Arab J. Biotech., Vol. 15, No. (2) July (2012):

Induction of systemic acquired resistance in watermelon

against watermelon mosaic virus-2

(Received: 04.11. 2012; Accepted: 28. 11 .2012)

Barakat O. S. *; Goda H. A. *; Mahmoud S. M.** and Emara Kh. S.*** *Department of Agricultural Microbiology, Faculty of Agriculture, Cairo University, Giza, Egypt

**Department of Plant Pathology, Agricultural Researches Center, Giza, Egypt

***Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza, Egypt

ABSTRACT

The current study was carried out to evaluate the efficiency of nine bioagents to induce the

phenomenon of systemic acquired resistance (SAR) against watermelon mosaic virus-2 (WMV-2) in

watermelon plants. The biotic inducers included four bacterial strains; Bacillus megathirium,

Bacillus polymyxa, Zymomonas mobilis ATCC 31823 and Zymomonos mobilis ATCC 10988, two

fungal strains; Trichoderma harizianum and Mycorrhiza fasciculatum, and three plant extracts;

garlic, black cumin and camphor oils. The induction of SAR against WMV-2 was evidenced

morphologically, chemically and histologically. The most effective bioagent for induction of SAR

was Mycorrhiza fasciculatum, since the highest reduction of virus infection (RI) and lowest disease

severity (DS) representing 64.6% and 23.5%, respectively, were recorded in plants treated with this

bioagent. In this respect, also the highest levels of total salicylic acid (SA); 1046.79 µg/100g,

antiviral protein content; 1.328 mg/g FW and peroxidase (POD) activity; 0.931 mg/g FW, were

detected in these plants. On the other hand, garlic oil had the lowest effect with decreasing RI and

increment in DS, resulting in reduction of SAR in watermelon plants. Ultrastructrual investigations

using transmission electron microscope (TEM) confirmed that Mycorrhiza, the most effective

treatment, initiated histological barriers as hypersensitivity (field immunity), gummosis and

forming of phenolic compounds. These structural resistance mechanisms may lead to hindering

virus movement inside living tissues. The treatment also may stimulate cells of susceptible host to

make organelles that enable them to withstand virus replication exhausting process for longer time.

Keywords: Watermelon, WMV-2, Biotic inducers, SAR, Salicylic acid, Peroxidase, Histology, TEM.

INTRODUCTION

atermelon (Citrullus lanatus

Thunb.) is a worldwide

economically important member in

family Cucurbitaceae. Fruit flesh varied in

colour; red, orange, yellow or white, among its

1200 varieties. Watermelon fruit contains 60%

flesh, of which 90% is a juice that contains 7

to 10% w/v sugars. Thus, over 50% of the

watermelon fruit is readily fermentable liquid

(Wayne et al., 2009). Watermelon is a source

of many vitamins, especially vitamin C. Also,

fruits with red flesh are significant source of

lycopene used as antioxidant and anticancer

agent (Giovannucci et al., 2002) and to reduce

the risk of heart attack (Figueroa et al., 2010).

In the field, watermelon is vulnerable to

numerous diseases caused by bacteria, fungi

and viruses. These diseases may infect its

foliage, roots or fruit. The viral diseases are a

major problem in cucurbits planting areas all

W

Arab J. Biotech., Vol. 15, No. (2) July (2012):

over the world. At least 25 viruses have been

reported to infect cucurbit plants naturally.

Viruses in the Potyviruses genus (Potyviridae

family), of which strain 2 of watermelon

mosaic potyvirus (WMV-2), are the major

virus group infecting cucurbits (Sidek et al.,

1999). The watermelon mosaic potyvirus-2

infection causes severe losses in crop yield

(Murphy et al., 2003). Watermelon is a

diagnostic species by symptoms of leaf

mosaic, mottling and fruit distortion (Branka

et al., 2002). WMV-2 has a wider host range

rather than cucurbits covering over 160

dicotyledonous species in 23 families. It is

readily mechanically transmissible, by at least

38 species of aphid belonging to 19 genera, in

a non-persistent manner (Baum, 1980).

Interactions between plants and

pathogens can lead either to a successful

infection (compatible response) or resistance

(incompatible response). Plants can defend

themselves against pathogens by a variety of

mechanisms that can be either constituted or

inducible (Abdel-Monaim, 2012). The

constituted mechanisms include an oxidative

burst (Kombrink and Schmelzer, 2001),

changes in cell wall composition that can

inhibit the penetration of pathogen, synthesis

of antimicrobial compounds (van Loon, 1997

and Hammerschmidt, 1999), hypersensitive

response (Goodmann and Novacky, 1994) and

systemic response (van Loon, 1997 and van

Loon and Strien, 1999). The induced

resistance (IR) could be developed by two

main mechanisms: systemic acquired

resistance (SAR) and induced systemic

resistance (ISR). SAR is a phenomenon by

which a plant activates its own defense

mechanisms under the influence of a bioagent

(may be bacteria, fungi or plant extracts),

physical or chemical injury (Dixon, 1995).

This resistance is developed with changes in

the biochemistry and physiology of the cell,

that is accompanied by structural

modifications in the plants, which act as

physical barriers to restrict pathogen

penetration (Waheed and Tehmina, 2011).

SAR is effective against a wide range of viral,

bacterial, and fungal pathogens (Sticher et al.,

1997 and Waheed and Tehmina, 2011).

The main objective of this work was to

evaluate some bacterial, fungal strains, and

plant oils for inducing SAR against WMV-2 in

watermelon, as an effective and

environmentally safe approach, in a trial to

fulfil lack of researches on this respect and to

overcome losses in watermelon yield.

MATERIALS AND METHODS

I. Source and inoculum preparation of

WMV-2

The WMV-2 was isolated from naturally

infected watermelon (Citrullus lanatus

Thunb.) plants showing typical symptoms

suspected to the virus including severe vein

clearing, green mosaic, yellow net, blisters,

malformation of fruits, and foliar distortion.

The infected plants were collected from Kafr

El-Dawar, El-Bahera Governorate.

The inoculum of infectious sap was

prepared by grinding 3g of the infected squash

leaves with 3ml of 0.1 M phosphate buffer, pH

7.2 and filtrating the resulting through two

layers of cheese-cloth (Noordam, 1973).

Fourteen plant species belonging to four

families (Table 1) were used to confirm the

presence of WMV-2 in the inoculum. The

leaves of tested plants were slightly dusted

with carborundum powder (600 mesh) and

mechanically inoculated with infectious sap.

After the inoculation, leaves were rinsed in tap

water for 10 min. Control plants were

inoculated with buffer only. The experiments

were performed under greenhouse conditions

with a temperature of 25- 28°C and the plants

were observed for symptoms development

(Khan and Monroe, 1963)

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Table (1): Plants used to confirm the presence of WMV-2 in the inoculum.

English name of plant Latin name of plant Family

Quinoa Chenopodium quinoa W. Chenopodiaceae

Gooses Foot Chenopodium amaranticolor C. and R.

Lamb’s Ouarters Chenopodium album L.

Watermelon Citrullus lanatus Thunb. Cucurbitaceae

Cantaloupe Cucumis melo L.

