994; Mckenry et al., 2001; Al-Sayed et al., 2005).

Both humic and fulvic acids significantly reduced

the numbers of the root-knot nematode in soil and on

roots of the susceptible rootstock, Flame seedless

and fulvic acid was more suppressive than humic. The

role of organic acids in suppressing the plant

parasitic nematodes populations was reported by Al-

Sayed et al., 1988; Al-Sayed et al., 2007 and Kesba and

El-Beltagi, 2008.

An opposite results were obtained in case of the

less susceptible rootstock, Freedom whereas treatment

with organic acids increased root galling and root

population, but suppressed soil and final population.

On Flame seedless/Freedom rootstock both acids

resulted in moderate increase in root populations,

and unsatisfactory reduction in the final population.

It seems that humic and fulvic acids somehow affect

the nematode fecundity and showed potential toxic

action against the free living stages in the soil.

The increase of proteins and fatty acids in root

tissues as a result of treating plants with organic

acids may enhance some biochemical compounds able to

retard nematode reproduction (Al-Sayed et al., 2005;

Kesba & Al-Shalaby, 2008).

Incompatible resistant interactions between

plant and pathogen are often determined by the

formation of reactive oxygen species (ROS) by the

pathogen (Baker & Orlandi, 1995; Montes et al., 2004).

Reactive oxygen species (ROS), such as hydrogen

peroxide (H2O2), is some of the most damaging

stressors in plants. Thus, H2O2 from the oxidative

stress plays a key role in the orchestration of a

localized hypersensitive response during the

expression of plant disease resistance (Levine et al.,

1994). ROS induced lipid peroxidation may be one of

the mechanisms accounting for cell death (Jabs,

1999). The results agreed with those facts and showed

that, with comparison to healthy plants of grape

rootstocks (Flame seedless, Flame seedless/Freedom

and Freedom) infection with (M. incognita or R. reniforms)

enhanced the content of MDA and H2O2. The results also

indicated that plants possess both enzymatic and non-

enzymatic antioxidant defense systems to counteract

ROS under nematode infections. The significant

increase of non-enzymatic antioxidant such as (GSH,

TAA and TPH) contents may be driven by enhancement of

MDA and H2O2 formation in the nematode infections.

However, GSH may play a protective role in scavenging

of singlet oxygen, peroxides and hydroxyl radicals

and is involved in recycling reduction of ascorbic

acid (AsA) in the ascorbate-glutathione cycle (Foyer,

1993). On the other hand, the significant increase in

the total soluble phenols (TPH) contents could be

affected as strong antioxidant natural products

induced under oxidative stress condition controlling

the oxidative damage. These data was in accordance

with (Goodman et al., 1967), who found that, multifold

increase of phenols after challenging with

elicitation may be due to the excess production of

H2O2 in elicited plant cells through increased

respiration (Farkas & Kiraly, 1962) or due to the

activation of hexose-monophosphate pathway, acetate

pathway and release of bound phenols by hydrolytic

enzymes. In addition, the nematode infection caused

marked increase in the activity of antioxidant

enzymes (APX, SOD, and CAT) which are involved in

scavenging excess ROS in plant cells (Yoshimura et al.,

2004). Catalase and Ascorbate peroxidase play an

essential role in scavenging from the H2O2 toxicity.

The combined action of CAT and SOD converts the toxic

superoxide radical (O2.−) and hydrogen peroxide (H2O2)

to water and molecular oxygen (O2), thus averting the

cellular damage under unfavorable conditions like

infection by nematodes (Asada, 1992; Zacheo & Bleve-

Zacheo, 1988). While the increase in polyphenol

oxidase (PPO) activity after nematodes infection may

be due to autooxidation of the total phenol substrate

which interact with H2O2 and may prevent nematode

spread to healthy tissue (Trudgill, 1991).

Treatments infected with M. incognita reduced the

contents of MDA and H2O2 in all rootstocks by

improving the contents of non-enzymatic antioxidants

(GSH, TAA and TPH) and increase the activities of PPO

and antioxidants enzymes (APX, SOD and CAT) and

reached its maximum induction at the higher dose (4

ml). In addition, our results indicated that the high

dose (4 ml) at Flame seedless/Freedom rootstock was

the best treatment that suitable plant treatments to

reduce the ROS, lipid peroxidation formation and

improve the induction of antioxidant defense system.

