Prevalence , mechanical transmission and impact of environmental conditions on cucumber mosaic disease in Sindh

Cucumber mosaic virus (Genus: Cucumovirus, Family: Bromoviridae) is major pathogen of cucumber (Cucumis sativus L.) in Pakistan and may cause up to 100 % losses. Therefore, this survey based study was planned to determine the disease intensity in fields, mechanical transmission for confirmation of virus and analysis of influence of environmental conditions on the disease development. The disease was monitored on 30 plants at each site in district Hyderabad, Tando Allahyar, Thatta and Shaheed Benazirabad. There were seven sites at each district for observations. Metrological data collected from Regional Agro Metrological Centre, ARI Tandojam Sindh was correlated with the disease intensity. The disease incidence was ranging, 4077, 3770, 63100 and 3090%, at district Hyderabad, Tando Allahyar, Thatta and Shaheed Benazirabad, respectively. District wise, there was highly significant variation among incidence of cucumber mosaic disease (Df= 3, F= 20.59, P= 0.0000). Maximum disease incidence was recorded at Thatta (81.0%). However, overall severity of cucumber mosaic in all the surveyed districts was 2-score. On mechanical inoculation, after 15 days necrotic lesions were only found on the leaves of Chenopodium amaranticolor inoculated with sap of symptomatic leaves. Maximum, minimum temperature (°C) and evaporation showed significant relation (r= 0.883, P= 0.0053, r= 0.664, P= 0.0482 and r= 0.827, P= 0.0119, respectively) only with the disease incidence but not with severity. Cloudiness was highly significantly related with disease severity (r= 0.975, P= 0.0002) but there was non-significant relationship with disease incidence (r= 0.600, P= 0.0705).


Introduction
Cucumber (Cucumis sativus L.) is a famous vegetable under family, Curcubitaceae.Cucumber is being grown thought out the world including Pakistan [1, 2].In Pakistan, during 2016 cropping season, the crop is cultivated in an area of about 3,482 ha with a total production of 56,204 tons [3].Cucumber production is adversely affected by biotic and abiotic factors.Among biotic factors, cucumber mosaic virus (CMV) (Genus: Cucumovirus, Family: Bromoviridae) is one of the devastating plant virus with wide host range, spread and causing huge production losses.Such features are considered to be key factors for the virus epidemics in host plants.CMV may cause up to 100% losses in susceptible host plant under favourable environmental conditions [4].The CMV's extensive host rang contains several species of crops, which play a role as a source of CMV, also for the spreading of virus the various crops.The reports proved that CMV infects about 1287 species of plants, which includes fruits, cucurbits, vegetables, ornamental, medicinal and weeds.The most important affected vegetables by CMV are included cucurbits, tomatoes and chillies [5,6].Plants can be attacked by the virus throughout cropping season but six to eightleaf stage is consider ideal for the attack.The virus may colonize vascular tissues, disturb chlorophyll and internode growth of plants if attack initiated in seedlings.New leaves are slightly mottled, wrinkled and distorted with a slight downward curling of the edges.On the older leaves greenishyellow lesions along with leaf veins are induced due to infection of the virus.Leaves are curled downward with wrinkled leaf lemina.Eventually, a yellow-green mottling develops.Occasionally, only the leaf tip turns yellowish without a sharply defined mottle.After infection, plant growth is stunted with shortened internodes.In lush growing crops, infected vines may wither before showing signs of distortion and may die within seven days.Few fruits are set once infection occurs and fruit that does set has a yellow-green mottle on the stem, gradually extending over the entire fruit surface, interspersed with dark green areas that are usually raised and wartlike.Occasionally, fruit becomes smooth, green-white and blunt at the end.Soggy pickles and bitter taste are the symptoms in cucumber infected by CMV [7].Therefore, this survey based study was planned to determine the disease intensity in fields, mechanical transmission for confirmation of virus and analysis of influence of environmental conditions on the disease development.

Materials and methods
Hyderabad, Tando Allahyar, Thatta and Shaheed Benazirabad district were selected for visual assessment of the disease under field conditions at commercial farmer fields.Fields were surveyed with the willingness of farmers to participate in the survey and easily feasibility to conduct observation for covering main cucumber growing areas.Fields were managed by growers as recommended production practices.Ten cucumber plants with three replications (N = 30) at each location were observed for the symptoms of the disease.The disease incidence percentage was calculated by dividing number of cucumber plants showing mosaic symptoms with total number of plants examined for the disease.Severity of the disease was measured by using 1-5 rating scale [4].Mechanical transmission of CMV was done on Chenopodium amaranticolor by rubbing the extract on wounded leaves.CMV infected leave tissues (5g) were collected and crushed in sterilized pestle and mortar in 10 ml phosphate buffer (2.4 gm of KH2PO4 and 5.4 gm of KH2PO4 in one litre water).Carborundum powder was used to provide tiny wounds for entry of the virus particles in the leaf tissues, then leaves were inoculated with prepared sap.After inoculation leaves tap water was applied to remove excess sap on wounds and plants were shifted in dark for symptoms development as described by [4].Plants were kept under observation until the symptom inducement.There was a control treatment which was treated with Carborundum powder but sap was not applied.Metrological data was collected from Regional Agro Metrological Centre, Agriculture Research Institute, Tandojam Sindh.Analysis of variance was performed to determine least significant difference among the disease parameters and aphid population α = 0.005.Test was performed using STATISTIX v. 8.1 software (Analytical Software).Correlation of the disease parameters with environmental factors was performed using PRISM v. 5.01 (GraphPad Software).