Squash Cucurbita maxima Duchesne

Pumpkin Squash Cucurbita moschata Duchesne

Cucumber Cucumis sativus L.

Gourds Cucurbita pepo L.

Upland Cotton Gossypium hirsutum L. Malvaceae

Okra Hibiscus esculentus L.

White Lupin Lupinus albus L. Fabaceae Pea Pisum sativum L.

Kidney Bean Phaseolus vulgaris L.

II. Biotic inducers

The biotic inducers used for induction

of the systemic acquired resistance in

watermelon against WMV-2 included four

bacterial, two fungal strains and three types of

plant oils.

1. Bacterial inducers

The bacterial cultures used including

Bacillus megathirium, Bacillus polymyxa,

Zymomonas mobilis ATCC 31823 and

Zymomonos mobilis ATCC 10988 were

obtained from Cairo MIRCEN, Faculty of

Agriculture, Ain Shams University. The Kings

Broth (2% peptone, 1% K2SO4, 0.14% MgCl2

and 0.05% formic acid) was used to prepare

the inoculum of B. megathirium and B.

polymyxa (Waksman, 1957). On the other

hand, ATCC Medium 948 Broth (2% glucose

and 0.5% yeast extract) was used to prepare

the inoculum of Zymomonas mobilis ATCC

31823 and Zymomonos mobilis ATCC 10988

(Atlas, 2004). Generally, the bacterial inocula

were prepared by inoculating 100 ml of each

medium with the bacterial culture, incubating

at 28˚C for 24 hr with shaking at 175 rpm and

followed by centrifugation at 6000 g for 10

min. The bacterial pellet was resuspended in

0.02 M phosphate buffer, pH 7.0, washed

twice with the same buffer then adjusted at

about mean density of 5×109 cfu/ml (Raupach

et al., 1996 and Allam, 2008).

2. Fungal inducers The fungal cultures used were

Trichoderma harizianum and Mycorrhiza

fasciculatum. T. harizianum was propagated in

Potato Dextrose Broth (20% potato infusion

and 2% dextrose) according to Waksman

(1957) by inoculating 100 ml of the medium,

incubating at 28˚C for 7 days with shaking at

110 rpm. The culture was harvested by

filtration through muslin cloth to exclude

mycelium growth, then centrifugation at 1000

g for 10 min and the pellets were resuspended

in 0.02 M phosphate buffer, pH 7.0, and mean

density was adjusted at about of 1010

spores/ml

according to Helmy et al. (2002) and Allam

(2008). Mycorrhiza was obtained from Plant

Pathology Dept., Agricultural Researches

Center. Thirty grams of Mycorrhiza were

inoculated to the surface of the soil without

any preparation.

3. Plant oil inducers

The plant oils used to induce the

systemic acquired resistance in watermelon

against WMV-2 were Allium sativum L.

(garlic), Nigella sativa L. (black cumin) and

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Cinnamomum camphora L. (camphor) oils.

The oils of these plants, purchased from

Harraz Company, were added to the virus

inocula at a ratio of 1:1v/v according to

Baranwal and Verma (1997) and Ali (2006 a).

III. Induction of SAR in watermelon against

WMV-2 A series of experiments was conducted

to test the ability of the selected biotic inducers

to protect watermelon from WMV-2 disease

development.

The susceptible watermelon (Citrullus

lanatus, cv. Giza 1) was used with eleven

treatments and three replicates for each

treatment using plastic pots (25 cm diameter x

30 cm depth) containing 2 Kg of soil. The

treatments included:

- Seed inoculation with Bacillus megathirium,

Bacillus polymyxa, Zymomonas mobilis

ATCC 31823 and Zymomonos mobilis

ATCC 10988. The sterilized seeds of

watermelon were soaked for 60 min in 100

ml of bacterial suspension at a density of

about 5×109 cfu / ml (Raupach et al., 1996;

Wei et al., 1996 and Allam, 2008).

- Seed inoculation with Trichoderma

harizianum. The sterilized seeds of

watermelon were soaked for 60 min in 100

ml of spore suspension at a density of about

1010

spores / ml (Wei et al., 1996; Helmy et

al., 2002 and Allam, 2008).

- Soil treatment with 30 g of Mycorrhiza

without any preparation.

- Cotyledons treatment with a mixture from

either garlic oil, black cumin oil or Camphor

oil with virus inocula (1:1 v/v) 4 weeks after

planting.

- WMV-2 non-inoculated control (healthy

control).

- WMV-2 inoculated control (infected

control).

Seeds were planted in pots. Four weeks

after planting, cotyledons were slightly

dusted with carborundum (600 mesh) and

inoculated with WMV2 inoculum. Healthy

control plants were inoculated with sterile

0.1 M phosphate buffer, pH 7.2. The

inoculated cotyledons were rinsed with water

and kept under greenhouse conditions (25-

28˚C). The symptomatic plants 14 days after

WMV2 inoculation were determined (Khan

and Monroe, 1963).

IV: Evidences of SAR induction

1. Morphological determinations

Fourteen days after WMV-2

inoculation, percentage of virus infection,

reduction of virus infection (RI) and disease

severity (DS) were determined. The

percentage of virus infection was determined

and calculated relatively to the infected

control. For determination of DS, all plants in

each treatment were examined weekly for

virus symptoms development. The symptoms

were recorded using the following rating scale:

0 = no symptoms, 2 = vein clearing, 4 = 50%

of leaves showing mosaic symptoms, 6 =

100% of leaves showing mosaic symptoms, 8

= 50% of leaves showing severe mosaic and

malformation and 10 = 100% of leaves

showing severe mosaic and malformation.

Disease severity values were calculated using

the following formula according to Yang et al.

(1996):

∑(disease grade × number of plants in each grade)

Disease severity (DS) = × 100

total number of plants × highest disease grade

Arab J. Biotech., Vol. 15, No. (2) July (2012):

2. Biochemical determinations The biochemical analysis included

quantification of total antiviral proteins,

peroxidase (POD) activity and salicylic acid

(SA).

Antiviral proteins analysis, including

total protein content and qualitative

electrophoretic sodium dodecyle sulphate

polyacrylamide gel electrophoresis (SDS-

PAGE), was performed in Central Lab. of

Biotechnology, Plant Pathology Department,

Agricultural Researches Center. The total

protein content was estimated according to the

method of Bradford (1976) by using bovine

serum albumin as a standard protein. Protein

content was adjusted to 2 mg / ml per sample.

Qualitative SDS-PAGE was performed

according to the method of Laemmili (1970)

with slight modification. In this modification,

TEMED (Tetramethylethylenediamine) was

reduced from 30 µl to 25 µl and also APS

(Ammonium persulphate) was reduced from

1.5 to 1.3 ml. Antiviral proteins among biotic

inducers were estimated with similarity

coefficient matrix based on SDS-PAGE bands

scored. The dendrogram was constructed

based on the dissimilarity matrix, using

UPGMA method.

Peroxidase activity was estimated

according to the method of Kochba et al.

(1977) in Central Lab. of Biotechnology, Plant

Pathology Department, Agricultural Research

Center. Peroxidase activity was estimated by

measuring the oxidation of pyrogallol to

pyrogallin in the presence of H2O2 at 425 nm.

