Incidence and distribution of Zucchini yellow mosaic virus (ZYMV) infecting Cucumber ( Cucumis sativus L) crop in Pothowar, Pakistan

Zucchini yellow mosaic virus (ZYMV) is one of the most destructive virus of cucurbits and found throughout the cucurbits growing areas of Pothowar region of Pakistan. Overall 300 samples were randomly collected from 40 cucumber growing fields from all over the pothowar region during two consecutive years (2015-16 and 2016-17). All the samples were screened by Double Antibody Sandwich- Enzyme Linked Immunosorbant Assay (DAS-ELISA). Relative incidence of ZYMV recorded in two consecutive years was 60.6% and 66% respectively. The highest disease incidence of ZYMV was 77% during 2015-16 in Chakwal while during 2016-17 highest disease incidence was 71% recorded from Rawalpindi. No district of pothowar region was found to free from viral infection. Among these samples weed flora was also collected where available and subjected to DAS-ELISA for conformation as alternate host of virus. Reaction plants were also used during experiment for virus conformation and for pathogenicity test. The core interest of this study was to explore the incidence of ZYMV infecting cucumber in pothowar region through serological assay.


Introduction
Cucumber (Cucumis sativa L) is one of most important crops belongs to family Cucurbitaceae and for over 3,000 years cultivated by man [1, 2]. It is a soft, succulent plant having high content of water and has large canopy of leaves which covers the fruit. The vines are grown on trellises or stakes. The fruit is roughly cylindrical, elongated having tapered ends, mostly used in unripe condition, usually eaten as salads or pickled and are in tropical region also stewed [3]. As for monetary importance, it has fourth positions after tomatoes, cabbage and onion in Asian continent [4] and in Western Europe, it is second most important vegetable crop after tomato [5]. Cucumber is enriched with nutrients and it has also contained, vitamin C, thiamine niacin, phosphorous, iron, calcium. The low production is hampered by biotic and abiotic factors and unavailability of resistant varieties. Among the biotic factors viral infection is a standout amongst the most critical reasons of ailment , wounds produce during mechanical weeding operations also enhance the transmission of ZYMV from plant-to-plant and it may also carried out by vertebrates like rabbits. However, there is no experimental evidence to favor these ideas. Diseased caused by plant viruses can be controlled by used of highly resistant varieties and by use of advance technique like nanotechnology. Present study was conducted to evaluate the prevelance and distribution of ZYMV in pothawar region by enzyme linked immuno-sorbent assay (ELISA).

Materials and methods
Overall forty cucumber fields were randomly visited in summer season during 2015 -16 and 2016-17 in four districts of pothwar region, viz Rawalpindi, Attock, Chakwal, Jhelum and capital territory (Fig.  1). Random sampling was done in such a way that leaf and fruit sample of cucumber exhibiting symptoms like yellow mosaic, necrosis, blister, distortion, fan-leaf appearance, shoestring, stunting was collected. Overall 300 samples were collected. Weeds (Datura spp, Kulfa, (Portulaca oleracea) Deela (Cyprus rotundus) were also collected from cucumber fields where they were available and subjected to DAS-ELISA to investigate as an alternate host of ZYMV in the fields. Along with weed flora some reaction plants are also tested for virus confirmation and symptoms development. However, in calculating the incidence data of the virus, they were not considered. All samples collected were placed in polythene bags, stored at 4 o C and subjected to DAS -ELISA for virus confirmation. Alkaline phosphatase-conjugated anti-ZYMV antibodies (Bioreba AG) were added and incubated overnight at 4 o C, after that incubation with p-nitrophenyl phosphate (MP Biomedicals, Inc. Ohio, USA) is done at room temperature for 1 h. Automatic ELISA Reader (HER-480 HT Company (Illford) Ltd., UK) is used to measure absorbance values (405 nm). Samples were considered significantly positive for ZYMV infection when the ELISA absorbance value was twice or higher than the average absorbance value of the healthy tissue as well as negative control. ZYMV ELISA kit was equipped with commercial positive and negative controls (Bioreba conditions. Symptoms development was investigated post inoculation after every two days up to one month.

Results
Total 40 fields were surveyed, and 300 plant samples were randomly collected during survey process in two consecutive years 2015-16 and 2016-17. All the samples collected, were subjected to DAS-ELISA and results of serological test showed that there was no pothowar district found free of ZYMV prevalence. Relative incidence of ZYMV was recorded in two consecutive years was 60.6% and 66% respectively as shown. (Fig. 2)

ZYMV prevalence during 2015-16 in pothowar region
Mostly cucumber crop is growing in all district of pothowar region and capital territory Islamabad throughout the season. During survey relative incidence of ZYMV was 66%, 65%, 50%, 77%, 57% recorded in Islamabad, Rawalpindi, Attock, Chakwal, and Jhelum respectively. The highest disease incidence was recorded 77% in Chakwal. (Fig. 3). During survey it is noted that ZYMV was present in all over the pothowar region. The basic reason of its persistence is its inoculum which remains in the fields and shifts from one location to another due to the transportation of infested materials by human activities whereas for the long range spreading of inoculum is caused by viruliferous aphids through wind to a new cultivated area. During survey aphid populations are also observed on infected plant. Unluckily, farmer in this region are not aware of how viruses are dispersing from one plant to another and do not know about management strategies to control virus transmission. It is also noted that favorable environmental conditions also aggravate aphid population.

ZYMV prevalence during 2016-17 in pothowar region
During 2016-17 highest disease incidence was recorded as 71% in Rawalpindi followed by 68% in Chakwal 64% in Islamabad, 58% in Attock as well as in Jhelum. (Fig. 4). During this year disease incidence increased in most of the areas of pothowar region. Increase in disease incidence is attributed to climatic conditions throughout the year. Day by day increase in temperature helps the virus vector (aphids) to proliferate and transmit the virus more rapidly.

Comparison of ZYMV prevelance in different areas of pothowar region during two consecutive year
Results shows that incidence of ZYMV mostly increase during 2016 as compare to 2015 in some districts. (Fig. 5) Increase in disease incidence is attributed to climatic conditions throughout the year. Day by day increase in temperature helps the virus vector (aphids) to proliferate and transmit the virus more rapidly. It is also attributed to lack resistant verities as well as poor management practices by the farmer and dis-functionality of agriculture extension department.     [25] ZYMV has highly devastating effect on cucurbits crop in this region so there is need to make strategies to control this notorious virus. Weeding to eradicate the virus source in fields and control of aphid vector may remain helpful to reduce the dissemination of virus. As compare to other viruses like CMV, reservoir of ZYMV is very few so it is easy to control through weeding [26] Application of insecticide also reduce the population of aphis vector [27,28]. Plastic mulches may also repel the aphis and may delay the spreading of virus [29,30] but it may also affect the growth of plants. One of the most important and environment friendly method to control the virus is development of resistance cultivar and certified seed.

Conclusion
In conclusion present study indicate that Zucchini yellow mosaic virus (ZYMV) prevail in throughout the pothwar region of Pakistan. There is need to develop management strategies against such catastrophic pathogen. There is need to develop resistance cultivar of vegetables against this virus.