It could be conclude that humic and fulvic acids

treatments may play an important role in antioxidant

defense system of plants, it is supposed that low

concentration might be a signal to induce the

expression of many antioxidative molecules and

enzymes and reduce the ROS in plant cells (Karasyova

et al., 2007).

ReferencesAbdulnabi, A.A., Emhemed, A.H., Hussein, G.D. and Biacs, P.A.

(1997). Determination of antioxidant vitamins in tomato.Food Chemistry, 60: 207-212.

Al-Sayed, A.A., Ahmed, S.S. and Montasser, S.A. (1988). Effects of some organic acids on egg hatchability of Meloidogyne incognita and Rotylenchulus reniformis. Annals of Agricultural Science, Moshtohor, Benha University, 26: 1325-1332.

Al-Sayed, A.A., Kheir, A.M., El-Naggar, H.I. and Kesba, H.H.(1999). Varietal reaction of some grape Rootstocks tothe infection with three plant parasitic nematodespecies. Journal of Agricultural Sciences, MansouraUniversity, 24: 7707-7718.

Al-Sayed, A.A., Kheir, A.M., El-Naggar, H.I. and Kesba, H.H. (2007). Organic management of Meloidogyne incognita on grapesin relation to host biochemistry. International Journal of Agricultural Research, 2: 776-785.

Al-Sayed, A.A., Kheir, A.M., El-Naggar, H.I. and Kesba, H.H. (2005). Could other Vitis species be helpful in nematode management in Egypt's sand soil viticultures? Bulletin of Faculty of Agriculture, Cairo University, 56: 393-406.

Anwar, S.A. and Mckenry, M.V. (2000). Penetration, developmentand reproduction of Meloidogyne arenaria on two new resistantVitis spp. Nematropica, 30: 9-17.

Anwar, S.A. and Mckenry, M.V. (2001). Susceptible ofgenetically transformed Freedom grape rootstocks toMeloidogyne arenaria Pt. Freedom. International Journal ofNematology, 11: 1-7.

Asada, K. (1992). Ascorbate peroxidase: a hydrogen peroxide-scavenging enzyme in plants. Physiologia Plantarum, 85: 235–241.

Asada, K. and Takahashi, M. (1987) Production and scavenging of active oxygen in photosynthesis. In: D. J. Kyle, C. B. Osmond and C. J. Arntzen (Eds.). Photoinhibition. Elsevier Science Publishers, Amsterdam, pp. 227-287.

Baker, C.J. and Orlandi, E.W. (1995). Active oxygen in plant pathogenesis. Annual Review of Phytopatholology, 33: 299–321.

Beauchamp, C. and Fridovich, I. (1971). Superoxide dismutase: improved assays and assay applicable to acrylamide gels.Analytcial Biochemistry, 44: 276–287.

Buffle, J. (1988). Complexation reactions in aquatic systems. Ellis Horwood Series, In Analytical Chemistry. Ellis Horwood Limited, Chichester, England.

Capaldi, D.J. and Taylor, K.E. (1983). A new peroxidase color reaction: oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with its formaldehydeazine application to glucose and choline oxidases. Analytcial Biochemistry, 129: 329–336.

Coseteng, M.Y. and Lee, C.Y. (1987). Change in apple polyphenol oxidase and polyphenol concentrations in relation to degree of browning. Journal of Food Science,52: 985-989.

Daneel, M.S., De Jager, K., Dreyer, S., Dekker, J. and Joubert, J.P. (2000). The influence of oxihumate on nematode control and on yield (Musa AAA, Cavendish subgroup). Acta Horticulture, 540: 441-452.

De Vos, C.H., Vonk, M.J., Vooijs, R. and Henk, S. (1992). Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucbalus. Plant Physiology, 98: 859-858.

Decker, E.A. (1997). Phenolics: prooxidants or antioxidants? Nutrition Review, 55: 396–398.

Dhindsa, R.S., Plumb-Dhindsa, P. and Thorpe, T.A. (1981). Leafsenescence: correlated with increased levels of membranepermeability and lipid peroxidation, and decrease levelsof superoxide dismutase and catalase. Journal of Experimental Botany, 32: 93–101.