Salicylic acid was determined in highly

inducible treatments (Mycorrhiza, T.

harizianum, B. megathirium and Zymomonas

mobilis ATCC 31823) and lowest inducible

treatment (garlic oil) which were selected

according to protein cluster analysis groups, in

addition to the infected control. SA was

measured at once in the treatments by a

method according to Raskin et al. (1989), with

one modification by Salem (2004) using

HPLC (SHIMADZO RF-10AXL

Fluorescence, HPLC Lab., Food Technology

Res. Institute, ARC). One hundred microliters

of each sample were injected into Dynamax

60A8. µm guord column (46 mm × 1.5 cm)

linked to 40˚C.

3. Ultrastructural studies

Histological investigations focused on

highly inducible treatments; Bacillus

megathirium, Zymomonas mobilis ATCC

31823 and Mycorrhiza fasciculatum, as well

as, garlic oil as the lowest inducible treatment,

in addition to healthy and infected controls.

These treatments were representatives to

cluster analysis groups. Leaf samples were

obtained from mid-point of plant stem, at age

of 2 weeks from infection. The work was done

in Transmitted Electron Microscope lab

(TEM) of CURP (Cairo University Research

Park). Slice tissue samples were processed for

TEM by fixation in glutaraldehyde and

osmium tetroxide, dehydrated in alcohol and

embedded in an epoxy resin. Microtome

sections prepared at approximately 75-90 µm

thickness with a Leica Ultracut (UCT)

ultramicrotome. Thin sections were stained

with uranyl acetate and lead citrate, then

examined by transmission electron microscope

JEOL (JEM-1400 TEM) at the candidate

magnification. Images were captured by CCD

camera model AMT, optronics camera with

1632 × 1632 pixel formate as side mount

configuration (Timothy and Kristen, 2010).

V: Statistical analysis

The experiment layout was a completely

randomized design (CRD). All percentages

were transformed to arcsine to be analyzed.

Data were subjected to convenient statistical

analysis methods for calculations of means

using MSTATC software. Mean separations

was estimated by calculating LSD values at

Arab J. Biotech., Vol. 15, No. (2) July (2012):

5% probability level according to Snedecor

and Cochran (1980).

RESULTS AND DISCUSSION

I. Host range and symptomatology of

watermelon mosaic virus

The presence of WMV-2 in the

prepared infectious sap was confirmed by

observing the viral infection symptoms that

developed on different host plants. According

to these symptoms, the plants were categorized

into four groups (Table, 2 and Figure, 1) as the

following:

- Group 1: Plants reacted only with local

symptoms. Chlorosis local lesions were

observed on the inoculated leaves of quinoa,

gooses foot and lamb’s quarters 5-7 days

after inoculation.

Group 2: Plants reacted only with systemic

symptoms. Symptoms varied according to

the plant species and ranged between mild

and severe mosaic, associated with blisters,

distortions and leaf deformity, alone or in

combinations. Generally, systemic

symptoms appeared 8-10 days after

inoculation. Most severe symptoms appeared

on cucurbitaceous species (watermelon,

cantaloupe, squash, pumpkin squash,

cucumber and gourds).

- Group 3: Plants reacted with local followed

by systemic symptoms. Kidney bean, okra,

pea and white lupin reacted first with local

symptoms followed by systemic mosaic.

- Group 4: Plants gave no symptoms: upland

cotton.

In agreement with these findings, Sidek

et al. (1994) reported that WMV-2 caused

systemic mosaic on pumpkin, melon,

watermelon, and cucumber, and chlorosis

local lesions on gooses foot and quinoa.

Hernàndez et al. (2000) reported that WMV-

2 could be identified according to the host

range on squash. In other studies, severe

mosaic disease accompanied by yellow spots

was observed on cucumber, okra, cantaloupe

and squash (Murphy et al., 2000; Crescenzi

et al., 2001; Fukumoto et al., 2003 and Raj

et al., 2008).

Table (2): Reaction of host range plants to WMV-2.

Group 1 Group 2 Group 3 Group 4

Plant Sym. Plant Sym. Plant Sym. Plant Sym.

Quinoa CLL Gourds MM,VC Kidney Bean MM Upland Cotton O

Gooses

Foot

CLL Watermelon SM,D,B Okra CLL,

MM

Lamb’s

Quarters

CLL Cantaloupe SM,VC White Lupin

Pea

SM

SM

Pumpkin Squash SM,D

Squash SM

Cucumber MM,D,B,VC

CLL means chlorosis local lesions, SM means severe mosaic, MM means mild mosaic, D means deformed leaves, B

means blisters, VC means vein clearing, O means no symptoms and Sym means symptoms

II. Induction of SAR by different biotic

inducers

Nine biotic inducers, including 4

bacterial, 2 fungal strains and 3 types of plant

oils, were used for induction of SAR in

watermelon plants against WMV-2 infection.

The efficiency of these bioagents was

evaluated morphologically by determining the

percentage of virus infection and DS,

biochemically by determining salicylic acid

Arab J. Biotech., Vol. 15, No. (2) July (2012):

level and antiviral proteins (total protein

content and qualitative protein) and peroxidase

activity, and histologically using the TEM.

1. Morphological determinations

The results in Table (3) and Figure (2)

show that all treatments have different

percentages of disease severity (DS), virus

infection and reduction of virus infection (RI).

The bacterial and fungal inducers were more

effective bioagents than plant oils to induce

the SAR in watermelon plants against WMV-

2. The most effective bacterial inducer was Z.

mobilis ATCC 31823, since the lowest

percentage of both virus infection (61.7%) and

DS (34%) was recorded in plants treated with

this bacterium. For fungal inducers, the results

reveal that, Mycorrhiza was more effective

than T. harizianum in inducing SAR in

watermelon against WMV-2. In this respect,

the lowest percentages of virus infection

(35.4%) and DS (23.5%) were determined in

plants treated with Mycorrhiza. In all

treatments, the highest percentage of DS

(78%) was recorded in plants treated with

garlic oil. From the previous results, it could

be concluded that, Mycorrhiza fasciculatum

was the most effective bioagent for induction

of SAR in watermelon against WMV-2, since

the highest reduction of RI and lowest DS

representing 64.6% and 23.5%, respectively,

were recorded in plants treated with this

bioagent. In this regard, Fahmy and Mohamed

(1984) found that, B. subtilis reduced the

number of local lesions produced on tobacco,

while Askora (2005) found that foliar

treatment of cucumber with Streptomyces

albovinaceus and Streptomyces sparsogenes

reduced mosaic symptoms by 95 and 100%,

respectively, caused by Zucchini yellow

mosaic virus (ZYMV). Kolase and Sawant

(2007) mentioned that T. harzianum induced

systemic resistance on tomato plants and

reduced tobacco mosaic virus (TMV)

symptoms.

Fig. (1): Symptoms on leaves of the host plants infected with WMV-2 sap. (1) Gourds (2) Kidney Bean (3)

Okra (4) Quinoa (5) Gooses Foot (6) Pumpkin Squash (7) Squash (8) White Lupin (9)

Watermelon (10) Cantaloupe (11) Cucumber (12) Lamb’s Quarters.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Fig. (2): Effect of different bio-inducers on infected watermelon plants.

