Natural infection of squash fruits ( Cucurbita pepo ) by Zucchini Yellow Mosaic potyvirus (ZYMV) in Alexandria governorate

An isolate of zucchini yellow mosaic virus (ZYMV) was obtained from naturally infected squash fruits were grown in Abees region, Alexandria governorate. Disease symptoms were Showing mosaic, yellowing and blistering and absis symptoms. The identiﬁ cation was based on the symptoms developed on diagnostic hosts and serological reactions with antisera to cucumber mosaic cucumovirus (CMV), watermelon mosaic potyvirus 2 (WMV-2) and ZYMV. Squash fruit isolate of ZYMV was transmitted by Aphis gossypii, aphis neri and Myzus persicae in non-persistent manner. The virus was puriﬁ ed by ultra-centrifugation and PEG. The puriﬁ ed virus had an ultraviolet absorption spectrum typical of a nucleoprotein with A260/280 and A280/260 being 1.1


Introduction
Zucchini yellow mosaic virus (ZYMV) is a member of family Potyviridae and is considered the most economically important virus attacking cucurbit plants under ield conditions [1]. The virus was isolated for the irst time in northern Italy [2] and latter from different countries [3][4][5][6][7][8]. In Egypt, ZYMV was isolated from naturally infected squash plants [9][10][11][12][13][14][15]. Squash plants exhibiting mosaic, yellowing, blister and stunt symptoms and squash fruits contain (abscess or Boils) were observed in Abees region, Alexandria governorate. The aims of present study was observed to isolate and identify the causal agent on the bases of symptomology, reactions of diagnostic hosts, serological reaction, puri ied the isolated virus by ultra-centrifugation methods and Measurement the yield of puri ied virus. Production of speci ic antiserum to the isolated virus was also carried out. The present study was directed to isolate and identify the causal agent on the bases of symptomology, reactions of diagnostic hosts, serological reaction, insect transmission, photometrical characters of the puri ied virus. Production of speci ic antiserum to the isolated virus was also carried out.

Materials and methods
Fruit samples from squash plants exhibiting mosaic, blister and absis or Boils symptoms were collected from Abees location (Figure 1), Alexandria governorate. Inoculum was prepared by grinding infected leaf tissue in a mortar and pestle, with small amount of 0.02 M phosphate buffer, pH7.0, containing 0.1% 2-mercaptoethanol, leaves of plants to be inoculated were irst dusted with carborundum (600 mesh) [16] and then inoculated with a freshly prepared inoculum using fore inger. The isolated virus was maintained in squash plants that served as a source of the virus for subsequent studies.

Abstract
An isolate of zucchini yellow mosaic virus (ZYMV) was obtained from naturally infected squash fruits were grown in Abees region, Alexandria governorate. Disease symptoms were Showing mosaic, yellowing and blistering and absis symptoms. The identifi cation was based on the symptoms developed on diagnostic hosts and serological reactions with antisera to cucumber mosaic cucumovirus (CMV), watermelon mosaic potyvirus 2 (WMV-2) and ZYMV. Squash fruit isolate of ZYMV was transmitted by Aphis gossypii, aphis neri and Myzus persicae in nonpersistent manner. The virus was purifi ed by ultra-centrifugation and PEG. The purifi ed virus had an ultraviolet absorption spectrum typical of a nucleoprotein with A260/280 and A280/260 being 1.1 and 0.91 respectively. The yield of purifi ed virus was 1.62 mg/100g infected leaf tissues. Specifi c antiserum was prepared and found to have a titer of 1:409600 as determined by indirect ELISA.   [11], Abdel Ghaffar, et al. [12], Prieto, et al. [19], Younes, [14], Fath-Allah and Ahmed, [15], Usher, et al. [20] and Vučurović et al. [8] were used as provisional identi ication of the isolated virus.

Modes of transmissions
ZYMV was studied for its transmissibility by different methods.

Mechanical transmission
Squash plants were infected and was used as a virus source, whereas, Chenopodium amaranticolor was an assay host. Hamza, et al. [16].