Results and discussion
The incidence and severity of cucumber mosaic disease In district Hyderabad, maximum the disease incidence 77.0, 73.0 and 63.0% was recorded at locations of Amar Town-1, Amar Town-2 and New Hyderabad City, respectively followed by Rahuki-2 and Channel (50.0%) (Figure 1).Rahuki-1 locality showed significantly lower (40.0%)incidence of cucumber mosaic disease followed by locality of Detha Stop (47.0%) (Figure 1).Ibrahim Shah-2 location of district Tando Allahyar showed significantly maximum 70.0% incidence of cucumber mosaic disease, followed by Khawaja Stop (67.0%),Mirpur Bypass (63.0%),Tando Allahyar and Bukera Road (57.0%).Minimum incidence of cucumber mosaic disease was recorded 37.0% at location of Keeria Shakh of district Tando Allahyar (Figure 2).There was significant variation in the disease incidence (%) at surveyed locations of district Thatta (Df= 6, F= 4.19, P= 0.0167).Cucumber crop grown nearby Bharosal Colony of Thatta was 100% infected with the virus (Figure 3).The disease incidence at Var was 90.0%, which was non-significantly differing with incidence recorded at Karimabad (83.0%).Minimum 63.0% incidence of cucumber mosaic was recorded at DC Gull Hassan Nahiyoo followed by locality of Ghulamullah (80.0%),Thatta (76.0%) and Mirpur Sakro (73.0%) (Figure 3).The cucumber mosaic disease incidence showed highly significant variation at surveyed locations of Shaheed Benazirabad (Df= 6, F= 31.27,P= 0.0000).The maximum disease incidence was recorded at Kazi Ahmed (90.0%), followed by Bhalu Ja Kuba-1 (70.0%) (Figure 4).Sahpur Jahania and Dolatpur Safan showed 50.0%incidence of the disease.Minimum incidence 30.0% was recorded at Bhalu Ja Kuba-2 followed by Deh Phulail and Chanesar Stop (37.0%) (Figure 4).District wise, there was highly significant variation among incidence of cucumber mosaic disease (Df= 3, F= 20.59, P= 0.0000).Maximum disease incidence was recorded at Thatta (81.0%) (Figure 5).However, incidence at district Shaheed Benazirabad (52.0%) was lower and nonsignificantly differs with Tando Allahyar (56.0%) and Hyderabad (57.0%) (Figure 5).In Hyderabad district the maximum severity 3-score was recorded at Amar Town-1 and minimum 1-severity score was determined at field of Rahuki-1, remaining showed 2-severity score (Figure 6).Mirpur Bypass locality of Tando Allahyar district showed maximum 3 severity score, whereas other all fields of the district showed 2 severity score (Figure 7).In district Thatta the maximum cucumber mosaic severity 3-score was recorded at Bharosal Colony and Karimabad, whereas at other remaining fields it was 2 score (Figure 8).Kazi Ahmed locality of district Shaheed Benazirabad showed maximum 3 severity score followed by Dolatpur Safan, Bhalu Ja Kuba-1 and Chanesar Stop with 2 severity score (Figure 9).The locality of Sahpur Jahania, Deh Phulail, and Bhalu Ja Kuba-2 showed minimum 1 severity score (Figure 9).Overall severity of cucumber mosaic in all the surveyed districts was 2score (Figure 10).It is concluded from our study that the cucumber mosaic disease is prevailing in all the sites surveyed in four districts of Sindh.The incidence percentage was varying site to site and some were showing nonsignificant variations in the incidence values.District wise, there was highly significant variation among incidence of cucumber mosaic disease (Df= 3, F= 20.59, P= 0.0000).Maximum disease incidence was recorded at Thatta.However, incidence at district Shaheed Benazirabad was lower, but non-significantly differing with Tando Allahyar and Hyderabad.Similarly variation in the disease severity at site to site was found in survey.Overall severity of cucumber mosaic in all the surveyed districts was 2-score.Similar, studies are conducted at different regions of the world [20-22].Iqbal [18] monitored and recorded CMV incidence in chili pepper in all over Pakistan during the year 2006-07.During survey 706 total number of samples were collected from various provinces (Baluchistan 207, Sindh 191, KPK 51 and Panjab 257) and has been tested through DAC-ELISA.The results of ELISA showed that CMV occurs all over the Pakistan with 44.7% of relative incidence.Sindh province found most effected with (51.8%) maximum incidence of CMV and Baluchistan province (17.47%) which is followed by Punjab and KPK with (8.5%) and (7.8%) incidence respectively.In Sindh and Baluchistan provinces there was not a single district free from CMV infection.Aphid has also been identified as positional vector of the virus [23-25].