Table (3): Effect of individual biotic inducers on watermelon resistance to WMV-2. Treatment % of infection % of RI

* % of DS

**

Infected control 100.00 0.00 84.00

B. polymyxa 70.90 29.10 57.50

B. megathirium 64.50 35.50 39.00

Z. mobilis ATCC 10988 67.30 32.70 46.00

Z. mobilis ATCC 31823 61.70 38.30 34.00

Mycorrhiza 35.40 64.60 23.50

T. harizianum 58.00 42.00 32.00

Garlic oil 80.60 19.40 78.00

Camphor oil 87.00 13.00 76.00

Black cumin oil 77.40 22.60 73.00

LSD0.05 0.46 0.47 0.47

*RI is reduction of virus infection **DS is Disease severity

2. Biochemical determinations

a. Detection of antiviral proteins

1. Total protein content

Total protein content was determined in

treated watermelon plants with individual

biotic inducers. Table (4) revealed that all used

biotic inducers increased total protein content

and enzyme activity in treated watermelon

plants.The treatment of infected plants with

Mycorrhiza induced the production of high

protein content (1.328 mg/g FW), while the

lowest content (0.905 mg/g FW) was produced

Arab J. Biotech., Vol. 15, No. (2) July (2012):

in plants treated with black cumin oil,

compared with healthy control (0.616 mg/g

FW).

Table (4): Effect of biotic inducers on protein content.

Treatment Protein content* Treatment Protein content

*

Healthy control 0.616 Z.mobilis ATCC 10988 1.083

Infected control 1.502 Black cumin oil 0.905

Mycorrhiza 1.328 Garlic oil 0.907

B. megathirium 1.202 Camphor oil 1.042

B.polymyxa 1.174 T. harizianum 1.251

Z.mobilis ATCC 31823 1.111

*Protein content was measured as mg/g FW LSD0.05 = 0.280

2. Qualitative analysis of antiviral proteins

a. SDS-PAGE and antiviral protein

analysis

The antiviral proteins were determined

qualitatively on the basis of molecular weight

and reproducibility of SDS-PAGE of the nine

inducers. Bands with the same mobility were

treated as identical fragments. Weak band with

negligible intensity and smear bands were both

excluded from final analysis. Figure (3)

demonstrates the SDS-PAGE profile obtained

with nine biotic inducers.

Table (5) illustrates the analysis of biotic

inducers SDS-PAGE. The total number of

antiviral proteins was 101 new bands. Eighty

nine bands of them were 8 of Z. mobilis ATCC

31823, 13 of Mycorrhiza, 10 of camphor oil,

10 of B. polymyxa, 10 of garlic oil, 6 of Z.

mobilis ATCC 10988, 10 of black cumin oil,

12 of T. harizianum and 10 bands of B.

megathirium. The molecular weight of newly

antiviral proteins ranged from 3 to 93 KDa. On

the other hand, the infected control induced 12

newly proteins compared with healthy control

having molecular weight that ranged from 3 to

95 KDa.

A group of plant-coded proteins induced

by different stress stimuli, named

“pathogenesis related proteins” (PRs) is

assigned as an important role in plant defense

against pathogenic constraints and in general

adaptation to stressful environment. This

assumption was driven from initial findings

that these proteins are commonly induced in

resistant plants, expressing a hypersensitive

necrotic response (HR) to pathogens. (Von

Loon, 1997 and Aglika, 2005).The systemic

response involves the production, in some

cases, of phytoalexins and of PRs (van Loon,

1997 and van Loon and Strien, 1999). While

phytoalexins are mainly characteristic of the

local response, PRs occur both locally and

systemically. Originally, PRs were detected

and defined as being absent in healthy plants

but accumulate in large amounts after infection

(van Loon and van Kammen, 1970). Some

PRs have chitinase or β-1,3 glucanase activity

(Legrand et al., 1987). Chitinases are a

functionally and structurally diverse group of

enzymes that can hydrolyze chitin and are

believed to contribute to the defense of plants

against certain fungal pathogens (Jackson and

Taylor, 1996).

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Fig.(3): SDS-PAGE gel electrophoresis of antiviral proteins. (M) Marker (1) Healthy control (2)

Infected control (3) Z. mobilis ATCC 31823 (4) Mycorrhiza (5) Camphor oil (6) B.

polymyxa (7) Garlic oil (8) Z. mobilis ATCC 10988 (9) Black cumin oil (10) T.

harizianum (11) B. megathirium.

b. Clusters analysis

The nine biotic inducers were divided

into 6 different clusters (Fig. 4), the selected

was the most distinct with the rest of the biotic

inducers failling under two major groups. The

first major group was separated into 2

subgroups. The first subgroup contains

Mycorrhiza and T. harizianum while the

second subgroup contains only B.megathirium.

The second major group was separated into 6

subgroups. The first subgroup contains only

infected control, the second subgroup contains

Allium sativum and Nigella sativa and the

third subgroup contains only Cinnamomum

camphora. While the fourth subgroup contains

only Z. mobilis ATCC 10988, the fifth

contains Z. mobilisATCC 31823 and B.

polymyxa. Finally, the sixth subgroup contains

only healthy control. These clusters show the

similarity coefficient between antiviral

proteins relationships among biotic inducers

treatments.

M 1 2 3 4 5 6 7 8 9 10 11

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Table (5): Numbers and molecular weight of antiviral proteins induced by different biotic

inducers.

Total 12 8 13 10 10 10 6 10 12 10

new

Bands

All bold numbers represent the new protein bands

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Fig.(4): Dendrogram showing antiviral proteins relationships among biotic inducers treatments.

b. Determination of peroxidase (POD)

activity

Table (6) shows that the POD activity

increased in all induced watermelon plants.

Mycorrhiza induced the highest activity of

0.931mg/g FW, while garlic oil induced the

lowest activity of 0.231mg/g FW. Peroxidase

activity was induced with different values

ranging from 0.910 to 0.337mg/g FW for T.

harizianum and Z. mobilis ATCC 10988,

respectively. Many authors reported that,

increased POD activity was associated with

induced systemic resistance in cucumber to a

variety of pathogens and tobacco to TMV

(Simons and Ross, 1970; Hammerschmidt et

al., 1982 and Lagrimini and Rothstein, 1987).

Yahraus et al. (1995) found that, in induced

tobacco, POD activity was higher than that

before challenge in uninfected leaves, and

increased more rapidly after challenge with

TMV than in controls. POD is also implicated

with hypersesitivity response (Bestwick et al.,

1998), lignin biosynthesis, ethylene production

and suberization (Quiroge et al., 2000).

Kandan et al. (2002) found that, tomato

spotted wilt virus (TSWV) was reduced in

tomato plants treated with P. fluorescens. The

POD activity also increased in tomato plants

induced by P. fluorescens compared with the

control.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Table (6): Effect of biotic inducers on peroxidase activity. Treatment POD (mg/g FW)

Healthy control 0.225

Infected control 0.949

B. megathirium 0.634

B.polymyxa 0.539

Z. mobilis ATCC 31823 0.549

Z. mobilis ATCC 10988 0.337

Mycorrhiza 0.931

T. harizianum 0.910

Black cumin oil 0.243

Garlic oil 0.231

Camphor oil 0.336

LSD 0.05 = 0.093

c. Quantification of total salicylic acid (SA)

Results on quantification of total SA

using HPLC in induced watermelon plants

(Table, 7) agreed with percentage of infection

and disease severity as the level of total SA

increased particularly in treated plants.