Aphid transmission
Three species of aphids were tested for their ability to transmit isolates of ZYMV. Apterous forms of three species of aphids; Aphis gossypii Glover, A. neri and Myzus persicae (These aphids were kindly identi ied by Dr. Hedaya Hamza, Entomology Departmet, Faculty of Agriculture, Alexandria University, Egypt). Apterous forms of aphids were starved for one hour then allowed to feed on ZYMV-infected squash leaves for 3-5 min. on infected squash attached leaves. Aphids were transferred in groups of ive to each of 10 healthy squash plants and allowed an inoculation period of 10 min. before they were killed by spraying by an insecticide. Inoculated plants were kept under greenhouse conditions and symptoms were observed for four weeks [21].
Indirect ELISA: Indirect ELISA was carried out as described by Abd El-Aziz and Younes, [23] extracts from infected and healthy squash fruits were diluted with coating buffer (0.05 M carbonate, pH 9.6) to 1:10. Wells were coated with antigens by adding 100 μl of to the bottom of the well and incubated for 3 hours at 37 °C or overnight at 4°C. The plates were rinsed three times by looding wells with (Phosphate buffer saline tween 20 (PBST)) for 3 minutes each. Antisera requiring cross-adsorption were diluted 1:500 with iltered extract from healthy tissues diluted 1:20 [24] in serum buffer (Phosphate buffer saline tween 20 (PBS-T) containing 2% soluble polyvinylpyrrolidone, 0.2% Bovine serum albumin (BSA)), and incubated for 45 min. at 37 °C. The precipitate, which had formed, was removed by centrifugation for 10 min. at 5000 rpm. 100 μl aliquots from the diluted antisera were added to each well, after which the plates were incubated at 37 °C for 2 hours or at 4 °C overnight, then washed as before. Goat anti-rabbit gamma globulin conjugated to alkaline phosphatase was diluted 1:15000 in serum buffer, and 100 μl were added to each well, followed by one hour incubation at 37 °C, and then washed as before. 100 μl of the enzyme substrate, 0.5 mg/ml paranitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8 were added to each well and incubated at room temperature (25 °C) for about 30 minutes. The enzyme activity was stopped by adding 50 μl of 3 M NaOH. The ELISA values measured by MeterTech E960 ELISA reader were expressed as absorbency at 405 nm and optical density of at least double that of the healthy control were considered positive.

Purifi cation
ZYMV was puri ied from systemically infected squash plants using the method described by Azzam and Makkouk [25] with some modi ications. Two hundreds grams of infected leaf tissues harvested three weeks after inoculation were mixed with 200 ml of an extraction buffer [0.5 M K 2 HPO 4, 0.002 M Na 2 SO 3 , 0.01 M Na-diethyldithiocarbamate (DIECA) and 0.01 M ethylenediamine-tetraacetate sodium salt (EDTA)] pH 8.5, and homogenized with a warring blender. The homogenate was iltered through 3 layers of cheesecloth and then 3% Triton X-100 was added (v/v). To the homogenate, a mixture of 25% chloroform and 25% carbon tetrachloride (v/v) was added slowly and stirred for 30 min. followed by low speed centrifugation (10000 rpm/30 min) at 4 °C. To the aqueous phase, 8% polyethyleneglycol (PEG), (mol. Wt. 6000) and 3% NaCl (w/v) were added and stirred for one hour at 4°C and subjected to 10000 rpm/30 min at 4 °C centrifugation using Beckman J-21 centrifuge and Type JA-14 rotor. The pellets were suspended in 0.05 M sodium citrate buffer, pH 7.5 and left overnight at 4 °C with occasional stirring. The resuspended pellet was centrifuged at 10000 rpm for 30 min at 4 °C. Further puri ication was obtained by a second precipitation with PEG, to the supernatant 8% PEG and 3% NaCl (w/v) were added and stirred for one hour at 4 °C and subjected to 30000 rpm/120 min centrifugation at 4 °C. The obtained pellets were resuspended in sodium citrate buffer and centrifuged at 8000 rpm/15 min (Figure 2). Virus presence was checked biologically by inoculating leaves of C. amaranticolor. U.V. absorption spectrum of the puri ied virus at a range of wave length 200-320 nm with 10 nm interval was recorded spectrophotometrically using Shimadzu UV-160 a spectrophotometer. A260/280 and A280/260 as well as virus concentration were estimated. The virus yield was calculated according to Noordam [26]  Antiserum production for ZYMV Antiserum to ZYMV was produced in New Zealand white rabbit by four weekly intramuscular injections with puri ied virus as Younes [14] with slid modi ications. For each injection, virus concentration at 0.65 mg/ml was emulsi ied with an equal volume of Freund's complete adjuvant in the irst one and for subsequent injections with Freund's incomplete adjuvant. The rabbit was bled 2 weeks after the ifth injection. The ZYMV antiserum obtained was stored in the presence of 0.05% sodium azide at -20 °C. The titer of ZYMV antiserum was determined by using indirect ELISA as described previously. Extracts from infected and healthy squash plants were diluted with coating buffer to 1:10. Serial dilutions of double fold up to 1:819200 of antiserum from cross-adsorption with iltered extracts from healthy tissues diluted 1:20 in serum buffer were used as [24].