Farkas, G. L. and Kiraly, Z. (1962). Role of phenolic compounds in the physiology of plant disease resistance.Phytopathology, 44: 105–150.

Foyer, C.H. (1993). Ascorbic acid. In: Alscher, R.G. and Hess, J.L. (Eds.). Antioxidants in higher plants. CRC press, Florida, USA. pp: 31-58.

Goodman, R. N., Kiraly, E. and Zaitlin, M. (1967). The biochemistry and physiology of infections in plant diseases. D.Van Nostri and Co., Princeton, N.J. , Inc. p. 534

Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16: 33–50.

Hodges, D.M., DeLong, J.M., Forney, C.F. and Prange, R.K. (1999). Improving the thiobarbituric acid-reactive-

substances assay for estimating lipid per-oxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 207: 604–611.

Hooper, D.J., Hallmann, J. and Subbotin, S.A. (2005). Methods for extraction, processing and detection of plant and soil nematodes. In: Luc, M., Sikora, R.A. and Bridge, J.(Eds). Plant parasitic nematodes in subtropical and tropical agriculture. Wallingford, UK, CABI Publishing, pp. 53-86.

Jabs, T. (1999). Reactive oxygen intermediates as mediators ofprogrammed cell death in plants and animals. BiochemicalPharmacology, 57: 231-245.

Jespersen, H.M., Kjaersgard, I.V.H., Ostergaard, L. and Welinder, K.G. (1997). From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase. Biochemical Journal, 326: 305–310.

Karasyova, T.A., Klose, E.O., Menzel, R. and Steinberg, C.E. (2007). Natural organic matter differently modulates growth of two closely related coccal green algal species. Environmental Science and Pollution Research – International, 14: 88-93.

Kesba, H.H. (2003). Integrated nematode management on grapes grown in sandiness soil. PhD Thesis, Faculty of Agriculture, Cairo University, 189 pp.

Kesba, H.H. and Al-Shalaby, Mona E.M. (2008). Survival and reproduction of Meloidogyne incognita on tomato as affected by humic acid. Nematology, 10: 243 – 249.

Kesba, H.H. and El-Beltagi, H.E.S. (2008). Biochemical changesin grape rootstocks resulted from humic acid treatments in relation to nematode infection. Egyptian Journal of Applied Sciences, Zagazig University, 23: 277-292.

Laughton, M.J., Halliwell, B., Evans, P.J. and Hoult, J.R.S. (1989). Antioxidant and prooxidant actions of the plant phenolics quercetin, gossypol and myricetin. BiochemicalPharmacology, 38: 2859–2865.

Levine, A., Tenhaken, R., Dixon, R. and Lamb, C. (1994). H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell, 79: 583-593.

Mckenry, M.V., Kretsch, J.O. and Anwar, S.A. (2001).Interactions of selected Vitis Rootstocks with endo-parasitic nematodes. American Journal of Enology andViticulture, 52: 310-316.

Metodiewa, D., Jaiswal, A.K., Cenas, N., Dickancait´e, E. and Segura-Aguilar, J. (1999). Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radical Biology and Medicine, 26: 107–116.

Montes, M.J., López-Braña, I. and Delibes, A. (2004). Root enzyme activities associated with resistance to Heteroderaavenae conferred by gene Cre7 in a wheat/ Aegilops triuncialis introgression line. Journal of Plant Physiolology, 161: 493–495.

MSTAT Version 4 (1987). Software program for the design and analysis of agronomic research experiments. Michigan, USA, Michigan State University.

Murgia, I., Tarantino, D., Vannini, C., Bracale, M., Carravieri, S. and Soave, C. (2004). Arabidopsis thaliana plants overexpressing thylakoidal ascorbate peroxidase show increased resistance to paraquat-induced photooxidative stress and to nitric oxide-induced cell death. The Plant Journal, 38: 940–953.

Nakano, Y. and Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 22: 867–880.

Nandi, B., Sukul, N.C. & Babu, S.P.S. (2000). Exogenous salicylic acid reduces Meloidogyne incognita infestation of tomato. Allelopathy Journal, 7: 285-288.

Neill, S., Desikan, R. and Hancock, J. (2002). Hydrogenperoxide signaling. Current Opinion in Plant Biology, 5:388–395.