The highest level of SA (1046.79

µg/100g) was recorded in plants treated with

Mycorrhiza followed by plants treated with T.

harizianum (872.70 µg/100g) and B.

megathirium (630.47 µg/100g), while the

lowest level (287.88 µg/100g) was recorded in

plants treated with garlic oil.

Table (7): Quantification of total SA in watermelon plants.

Treatment Total SA (µg/100g FW)

Infected control 1200.33

Mycorrhiza 1046.79

T. harizianum 872.70

B. megathirium 630.47

Z.mobilis ATCC 31823 524.85

Garlic oil 287.88

LSD 0.05 = 0.40

Salicylic acid (SA) is an important signal

involved in the activation of plant defense

responses against abiotic and biotic stresses

(Fragnière et al., 2011). Local resistance (LR)

and SAR are generally accompanied by

elevated levels of endogenous SA (Malamy et

al., 1992). There is strong evidence that SA

plays a central role in LR and SAR signaling

(Malamy et al., 1990; Métraux et al., 1990;

Rasmussen et al., 1991; Malamy et al., 1992;

Dorey et al., 1998 and Durner and Klessig.,

1996). White (1979) was the first to be

interested in the role of endogenous SA in

plant defense resistance to TMV in tobacco.

Yalpani et al. (1991) and Enydi et al. (1992)

reported that in TMV-infected tobacco, the

endogenous level of SA in infected as well as

in uninfected leaves are sufficient to induce

resistance and PR-proteins. Malamy et al.

(1990) showed that the development of

hypersensitivity reaction (HR) and SAR is a

complex of a dramatic increase in the level of

endogenous SA in the inoculated leaves and in

the systemically protected tissue.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

3. Ultrastructural studies

TEM was used as a sharp tool to

investigate watermelon plant inner

modifications in cells and tissues structure in

response to treatments with experimental

resistance inducers.

Watermelon leaf is usually dorsiventral.

Hairs include the following types: (1) simple,

unicellular or uniseriate, (2) glandular hairs

with uniseriate stalks of variable length and

spherical or disk-shaped heads. Cells of the

epidermis are particularly large in Japanese

species of Citrullus. Stomata present on both

surfaces; ranunculaceous. Midrib is provided

with a ring of bundles in species of Citrullus.

The bicollateral bundle of the Cucurbitaceae is

a compound structure consisting of attachment

of two distinct vascular bundles, of which the

innermost has lost the xylem.

Ultrastructural symptoms of infection

with WMV-2 in watermelon blades presented

in Plate (1) show that, at the same age (after 2

weeks from infection), healthy control cells

maintain their functionable organelles,

whereas, infected plants treated with plant oils

endure severe deterioration in cell organelles;

nucleus burst, vacuoles vanished, cytoplasm

scattered and cell wall breaks down, so, cells

fused together. Treatment with Mycorrhiza, as

shown in Plate (1), gave prolonging in

responses as follows:

1. Organelles enlarged due to augmented

synthetic processes of virus RNA (WMV-2

virion is non-enveloped with a filamentous

rod shaped nucleocapsid, 750-780 nm long

and 11-20 nm in diameter, with nucleic acid

of RNA). That may occur in the cytoplasm,

nuclei, chloroplasts, Golgi apparatus, cell

vacuoles or more rarely in unusual sites.

2. Chloroplasts with prominent starch grains,

as counter-effect of virus damages to

enzymes responsible of carbohydrates

metabolism inside green plastids, which led

plastids to disrupt.

3. Appearing of nuclear and cytoplasmic

inclusions; ultrastructural studies, using

TEM, led to the separation of potyviruses, on

the basis of differences in morphology of the

cylindrical (pinewheal) inclusions, into three

subdivisions, from which subdivision III has

WMV-2 which induces tubes and laminated

aggregates inclusions, in the cytoplasm of

infected host cells, immunologically distinct

from the viral capsid protein and host

proteins. In cross section, the tubular

inclusions appear as scrolls. The laminated

aggregates are usually observed in

negatively stained preparations as roughly

triangular or rectangular plates compressed

together for part or all of their length.

4. Finally, organelles aggregate.

The aforementioned results indicated

that, Mycorrhiza treatment made it possible,

may be for its preferable nutritional

(fertilizing) effect, for the infected plants with

WMV-2, to withstand for a longer period

practicing the replication active process, which

forced organelles to work to their maximum

limits until they collapse.

Table (8) and Fig. (5) revealed that,

linking blade thickness and their components

as growth indicators, for resistance inducing

treatments (which had some growth promoting

effects), with host resistance is less reliable,

since they exhibit inconsistent trend. It seems

that combination of inoculums (includes a

pathogen; WMV-2 for instance) invasion may

promote the resistance although it suppress

growth. It seems to be logic since measured

chemical responses to infection reach the

climax (after infected control) in the lowest

growth plants; treated with Mycorrhiza.

Whereas plants treated with Allium sativum oil

reflect the lowest chemical responses to

infection, although they exceeded the former

mentioned treatment in growth. Taking former

results in consideration, with knowing that

viruses are biotrophics (obligatory parasites on

Arab J. Biotech., Vol. 15, No. (2) July (2012):

living cells), must lead to investigate active / dynamic resistance barriers.

Plate (1): Electron micrographs of deterioration stages due to WMV-2 inoculation in watermelon leaf

cells, after 2 weeks of infection; A: healthy cell (control); 1-nucleus, 2-chloroplast, 3-

mitochonderion, 4-dictyosomes, B: infected cell treated with Mycorrhiza: 1- vacuoles split and

organelles enlarged, 2-chloroplasts with prominent starch grains 3- nuclear inclusion, 4-

cytoplasm inclusion, 5- organelles aggregate, C: infected cell treated with plant extract (Allium

sativum L.); 1- nucleus burst, vacuoles vanished and cytoplasm scattered, 2-cell wall breaks

down, 3-cells fused together.

Table (8): Watermelon lamina measurements (µ) after 2 weeks of infection. Upper

cuticle

Upper

epidermis

Palisade

tissue

Spongy

tissue

Lower

epidermis

Lower

cuticle

Blade

thickness

Healthy plant 5.56 13.89 44.44 69.44 11.11 2.78 147.22

Infected plant with WMV2 8.34 11.11 38.89 44.44 11.11 5.56 119.45

Plants treated with Mycorrhiza 2.78 8.34 19.45 41.76 8.34 2.78 83.45

Plants treated with garlic oil 1.39 8.34 25 47.22 8.34 1.39 91.68

Plants treated with Z. mobilis

ATCC31823

8.34 8.34 25 50 11.11 2.78 105.57

Plants treated with B. megathirium 4.17 11.11 16.67 44.44 6.94 2.78 86.11

A3

A2

A1

B2

B1

A4

B5

B4

B3

C1

C2

C3

Arab J. Biotech., Vol. 15, No. (2) July (2012):

As seen in Fig. (6), it could be stated

that, in response to both WMV-2 and

Mycorrhiza treatment, both biochemical and

structural resistance mechanisms were

initiated. Biochemical mechanisms are

represented in hypersensitivity (field

immunity) symptoms as contents aggregate in

only epidermal cells, confronting pathogen

invasion till the cells black out and die out,

also forming phenolic compounds and around

infected tissues to fill the cell till it dies. The

latter represented in gummosis; depositing

carbohydrates (poly-saccharides) that are hard

Fig. (5): Electron micrographs of transverse sections in watermelon leaf blades, after 2 weeks of

infection A. Healthy plant (control) B. Infected plant with WMV-2

C. Infected plant treated with Mycorrhiza D. Infected plant treated with garlic oil

E. Infected plant treated with Z. mobilis

ATCC31823

F. Infected plant treated with B. megathirium

Bar beneath every image represents scale in microns.

