IDENTIFICATION OF CUCUMBER MOSAIC VIRUS (CMV) ON ...

© by PSP Volume 24 – No 2. 2015

Fresenius Environmental Bulletin

IDENTIFICATION OF CUCUMBER MOSAIC VIRUS (CMV) ON SQUASH (CUCURBITA PEPO L.) CULTIVARS IN LAKES REGION OF TURKEY Nejla Yardımcı1,*, Handan Çulal Kılıç1 and Aynur Kör1 1

Süleyman Demirel University Faculty of Agriculture Department of Plant Protection, Isparta 32260, Turkey

ABSTRACT Lakes Region is an important area in cucurbit production of Turkey. During surveys conducted in the 20112012 growing season, field-grown squash plants with symptoms of mosaic, chlorotic mottling, vein banding, blistering and leaf malformation were observed. A total of 268 squash samples were collected from various locations in this region. Cucumber mosaic virus (CMV), was detected in squash, using enzyme-linked immunosorbent assay (ELISA), double–stranded RNA (dsRNA) analysis and Reverse transcription polymerase chain reaction (RT-PCR) methods. ELISA tests showed that among 268 field samples tested, 54 were infected with CMV. ELISA-negative samples were tested by RT-PCR for CMV. All samples were positive for CMV. As a result of dsRNA analysis, specific 3 CMV RNA profiles were obtained. KEYWORDS: Cucurbits, Cucumber mosaic virus, ELISA, RT- PCR, dsRNA.

1. INTRODUCTION Lakes region of Turkey is an important area for vegetable production due to its suitable climate and soil conditions. In this region, various vegetables are produced in small family gardens or large commercial areas. Squash is an important crop in this region. It is affected by many biotic and abiotic factors. Among these, virus diseases are also important and cause economic losses. Large number of virus diseases were reported on cucurbit crops in the world. Among these, Cucumber mosaic virus (CMV) is the most destructive and widespread virus disease of cucurbits [1]. CMV is a plant virus with the broadest host range in nature, infecting more than 1000 species in about 80 monocotyledonous and dicotyledonous plant families and causes epidemics with important yield losses in many crops, par* Corresponding author

ticularly vegetable crops in temperate regions [2, 3]. CMV causes a systemic infection in most host plants, but may remain symptomless in some crops [4]. Symptoms of cucumber mosaic can vary greatly depending on the host, virus strains, presence or absence of virus satellite RNAs. Symptoms ranged from yellowing, mosaic, malformation on the leaves to ring-spots or line patterns. Due to early infections of CMV, severe foliar mosaic symptoms and fruit deformations is observed on squash, and especially the latter symptom has negative effects on marketability [5, 6, 7]. Aphids transmit CMV to many weeds, flowers and other plants. More than 80 aphid species, including Myzus persicae (Sulz.) and Aphis gossypii (Glover.), are capable of transmitting the virus in a nonpersistent, stylet-borne manner. The virus can also be transmitted through seeds or bulbs, dodder and grafting in some plant species [4, 8]. In addition, virus can be transmitted by mechanically on workers' hands or on tools [4]. Some cucurbit viruses has previously been detected in different regions of Turkey [9, 10, 11 12], but there is not a study on CMV in squash production areas in the Lakes region. This is the first report of CMV infecting squash plants in Lakes region in Turkey. Even though the region is an important vegetable production area, information about viral diseases in this region is scarce. We have initiated a virus-screening program to determine the common viral diseases in squash fields in the Lakes region of Turkey.

2. MATERIALS AND METHODS 2.1 Field survey

Survey was conducted in 2011-2012, in squash fields of Lakes region. A total of 268 squash plants exhibiting virus-like symptoms were collected from 11 locations during surveys in this region. Symptoms of plants were recorded and then were stored in a freezer at -200 C until detection tests were done.