Peltzer, D., Dreyer, E. and Polle, A. (2002). Differential temperature dependencies of antioxidative enzymes in twocontrasting species: Fagus sylvatica and Coleus blumei. Plant Physiology and Biochemistry, 40: 141–150.

Reed, J.D. (1995). Nutritional toxicology of tannins and related polyphenols in forage legumes. Journal of AnimalScience, 73: 1516–1528.

Reymond, P. and Farmer, E.E. (1998). Jasmonate and salicyclateas global signals for defense gene expression. Current Opinion in Plant Biology, 1: 404–411.

Sarowar S., Kim E.N., Young, J.K., Sung, H.O., Ki, D.K., Byung, K.H. and Jeong, S. (2005). Overexpression of a pepper ascorbate peroxidase-like1 gene in tobacco plantsenhances tolerance to oxidative stress and pathogens. Plant Science, 169: 55–63.

Saur, M.R. (1977). Nematode resistant grape rootstocks.Australian Dried Fruits News, 5: 10-14.

Stackhouse, R.A. and Benson, W.H. (1988). The influence of humic acid on the toxicity and bioavailability of selected trace metals. Aquatic Toxicology, 13: 99–108.

Trudgill, D.L. (1991). Resistance to and tolerance of plant parasitic nematodes in plants. Annual Review of Phytopathology, 29: 167–192.

Vaughn, K.C. and Duke, S.O. (1984). Function of polyphenol oxidase in higher plants. Physiologia Plantarum, 60: 106–112.

Waetzig, G.H., Sobczak, M. and Grundler, F.M.W. (1999). Localization of hydrogen peroxide during the defence response of Arabidopsis thaliana against the plant parasitic nematode Heterodera glycines. Nematology, 1: 681– 686.

Walker, G.E. (1997). Effects of Meloidogyne spp and Rhizoctoniasolani on the growth of grapevine rootlings. Journal ofNematology, 29: 1-9.

Walker, M.A., Ferris, H. and Eyre, M. (1994). Resistance inVitis and Muscadinia species to Meloidogyne incognita. PlantDisease, 78: 1055-1058.

Yoshimura, K., Miyao, K., Gaber, A., Takeda, T., Kanaboshi, H., Miyasaka, H. and Shigeoka, S. (2004). Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol. Plant Journal, 37: 21–33.

Zacheo, G. and Bleve-Zacheo, T. (1988). Involvement of superoxide dismutases and superoxide radicals in the susceptibility and resistance of tomato plants to Meloidogyne incognita attack. Physiological and Molecular Plant Pathology,  32: 313–322.

Zaki, M.J., Javad, S., Abid, M., Khan, H. and Moinuddin, M. (2004). Evaluation of some chemicals against root-knot nematode, Meloidogyne incognita. International Journal of Biology and Biotechnology, 1: 613-618.

Zhang, X. and Schmidt, R.E. (2000). Hormone-containing products impact on antioxidant status of tall fescue and creeping bentgrass subjected to drought. Crop Science, 40: 1344-1349.

Zieslin, N. and Ben-Zaken, R. (1993). Peroxidase activity and presence of phenolic substances in peduncles of rose flowers. Plant Physiology and Biochemistry, 31: 333–340.

Table (1): Reproductivity of M. incognita, R. reniformis and T. semipenetrans on grape rootstocks.

Rootstocks Galls

Embedded

stages

Finalpopulation

Pf/PiEggs/Eggmas

sM. incognita

Flameseedless 1865 a 1958 a 13488 2.70

a 187 a

Flameseedless

/Freedom

1062 b 1115 b 11215 2.24b 161 b

Freedom 472 c 496 c 7526 1.51c 154 b

R. reniformisFlame

seedless - 1220 a 16830 3.37a 130 a

Flameseedless

/Freedom

- 743 b 10713 2.14b 100 b

Freedom - 429 c 6984 1.40c 65 c

T. semipenetransFlame

seedless - 217 a 5912 1.18a 66 a

Flameseedless

/Freedom

- 197 a 4502 0.90b 47 b

Freedom - 186 a 4056 0.81b 26 c

Means followed by the same letter(s) within a column in each block are not significantly different (P ≤ 0.05) according to

Duncans׳ multiple range test. Final population= embedded stages + soil population.