Pathogenicity test for confirmation of viral infection
Pathogenicity test was performed on Chenopodium amarenticolor indicator plants by mechanical inoculation method.After about one month of sowing plants were rubbed with sap of symptomatic plants collected from field.In control treatment the plants were rubbed with carborandum powder but sap was not applied (Figure 11).After 15 days of inoculation necrotic lesions were found on the leaves of inoculated with sap but in control plants leaves were free of necrotic lesions (Figure 12).Such results are in accordance with [10, 26] who performed mechanical transmission of CMV on indicator plant (C.amarenticolor) which was grown in an insect proof net house using five inches earthen pot.The soil used in the pot is amended with manure and fertilizer as per recommendation.Three different buffers namely 0.01M potassium phosphate buffer (pH 7.1), sodium phosphate buffer (pH 7.1) and sodium potassium phosphate buffer (pH 7.1) are used to extract sap of infected chilli leaves in the inoculation test.In each case, 5g fresh leaves collected from infected chilli plants is macerated using 50 ml aforesaid buffers for sap extraction.On inoculation, necrotic lesions were observed on inoculated plants.

Correlation between cucumber mosaic viral disease and environmental factors
Impact of environmental conditions like minimum and maximum temperature (℃), relative humidity (%), cloudiness and evaporation on cucumber mosaic disease was tested.Minimum Temperature (℃) showed significant correlation with the disease incidence (r 2 = 0.664, P= 0.0482) but it was non-significantly correlated with severity of cucumber mosaic disease (Table 1).Similarly, maximum temperature (℃) showed significant correlation with the disease incidence (r 2 = 0.883, P= 0.0053) but non-significant with the disease severity (r 2 = 0.148, P= 0.4522) (Table 1).Cloudiness was highly significantly correlated with disease severity (r 2 = 0.975, P= 0.0002) but there was no relationship with disease incidence (r 2 = 0.600, P= 0.0705) (Table 1).The disease incidence (%) was dependent with evaporation (r 2 = 0.827, P= 0.0119) but not with severity (r 2 = 0.599, P= 0.0708) (Table 1).Our results showed that minimum and maximum temperature (℃) were significantly correlated with the disease incidence but it was non-significantly correlated with severity of cucumber mosaic disease.Cloudiness was highly significantly correlated with disease severity but there was no relationship with disease incidence.The disease incidence (%) was dependent with evaporation but not with severity.Previously, Khan et al. [19] in a field study showed impact of environmental conditions on development of CMV that relative humidity and rainfall are important factor for the disease development while temperature is negatively correlated with disease development.Similarly, Imran et al. [27] showed increase in ToMV severity with temperature but decreased with increase in relative humidity.Such studies are also conducted by various authors for many different viral diseases, like Zeshan et al. [28] showed direct relationship of TLCV with temperature but it was not increasing with increase in relative humidity, while rainfall and wind speed were not related with disease development.Likewise, Haider et al. [29] assessed relationship of epidemiological factors among whitefly population and tomato leaf curl virus disease (TLCVD) incidence was studied on six tomato varieties.Epidemiological factors have significant role for the development of whitefly and TLCVD incidence during 2016.Temperature (maximum and minimum) had positively significant relationship for whitefly population and disease incidence.With increase in maximum temperature whitefly population showed 77-99% variability while with minimum temperature it was 66-99% variability.TLCVD incidence showed 84-96% variability with minimum temperature while 86-96% with maximum temperature.Relative humidity had negatively significant relationship with whitefly population and disease incidence.With increase in relative humidity whitefly population and TLCVD incidence decreased.Relative humidity showed 75-96% and 83-93% variability in case of whitefly population and disease incidence respectively.Wind speed and rainfall have very poor relationship with vector population and disease incidence.

Table 1 . Correlation of environmental conditions with the incidence and severity of cucumber mosaic disease Disease parameters
Necrotic lesions on inoculated leaves of indicator plant Chenopodium amarenticolor were produced after 15 days of mechanical inoculation.Impact of environmental conditions like minimum and maximum temperature and evaporation showed significant correlation with the cucumber mosaic disease incidence.Cloudiness was highly significantly correlated with disease severity, whereas relative humidity was not related to the disease development.