E

C

F

D

B A

Arab J. Biotech., Vol. 15, No. (2) July (2012):

to be analyzed, in intercellular spaces on the

edges of infection spots.

B

D

A

C

E

Fig. (6): Electron micrographs of transverse sections in watermelon blades infected with WMV-2

and treated with Mycorrhiza showing dynamic resistance mechanism symptoms, after 2

weeks of infection

A – B: hypersensitivity symptoms; aggregation of cell contents confronting infection,

C – D: forming phenolic compounds to fill the cell till it dies, E: gummosis; depositing carbohydrates in

intercellular spaces

Bar beneath every image represents scale in microns.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Finally, It could be concluded that, due

to Mycorrhiza (the most effective) treatment,

afforementioned barriers may lead to hinder

virus movement inside living tissues, treatment

also may stimulate cells of susceptible host to

make organelles withstand virus replication

exhausting process.

Previous results are in harmony with

those described by Metcalfe and Chalk (1950),

Baum (1980), Hassan (1994), Mahrotra

(1997), Abd El Hak et al. (1999), Khidr

(2002), Awad (2005), Hassan (2005), Ali

(2006 b), and Zayed and Kash (2009).

CONCLUSION

Morphological investigation indicated

that, Mycorrhiza treatment was the most

effective bioinducer for eliminating WMV-2

impact in infected watermelon plants, where

plant oils has weak effect as inducers of SAR.

Former chemical investigation confirmed that,

watermelon plants treated with Mycorrhiza

then inoculated with WMV-2 gave highest

resistance chemical indicators close to infected

control values, whereas, infected plants treated

with garlic oil gave remote values than

infected control. The mystery of these

contradicting results solved by comparing

antiviral protein bands obtained from SDS-

PAGE study, which revealed that, bands do

not compatible among treatments. Thus, it

could be concluded that, protein contents

among treatments belong to variety of kinds.

Detecting histological symptoms of infection

and resistance mechanisms confirmed that

Mycorrhiza treatment allows infected plants to

withstand viral activities for longer period.

This durability resulted, mainly, from dynamic

resistance barriers; biochemical and structural.

REFERENCES

Abdel - Hak, T.; Elewa, I.; Barakat, F.M.

and Eisa, N.A. (1999). Plant Diseases and

Their Control. Anglo Bookshop. (In Arabic).

414p.

Abdel - Monaim, M. F. (2012). Induced

systemic resistance in tomato plants against

Fusarium wilt disease. Int. Res. J.

Microbiol., 3 (1): 14 – 23.

Aglika, E. (2005). Pathogenesis-related

proteins: Research process in the last 15

Years. Gen. Appl. Plant Physiol., 31(1-

2):105-124.

Ali, H.H.M. (2006a). Studies on plant virus

inhibitors. M.Sc. Thesis. Fac. of Agric.,

Cairo.Univ., Cairo, Egypt, 83p.

Ali, M. M., (2006b). Plant Pathol. Osoris

Bookshop. (In Arabic). 621p.

Allam, A.E.M.(2008). Effect of antiviral

proteins produced by bacterial and fungal

isolates on some viruses infecting vegetable

crops. M.Sc. Thesis, Fac. of Agric., Ain

Shams.Univ., Cairo, Egypt, 46p.

Askora, A.A. (2005). Antiphytoviral studies

from certain actinomycetal isolates. M.Sc.

Thesis, Fac. Sci., Zagazig Univ., Zagazig,

Egypt, 189 p.

Atlas. R. M. (2004). Handbook of

Microbiological Media. 3rd

ed. CRC. Boca

Raton, London, New York, Washington, D.

C. 2051p.

Awad, M.A. (2005). Viral Plant Diseases and

Their Causes. Part 1. Al Dar Al Arabia. (In

Arabic). 448p.

Baranwal, V.K. and Verma, H.N. (1997). Characteristics of virus inhibitor from the

leaf extract of Celosia cristata. Plant Pathol.,

46(4):523-529.

Baum, R.H. (1980). Purification, partial

characterization and serology of the capsid

and cylindrical inclusion proteins of four

isolates of watermelon mosaic virus. Ph.D.

Thesis, University of California, USA. 107p.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Bestwick, C.S.; Brown, I.R. and Mansfield,

J.W. (1998). Localized changes in

peroxidase activity accompany hydrogen

peroxidase generation during the

development of a non host hypersensitive

reaction in lettuce. Plant Physiol., 118:1068-

1078.

Bradford, M.M. (1976). A rapid and sensitive

method for the quantification of microgram

quantities of protein utilizing the principle of

protein dye binding. Anal. Bioch., 72: 248-

254.

Branka, B.K.; Janoš, B.B.; Nataša, D.D.;

Ivana, M.V.; Nikolaos, I.K. and Chryssa,

C.P. (2002). Identification of viruses

infecting pumpkins (Cucurbita pepo L.).

Matica Srpska Novi Sad, 103: 67-79.

Crescenzi, A.; Fanigliulo, S.; Comes, V.;

Masenga, R.P. and Pizzolla, P. (2001).

Necrosis of watermelon caused by

watermelon mosaic virus. J.plant pathol., 83

(3).

Dixon, R.A. (1995). The phytoalexin

response: Elicitation, signalling and control

of host gene expression. Biol. Can. Philos.

Soc., 61: 239-292.

Dorey, S.; Baillieul, F.; Saindrenan, P.;

Fritig, B. and Kauffmann, S. (1998).

Tobacco class I and II catalases are

differentially expressed during elicitor-

induced hypersensitive cell death and

localized acquired resistance. Mol. Plant-

Microbe Interact, 11:1102–1109.

Durner, J. and Klessig, D.F. (1996). Salicylic

acid is a modulator of tobacco and

mammalian catalases. J. Biol. Chem., 271:

28492–28501.

Enyedi, A.J.; Yalpani, N.; Silverman, P. and

Raskin, I. (1992). Localization, conjugation

and function of salicylic acid in tobacco

during the hypersensitive reaction to tobacco

mosaic virus. Proc. Natl. Acad. Sci. USA.,

89: 2480-2484.

Fahmy, F.G. and Mohamed, M.S. (1984).

Effect of onion root exudates, sulphide

compounds and culture filtrates of certain

microorganisms on infectivity of tobacco

mosaic and potato viruses. Egypt. J.

Phytopathol., 16(1-2): 65-69.