Table (2): Reproductivity of M. incognita on grape rootstocks as influenced by addition of humic and fulvic acids.

Rootstocks

Treatments

Dose(ml/pot)

Galls Embeddedstages

Finalpopulatio

nPf/Pi Eggs/

Eggmass

Flame

seedle

ss

Humicacid

2 1763 b 1850 b 12740 2.55b 176 d

4 1537 c 1583 c 11083 2.22c 155 ef

Fulvicacid

2 1319 d 1385 d 10265 2.05de 132 hi

41119ef 1175 e 8095 1.62

f 112 j

Inoculated only 1865 a 1958 a 13488 2.70a 187 cd

Fla

me see Humic

acid2 1134 e 1166 e 10436 2.09

d209 a

dles

s /

Freedo

m 41113efg 1149 ef 9969 1.99

de 201 ab

Fulvicacid

21092efg 1171 e 9771 1.95

e 193 bc

41071fg 1193 e 9573 1.92

e 185 cd

Inoculated only 1062 g 1115 f 11215 2.24c 161 e

Freedom

Humicacid

2 684 h 958 g 7288 1.46g 139 gh

4 590 i 767 h 6387 1.28h 123 ij

Fulvicacid

2 660 h 792 h 5712 1.14i 146 fg

4 566 i 623 i 5193 1.04i 116 j

Inoculated only 472 j 496 j 7526 1.51f 154 ef

Means followed by the same letter(s) within a column in each block are not

significantly different (P ≤ 0.05) according to Duncans׳ multiple range test. Final population= embedded stages + soil population.

Table (3): Effect of M. incognita, R. reniformis or T. semipenetrans on grape rootcontents of non enzymatic antioxidants (oxidative burst) and antioxidant defense enzymes.

Rootstocks

Nematodespecies

Non enzymatic antioxidants Antioxidant enzymesMDA

(μ mol/gFW)

H2O2(μ mol/g

FW)

GSH(μ mol/g

FW)

TAA(mg/g FW)

TPH(mg/g FW)

APX(unit/mgprotein)

SOD(unit/mgprotein)

CAT(unit/mgprotein)

PPO(unit/mgprotein)

Flame se

edless

M. incognita6.33bc

180.19c 5.61 h 9.18 cd 5.11 f

13.97hi

183.44h

46.47fg 9.70 i

R. reniformis 5.29 d125.93

i 9.67 c15.05

a 8.54 b31.52

b284.56

b 75.87 a44.80

a

T.semipenetrans 4.23 e

137.75g 6.88 f 7.90 d 6.14 d

20.47e

210.60e 55.05 d

24.51de

Healthy2.55fg

80.06k 2.81 j 3.64 e 1.18 j 8.02 j

121.24k 17.77 l 2.52 kl

Flame

seed

less /

Free

dom

M. incognita 7.88 a202.98

a 5.96 g 9.53 cd 4.28 h13.27

i177.88

i 42.24 h11.15

hi

R. reniformis6.17bcd

151.82e

10.54a

12.04bc 8.23 c

23.47c

243.88c

58.43cd

29.18c

T.semipenetrans

6.32bc

159.66d 7.43 d 9.71 cd 5.63 e

16.53f

189.79g

45.30gh

18.15f

Healthy 2.85 f85.81

j 3.11 i 2.61 e 1.21 j 7.22 k117.51

l 21.26 k 3.17 j

Free

dom M. incognita 6.60 b 197.40

b5.96 g 9.95 cd 4.85 g 14.26

g199.90

f38.16 i 13.19

gh

R. reniformis5.38cd

135.42h 9.98 b

13.94ab 8.95 a

35.42a

297.50a 69.28 b

38.03b

T.semipenetrans

5.40cd

149.77f 7.17 e

10.75cd 6.21 d

22.36d

223.83d 48.35 e

23.05e

Healthy 1.89 g85.60

j 2.67 j 3.06 e 1.31 i 7.25 k136.42

j 24.91 j 2.28 l

Means followed by the same letter(s) within a column in each block are not significantly different (P ≤

0.05) according to Duncans׳ multiple range test. MDA= lipid peroxidation, H2O2 = hydrogen peroxide, GSH= glutathione, TAA= total ascorbic acid, TPH= total phenols, APX= ascorbat peroxidase, SOD= superoxide dismutase, CAT= catalase, PPO= polyphenol oxidase.