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2.2 DAS-ELISA

All samples were tested by ELISA for the presence of CMV using specific ELISA detection kit (Agdia, USA). Absorbance values of alkaline phosphatase were measured at 405 nm with microplate reader (EL X 800 universal Microplate Reader Bio-Tek Instruments, Inc.B-2610, Wilrijk, Belgium). Samples with absorbance values greater than twice the mean absorbance value obtained from healthy controls were considered positive for virus.

from squash plants. During the field surveys, virus-like symptoms including yellowing, mosaic pattern of light and dark green, yellow spotting, malformation symptoms on the leaves, ring-spots or line patterns on leaves or fruits were observed (Figs. 1-4) .

2.3 RT-PCR

In the ELISA study, 35 negative samples close to the positive value were tested by RT-PCR for presence of CMV. Total RNA was extracted from fresh leaves of squash samples. RT-PCR was performed using a One Step RT-PCR Kit (Bio Basic, Canada Inc). RT-PCR was performed in a 50 μl reaction mixture containing, 21μl H2O, 25 μl 2x1 PrimeScript One Step RT-PCR buffer (containing dNTP mixture, One step Enhancer solution), 2 μl Prime Script 1 step enzyme mix, 1 μl 20 Μm primers. RT-PCR of CMV coat protein gene portion of approximately 678 bp were amplified [13].

FIGURE 1 - Mosaic symptoms observed on CMV-infected squash leaves.

Primer I: F- 5-TTGAGTCGAGTCATGGACAAATC-3; Primer II: R-5-AACACGGAATCAGACTGGGAG-3. Thermocycling program was carried out as follows: 50ºC for 30 min, 94ºC for 2 min, then 30 cycles of 94ºC for 30 second, 55ºC for 30 second and 72ºC for 1 min, followed by 72 ºC for 3 min. PCR products were separated in 1% agarose gel by electrophoresis, stained with 0.5 μg/ml ethidium bromide solution. DNA markers (1 Kb DNA ladder, TAKARA) were used in each electrophoretic run. 2.4 DsRNA analysis

A previously reported dsRNA extraction method was modified [14]. Briefly, about 5 g leaf tissue were first grounded in a mortar and homogenized in 10 ml of STE buffer (0.1 MNaCl, 0.05 M Tris -HCl, 0.1 mM EDTA, pH 6.8), then were mixed with 10 ml phenol, chloroform-pentanol (25:1 v:v),with 2% bentonit and 10% SDS. The mixture was centrifuged at 10,000 rpm for 20 min, 95% ethanol was added into the supernatant and after overnight incubation at 4ºC it was fractionated on Whatman CF-11 cellulose columns in the presence of ethanol to select dsRNA. Then, dsRNAs bound cellulose columns were eluted from the cellulose in ethanol free STE buffer, concentrated by ethanol precipitation, resuspended in 50 μl sterile water and were stored at − 80ºC.

FIGURE 2 - Yellow spotting symptoms observed in squash leaves.

3. RESULTS 3.1 Field survey

Survey was conducted in 11 different districts of Lakes-Region. A total of 268 leaf samples were collected

FIGURE 3 - Ring-spots and deformation observed in squash fruit.

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were negative in DAS-ELISA. In RT-PCR positive samples produced expected amplification size of band with 678 bp as observed on agarose gel electrophoresis (Fig. 5, 6)

FIGURE 4 - Blister, roughness and deformation observed in squash fruits.

For the control of virus diseases, primarily their diagnosis is required. Various methods are used for this purpose. PCR technique is much more sensitive than other methods [15, 16]. In this study, ELISA, dsRNA analysis and RT-PCR methods are used for CMV diagnosis.

FIGURE 5 - Agarose gel electrophoretic stained by ethedium bromide analysis of RT-PCR amplification of CMV cp gene from infected squash. M: Marker 1 kb DNA ladder (TAKARA). P: Positive control, N: Negative control, Lane1-11 infected field samples.