Figueroa , A; Marcos, A.; Sanchez, G.;

Penelope, M.; Perkins,V.; Bahram, H. and

Arjmand, I. (2010). Effects of watermelon

supplementation on aortic blood pressure

and wave reflection in individuals with

prehypertension. Am. J. Hypertens., 10:10-

38.

Fragnière, C.; Serrano, M.; Abou-Mansour,

E.; Métraux, J.P. and L'Haridon, F.

(2011). Salicylic acid and its location in

response to biotic and abiotic stress. FEBS

Letters, 585:1847-1852.

Fukumoto, F.; Masuda, Y. and Hanada, K.

(2003). Pea tissue necrosis induced by

cucumber mosaic virus alone or together

with watermelon mosaic virus. Plant Dis.,

27: 324-328.

Giovannucci, E.; Rimm, E. B.; Liu, Y.;

Stampfer, M. J. and Willett, W. C. (2002). A prospective study of tomato products,

lycopene, and prostate cancer risk. J. Natl.

Cancer Inst., 94: 391-398.

Goodmann, R.N. and Novacky, A.J. (1994).

The bacteria-induced hypersensitive

reaction. Amer. Phyto. Pathol. Soc., pp117-

173.

Hammerschmidt, R. (1999). Induced disease

resistance: how do induced plants stop

pathogens? Phys. and Mol. Plant Pathol.,

55(2): 77-84.

Hammerschmidt, R.; Nuckles, E.M. and

Kuc, J. (1982). Association of enhanced

peroxidase activity with induced systemic

resistance of cucumber to Colletotrichum

lagenarium. Physiol. Plant Pathol., 20: 73-

82.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Hassan, A.A. (1994). Plant Breeding for

Diseases and Pests Resistance. Al Dar Al

Arabia. (In Arabic). 378p.

Hassan, A.A. (2005). Plant Breeding

Methods. Al Dar Al Arabia. (In Arabic).

393p.

Helmy, E.; Yomna, I. and Maklad, F.M.

(2002). Induced systemic resistance in

cucumber plants under plastic houses against

cucumber mosaic virus using biotic inducers.

Egypt. J. Hort., 29(1):1-6.

Hernάndez, J.R.; Ramallo, J.C. and Weth,

S. (2000). Watermelon mosaic-2 potyvirus

on winter squash in Tucuman, Argentia.

Fitopatologia, 35(2): 91-97.

Jackson, A.O. and Taylor, C.B. (1996).

Plant–microbe interactions: life and death at

the interface. The Plant Cell, 8: 1651–1668.

Kandan, A.; Commare, R.R.; Nandakumar,

R.; Ramiah, M.; Raguchander, T. and

Samiyappan, R. (2002). Induction of

phenylpropanoid metabolism by

Pseudomonas fluorescens against tomato

spotted wilt virus in tomato. Folia

Microbiologica, 47(2):121-129.

Khan, R.B. and Monroe, R.I. (1963). Detection of tobacco veinal necrosis (strain

of Potato virus Y) in Solanum carensaii and

S.andigenum introduced into the United

States. Phytopathology, 53:1356-1359.

Khidr, M. O. (2002). Principles and Methods

of Plant Breeding. Muhira for Printing and

Publishing. 377p.

Kochba, J.; Lavee, S. and Spiege-Roy, P.

(1977). Difference in peroxidase activity and

isozymes in embryogenic and non-

embryogenic 'sharr outi' orange ovular callus

lines. Plant Cell Physiol., 18(46): 3- 7.

Kolase, S.V. and Sawant, D.M. (2007).

Isolation and efficacy of antiviral principles

from Trichoderma spp. Against Tobacco

mosaic virus (TMV) on tomato. Journal of

Maharashtra Agriculture Universities,

32(1):108-11. (English abstract).

Kombrink, E. and Schmelzer, E. (2001). The

hypersensitive response and its role in local

and systemic disease resistance. Eur. J. of

Plant Pathol. (EJPP), 107:69–78.

Laemmili, U.K. (1970). Cleavage of structural

proteins during the assembly of the head of

Bacteriophage T4. Nature, 227: 680-685.

Lagrimini, L.M. and Rothstein, S. (1987).

Tissue-specificity of tobacco peroxidase

isozyme and their induction by wounding

and tobacco mosaic virus infection. Plant

Physiol., 84: 438-442.

Legrand, M.; Kauffmann, S.; Geoffroy, P.

and Fritig, B. (1987). Biological function of

pathogenesis- related proteins: Four tobacco

pathogenesis-related proteins are chitinases.

Proc. Natl. Acad. Sci. USA, 84: 6750-6754.

Mahrotra, R.S. (1997). Plant Pathology.

Arabic Institute of Development. 1383p.

Malamy, J.; Carr, J.P.; Klessig, D.F. and

Raskin, I. (1990). Salicylic acid: a likely

endogenous signal in the resistance response

of tobacco to viral infection. Science,

250:1002–1004.

Malamy, J.; Hennig, J. and Klessig, D.F.

(1992). Temperature-dependent induction of

salicylic acid and its conjugates during the

resistance response to tobacco mosaic virus

infection. Plant Cell, 4: 359-366.

Metcalfe, C.R. and Chalk, L. (1950). Anatomy of the Dicotyledons. Vol. 1. The

Clarendon Press. 725p

Métraux, J.P.; Signer, H.; Ryals, J.; Ward,

E.; Wyss, M.; Gaudin, J.; Raschdorf, K.;

Schmid, E.; Blum, W. and Inverardi, B.

(1990). Increase in salicylic acid at the onset

of systemic acquired resistance. Science,

250:1004–1006.

Murphy, J.F.; Ruhui, L.I; Joseph, M.;

Kemble, M.; Baltikauski, D.P.; Gary, G.

and Beauchamp, R.R. (2000). Viruses

identified in commercial pumpkin and

watermelon. Highlights of Agric. Res.,

47(1): 5-23.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Murphy, J.F.; Reddy, M.S.; Ryu, C.M.;

Kloepper, J.W. and Li, R. (2003).

Rhizobacteria mediated growth promotion of

tomato leads to protection against cucumber

mosaic virus. Phytopathology, 93:1301-

1307.

Noordam, D. (1973). Identification of Plant

Viruses: Methods and Experiments. Center

for Agriculture Publishing and

Documentation, Wageningen, the

Netherlands, 207p.

Quiroge, M.; Guerrero, C.; Botella, M.A.;

Barcelo, A.; Amaya, I.; Madina, M.I.;

Alanso, F.J.; Forchetti, S.M.; Tigier, H.

and Valpuesta, V. (2000). A tomato

peroxidase involved in the synthesis of

lignin and suberin. Plant Physiol., 122:1119-

1128.

Raj, S.K.; Kumar, S.; Snehi, S.K. and

Pathre, U. (2008). First report of cucumber

mosaic virus on Jatropha curcas in India.

Plant Dis., 92(1):171.

Raskin, I.; Turner, I.M. and Melander,

W.R. (1989). Regulation of heat production

in the inflorescences of an Arum lily by

endogenous salicylic acid. Proc. Natl. Acad.

Sci. USA., 86: 2214-2218.

Rasmussen, J.B.; Hammerschmidt, R. and

Zook, M.N. (1991). Systemic induction of

salicylic acid accumulation in cucumber

after inoculation with Pseudomonas syringae

pv. syringae. Plant Physiol., 97:1342–1347.