Table (4): Effect of humic and fulvic acids on infected grape root contentsof non enzymatic antioxidants (oxidative burst) and antioxidant defense enzymes.

Rootstocks

Treatments

Dose(ml/pot)

Non enzymatic antioxidants Antioxidant enzymesMDA

(μ mol/gFW)

H2O2(μ mol/g

FW)

GSH(μ mol/g

FW)

TAA(mg/gFW)

TPH(mg/g FW)

APX(unit/mgprotein)

SOD(unit/mgprotein)

CAT(unit/mgprotein)

PPO(unit/mgprotein)

Flam

e seedless

Humicacid

2 4.22 d110.61

h12.21

d19.87

d 9.75 ef14.67

hi201.78

j41.82

ef15.52

h

4 3.77 f123.62

d16.00

a21.15

b10.38

cd34.93

e220.13

f 88.29 a43.65

a

Fulvicacid

2 4.00 e104.10

j11.79

de20.51

c10.06

de14.32

hij210.96

h39.50

fg16.01

h

4 3.55 g117.11

f15.16

b21.79

a10.69

c32.13

f293.50

c 90.62 a34.14

d

Inoculated only 6.33 c180.19

c 5.61 i9.18

j 5.11 i13.97

ij183.44

m 46.47 d 9.70 k

Healthy 2.55 k80.06

o 2.81 j3.64

k 1.18 k 8.02 k121.24

p 17.77 k 2.52 l

Flam

e seed

less

/ Fre

edom Humic

acid

2 3.28 h102.97

k12.22

d10.48

h 8.99 g14.60

hi204.56

i38.02

gh16.73

gh

42.99ij

94.39m 6.56 h

14.36e

13.27b

43.84a

222.35e 80.26 b

43.49a

Fulvicacid

23.14hi

107.26i

13.11c

10.01hi 9.42 fg

15.92g

195.67l 35.90 h

17.28fg

4 2.89 j98.68

l 7.85 g13.70

f13.70

ab38.48

d213.46

g 82.37 b44.04

a

Inoculated only 7.88 a202.98

a 5.96 hi 9.5 ij 4.28 j13.27

j177.88

n 42.24 e11.15

j

Healthy 2.85 j85.81

n 3.11 j2.61

l 1.21 k 7.22 k117.51

q 21.26 j 3.17 l

Freedom

Humicacid

22.27lm

111.28g

11.32ef

9.49ij 7.28 h

14.97ghi

219.89f

41.98ef

19.13e

42.08mn

98.44l 6.64 h

13.77f

13.58ab

41.35b

339.88a 72.50 c

38.25c

Fulvicacid

22.36kl

119.84e

10.73f

9.18j 7.52 h

15.26gh

229.89d

40.07efg

18.47ef

42.17lm

102.72k 6.05 hi

13.01g

14.07a

39.93c

329.88b 74.41 c

40.89b

Inoculated only 6.60 b197.40

b 5.96 hi9.95hi 4.85 ij

14.26hij

199.90k

38.16gh

13.19i

Healthy 1.89 n85.60

n 2.67 j3.06

l 1.31 k 7.25 k136.42

o 24.91 i 2.28 l

Means followed by the same letter(s) within a column in each block are not significantly different (P ≤

0.05) according to Duncans׳ multiple range test. MDA= lipid peroxidation, H2O2 = hydrogen peroxide, GSH= glutathione, TAA= total ascorbic acid, TPH= total phenols, APX= ascorbat peroxidase, SOD= superoxide dismutase, CAT= catalase, PPO= polyphenol oxidase.

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ور م�ن� ك�ل م�ن� ذ� وى ال�ج� أدة� م�حي, ي& ة� ل�ز� ص�اث�� اط TPH و TAA وGSHالا* س% أدة� ن�� ي& antioxidant enzymes ور�

.PPOو CAT وSOD و APXل�كل م�ن� ب% أت� ح�ن اي&� ب� اومة� ال�ن� ن� م�ق� حسي ي ت�� ك�5 دورًا ف� ب ول�ف� و ح�ام�ض� ال�ف� وم�ك� ا$ كل م�ن� ح�ام�ض� ال�هي وك�ان��ت� ل�لمعام�لة� ب��

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29