3.2 Detection of CMV by DAS-ELISA

All squash leaf samples were tested for CMV using commercially available DAS-ELISA kit. The results showed that 54 out of 268 samples (20.15 %) were infected with CMV. ELISA test showed the presence of CMV infection in the Lakes region in Turkey. ELISA is widely used for the detection of plant viruses [17]. Even though ELISA is a reliable test for routine diagnostic screening, it has low sensitivity comparing to RT-PCR [15, 18, 19]. 3.3 Detection of CMV by RT-PCR assay

Total RNAs were extracted from fresh leaves of 35 squash samples and then tested by RT-PCR. All tested samples (35) were determined as positive. These 35 samples

FIGURE 6 - Agarose gel electrophoretic stained by ethedium bromide analysis of RT-PCR amplification of CMV cp gene from infected squash. M: Marker 1 kb DNA ladder (TAKARA). P: Positive control, N: Negative control, Lane1-12, infected samples.

RNA 1 RNA 2 RNA 3

FIGURE 7 - CMV dsRNA analysis on agarose gel by electophoresis Lane 1, Nicotiana tabacum Xanthii, Lane 2. Squash sample, Lane 3. Healthy plant, M. Marker (Lambda DNA Hind III ladder).

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3.4 DsRNA analysis

REFERENCES

RT-PCR positive leaf samples were used as inoculum and applied to the indicator plants. Showing systemic infections Nicotiana tabacum Xanthii plants were used in dsRNA isolation and analysis studies. For this purpose, as previously reported dsRNA isolation method [14] was modified and used for dsRNA isolation from samples. Virus specific RNAs were analyzed on 1.2 % agarose gel by electophoresis at 100 volt for 2.5-3 hr. As a result of dsRNA analysis, specific 3 CMV RNA profiles were obtained (Fig. 7).

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4. DISCUSSION AND CONCLUSION Several viral like sympotms were observed during the survey including; yellowing, mosaic, line patterns, malformation and ring-spot symptoms on the leaves and/or fruits. Observed symptoms inducated that squash plants in the visited fields might be infected with CMV and/or other viruses. Similar symptoms were previously reported in different studies [3-6]. ELISA and RT-PCR results revealed that CMV was present in the Lakes region. In this study as a result of the ELISA test, CMV was detected in 20.15 % of the tested samples. It was reported in the previous studies performed in other regions of Turkey that CMV was found in vegetable leaf and seed samples in different rates. CMV infection rates were reported as 20.6% on cucurbits in Samsun province, 19.9% on watermelon and 7% on melon plants in Thrace region, 36.8% on cucumber seed samples and 18.5% on squash and melon seeds in the Aegean region [10, 20, 21]. In order to confirm the presence of CMV, RT-PCR was carried out on 35 ELISA negative samples. Samples were tested by RT-PCR, and all were found positive for CMV. Expected size of band with 678 bp was observed markedly on agarose gel electrophoresis (Fig. 5, 6, 7). This results indicate that RT-PCR is more sensitive and specific than DAS-ELISA test. The results obtained in this study show the successful use of RT-PCR as a rapid assay for direct detection of CMV in infected tissues of squash samples. Sensitivity of RT-PCR lead many researchers to relay on it in viruses diagnosis [15, 18, 19]. In this study, serological and molecular assays revealed that CMV was present in squash plants produced in this region. Biological, serological and molecular assays have generally been used for identification of vegetable viruses. Although ELISA is the preferred assay for routine virus detection, RT-PCR has increasingly been used for detection and identification of viruses due to higher level of sensitivity [18, 19, 22]. According to our knowledge, CMV was not previously detected in any cucurbit crops in the Lakes region. Therefore, this study presents the first report of CMV in Cucurbita pepo L. fields in the Lakes region of Turkey.

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Received: January 30, 2014 Revised: May 06, 2014; June 09, 2014 Accepted: August 04, 2014

CORRESPONDING AUTHOR Dr. Nejla Yardimci Süleyman Demirel University Faculty of Agriculture Department of Plant Protection Isparta 32260 TURKEY Phone: +902462114875 Fax: +902462114885 E-mail: [email protected] FEB/ Vol 24/ No 2/ 2015 – pages 417 - 421

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