Raupach, G.S.; Liu, L.; Murphy, J.F.;

Tuzun, S. and Kloepper, J.W. (1996).

Induced systemic resistance in cucumber and

tomato against cucumber mosaic

cucumovirus using plant growth promoting

rhizobacteria (PGPR). Plant Dis., 80: 891-

894.

Salem, E.A. (2004). Induction of salicylic acid

by some chemical substance used to control

virus infection under protected Agriculture.

M.Sc. Thesis, Fac. of Agric., Ain

Shams.Univ., Cairo, Egypt, 118p.

Sidek, Z.; Okui, H. and Sako, N. (1994).

Watermelon mosaic virus-2 isolated in

Malayssia. Annals Phytopathol. Soc. Japan,

60(6): 738-739.

Sidek, Z.; Barin, J. and Sulaiman, I. (1999).

Weed hosts of cucurbit viruses. Agro-

Search, 6(1): 1-3.

Simons, T.J. and Ross, A.F. (1970).

Enhanced peroxidase activity associated

with induction of resistance to tobacco

mosaic virus in hypersensitive tobacco.

Phytopathology, 60: 383-384.

Snedecor, G.W. and Cochran, W.G. (1980).

Statistical methods. 6th

ed. The Iowa State

Univ. Press. Amer. Iowa, USA, 593p.

Sticher, L.; Mauchani, B. and Métraux, J.P.

(1997). Systemic acquired resistance.

Phytopathol., 35: 235–270.

Timothy, P. and Kristen, A.L. (2010).

Biology Department Frostburg State

University. Newsletter, 23(1):1-3.

Van Loon, L.C. (1997). Induced resistance in

plants and the role of pathogenesis related

proteins. Eur. J. of Plant Pathol., 103:753–

765.

Van Loon, L.C. and Kammen, A. (1970).

Polyacrylamide disc electrophoresis of the

soluble leaf proteins from Nicotiana

tabacum var. ‘Samsun’ and ‘Samsun NN’ II.

Changes in protein constitution after

infection with tobacco mosaic virus.

Virology, 40:199–211.

Van Loon, L.C. and Strien, E.A. (1999). The

families of pathogenesis related proteins,

their activities, and comparative analysis of

PR1 type proteins. Phys. and Mol. Plant

Pathol., 55: 85–97.

Waheed, A. and Tehmina, A. (2011). Use of

bioagents and synthetic chemicals for

induction of systemic resistance in tomato

against diseases. Int. Res. J. of Agric. Sci.

and Soil Sci., 1(8): 286-292.

Arab J. Biotech., Vol. 15, No. (2) July (2012):

Waksman, S.M. (1957). Soil microbiology.

John. and Sons. New York and London. 253

p.

Wayne, W.F.; Benny, D.B. and Vincent,

M.R. (2009). Watermelon juice: a promising

feedstock supplement, diluent, and nitrogen

supplement for ethanol biofuel production.

U.S. Dep. of Agric. Sci., magazine (USDA-

ARS), 10: 2-18.

Wei, G.; Kloepper, J.W. and Tuzun, S.

(1996). Induced systemic resistance to

cucumber diseases and increased plant

growth by plant growth-promoting

rhizobacteria under field conditions.

Phytopathology, 86: 221-24.

White, R.F. (1979). Acetylsalicylic acid

(aspirin) induces resistance to tobacco

mosaic virus in tobacco. Virology, 9: 410-

412.

Yahraus, T.; Chandra, S.; Legendre, L. and

Low, P. (1995). Evidence for a mechanically

induced oxidative burst. Plant Physiol., 109:

1259–1266.

Yalpani, N.; Silverman, P.; Wilson, T.M.A.;

Kleier, D.A. and Raskin, I. (1991).

Salicylic acid is a systemic signal and an

inducer of pathogenesis-related proteins in

virus-infected tobacco. Plant Cell, 3: 809–

818.

Yang, X.; Liangyi, K. and Tien, P. (1996). Resistance of tomato infected with cucumber

mosaic virus satellite RNA to potato spindle

Tuber viroid. Ann. Appl. Biol., 129:543-551.

Zayed, K.A. and Kash, K. (2009). Genetics

and Plant Pathology. Academic Bookshop.

(In Arabic). 355p.

الملخص العربي

حث المقاومه الجهازية المكتسبه لنبات البطيخ ضد فيروس موزاييك

2-البطيخ

***

خالد سعد عماره، **

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حنان عبداللطيف جوده، * الفت سيد بركات

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مصر -ةالجيز -ةجامعه القاهر -ةكليه الزراع نبات الزراعيالقسم - ***

يروس ڤكساب نباتات البطيخ مقاومة جهازية ضد إمحفزات حيوية علي 9ختبار مقدره إجريت هذه الدراسة بغرض أ

Bacillus و Bacillus megathirium هي أربع سالالت من البكتريا هي المحفزات الحيوية ههذوكانت . 2-موزاييك البطيخ

polymyxa و Zymomonas mobilis ATCC 31823 و Zymomonos mobilis ATCC 10988 ضافة الي ، باإل

نواع من أثالثة وايضا ، Mycorrhiza fasciculatum و Trichoderma harizianum: ساللتين من الفطريات وهما

ستدالل علي تكوين المقاومه الجهازية المكتسبة في ولقد تم اإل. ور و زيت حبه البركةزيت الكاف، زيت الثوم: اتية وهيت النبالزيو

يزا كانت أفضل المحفزات الحيوية إلكساب أن الميكوروضحت النتائج أوقد .النباتات المعامله مورفولوجياً وكيميائياً وهستولوجياً

ةيروس وشدڤصابه بالاإل ةتقليل نسبالمعاملة الى هحيث أدت هذ2 –يروس موزاييك البطيخ ڤجهازية ضد نباتات البطيخ مقاومة

6464.99 )مستوي حامض الساليسيلك ةالي زياددت أكما ، علي التوالي، ٪ 22.5و ٪ 46.4 صابه بنسبه مظاهر اإل

نزيم البيروكسيديزإوكذلك نشاط (ام وزن رطبجر/مجم 6.223 ) والمحتوي البروتيني( جرام وزن رطب644/ ميكروجرام

ستخدام إصابه بمظاهراإل ةيروس وشدڤصابه بالاإل ةفقد لوحظ زيادة نسب، خر وعلي الوجه اآل (.جرام وزن رطب/مجم4.926)

ستخدام إجي بتولوأكد الفحص الهسولقد .في نباتات البطيخ الي تقليل المقاومة الجهازيةدي أالزيوت النباتيه و خاصة زيت الثوم مما

يزا أفضل المعامالت في تحفيز تكوين العوائق الهستولوجية كمقاومة جهازية مثل ان الميكور لكتروني النافذالميكروسكوب اإل

يروس داخل ڤإلي إعاقة حركه الوتكوين الصموغ والمركبات الفينولية، وكل هذه الحواجز تؤدي ( مناعة الحقل)الحساسية الفائقة

.يروس بداخلها لمدة أطولڤة، وأيضاً ربما تحث المعاملة خاليا العائل المصاب كي تتحمل عضياتها تضاعف الاألنسجة الحي