Reactions of diagnostic hosts
Diagnostic hosts reacted with symptoms similar to those produced by ZYMV. The virus induced chlorotic local lesions without systemic spread on C. amaranticolor, (Figure 3d) and mosaic, yellowing, blistering and deformation on squash Eskandrani (Figure 3c). These symptoms are similar to those observed on naturally infected squash plants ( Figure  3b). Squash c.v. Eskandrani showed severe yellow mosaic and malformation with blistering and shoe string battern and necrotic local lesion observed on inoculated cotyledons (Figure 3a)

Serological reaction
ZYMV antiserum positively reacted with the isolated virus when used at a dilution of 1:500 in indirect ELISA. No positive reaction was detected with antisera to CMV and WMV-2 as determined by indirect ELISA (Table 1).

Aphid transmission
The virus was transmitted none persistently with Aphis gossypii Glover, A. neri and Myzus persicae with average transmission rates of 60%, 70% and 90% respectively when ten viruliferous aphids were used on each test plant ( Table 2).

Photometrical characters and yield of the purifi ed virus
The Squash fruits isolate of ZYMV was puri ied by ultracentrifugation and PEG. The absorption spectrum of the puri ied virus isolate determined through Jon Way 6405 UV/VIS spectrophotometer, was typical for nucleoprotein ( Figure 4). The U.V. absorption spectrum of the puri ied virus preparation revealed typical spectrum of nucleoprotein. The ratios of A260/280 and A280/260 were 1.1 and 0.91 respectively. Concentration of the virus in the preparation was estimated using an extinction coef icient of E 260 0.1% = 2.4. The yield of the puri ied virus was about 1.62 mg/100 g fresh weight of squash leaves. Inoculum prepared from such puri ied virus induced numerous local lesions on inoculated leaves of C. amaranticolor were observed.

Production of ZYMV antiserum
The antiserum against ZYMV was produced. Its titer was determined by indirect ELISA. Positive ELISA values were obtained up to dilutions of 1:409600 and not with 1:819200 ( Figure 5).

Discussion
On the principle of symptoms developed on diagnostic hosts and serological reactions with antisera to CMV, WMV-2 and ZYMV were detected by indirect ELISA. The virus isolated from naturally infected Squash plants, grown in Abees Region, Alexandria governorate, with the symptoms of mosaic, yellowing, blisters and stunt symptoms, was identi ied as ZYMV. In Egypt, ZYMV was isolated from naturally infected squash plants [9][10][11][12][13][14][15]. ZYMV showed severe mosaic, yellowing, blistering and leaf deformation on most of cucurbits tested. ZYMV was readily sap transmissible to a limited range of hosts; these results are in harmony with the inding of Lisa and Lecoq and Purciful, et al. [1,28]. Gomphrena globosa reacted negativily with ZYMV isolated from squash, the same was found by Al-Shahwan and Younes, 2003 [7,14]. ZYMV is ef iciently transmitted from plant to plant by aphids in a non-persistent manner [2]. The transmission ef iciency varies between different aphid species [21] by Aphis gossypii [2,11,12,29]. The present study showed also that, in addition to Aphis gossypii, the squash isolate of ZYMV is transmited by Aphis neri and Myzus persicae. The puri ication of the isolated virus was carried out to determine the photometrical characters of the puri ied virus and to prepare speci ic antiserum. The yield of the virus as well as its speci ic photometrical data such as A260/280 and A 280/260 fall in the range reported for ZYMV [2,11,12,14,19,30,31], however, the slight differences in absorbance ratios as well as yield of the virus of ours and theirs may be attributed to the method used in puri ication as precipitation by PEG and ellipsis of inal step for further puri ication such as ultracentrifugation or density gradient centrifugation [4,19,31,32]. Antiserum against the isolated ZYMV was produced and found to have titer of 1: 4096 X 10 5 Figure 4: Ultra Violet absorption spectrum of ZYMV purifi ed from Cucurbit pepo infected leaves.   as determined by indirect ELISA [14,33]. While, Bhargavi, et al. [31] was performed DAC-ELISA and the positive reaction up to 1/5000 titre of ZYMV antiserum was observed.

Conclusion
Squash is one of the important crops of cucurbitaceae grown in northern of Egypt. During ield survey stunting of plants, yellow mosaic, blistering of the leaves and fruit deformation was observed. Both the natural symptoms on squash and arti icial induced symptoms on squash seedlings were similar. In indirect ELISA with ZYMV antiserum it was reacted positively. Puri ication of ZYMV from infected squash leaves was puri ied by ultra-centrifugation with PEG. A good yield was gained from the infected leaves. A highly titer was observed by indirect ELISA.