Incidence of Escherichia coli in Vegetable Crops and Soil Profile Drip Irrigated with Primarily Treated Municipal Wastewater in a Semi-Arid Peri Urban Area

This study investigates the incidence of Escherichia coli in cauliflower, bitter gourd and soil profile drip-irrigated with municipal wastewater in a semi-arid peri-urban area in India. There were four treatments: drip irrigation with primarily treated municipal wastewater through inline (non-pressure compensating) surface drip (T1), inline subsurface drip (T2), bioline (pressure compensating) subsurface drip (T3) and bioline surface drip (T4). Results revealed that T1 had the highest concentration of E.coli (35 ± 2.66 and 25 ± 2.26 colony forming unit (CFU) g−1) and T3 had the lowest concentration of E. coli (29 ± 2.29 and 18.9 ± 2.04 CFU g−1) for cauliflower and bitter gourd, respectively. In bitter gourd top fruits (1 m above the ground level), the E. coli count was significantly lower (p < 0.05) than in the surface level fruits. There was also a considerable reduction of E. coli counts in bioline drip lateral as compared to the inline drip. A higher concentration of E. coli (470 ± 70.5 and 410 ± 36.9 CFU g−1 soil) was also found in the top soil (0–0.15 m) in T1 treatment, while the minimum (154 ± 13.86 and 95 ± 14.25 CFU g−1) was observed in T3. Hence, bioline drip lateral may be a better option for wastewater irrigation as compared to inline drip to reduce microbial contamination of crop and soil.


Introduction
Substantial attention is being given to the microbial quality of irrigation water because of the increased incidence of gastrointestinal illness caused by contaminated agricultural produce [1]. Various scientific guidelines and standards have been suggested for microbial water quality to minimize the risks associated with irrigation waters [2][3][4][5][6][7]. In developing countries, the presence of pathogens is of primary concern for the wastewater irrigation [8] and has long been considered as a source of risk in the reuse of the effluent water [9]. The urban and semi-urban areas, particularly those lying along the courses of urban drainage systems in the developing countries [10], are becoming main

Location of Experimental Field
The study was conducted in 2010-2011 at the Precision Farming Development Centre (PFDC), Water Technology Centre (WTC), Indian Agricultural Research Institute (IARI), Pusa, New Delhi, India, which is located at 28°37'22" N and 77°8'45" E longitude covering an area of about 475 ha at on average elevation of 230 m above mean sea level. A location map of the experimental site is shown in Figure 1.

Climatic Condition
The climate of the study area is semi-arid and characterized by a hot summer and cold winter. The temperature of the study area varies from 7.6 °C to 45.2 °C with an average annual temperature of 24 °C. The mean annual rainfall based on a 100 year record is 790 mm. About 80% of the annual rainfall is received during the monsoon (June-September).

Soil Properties
Soil samples were collected up to a depth of 0.45 m in 0.15 m intervals. The hydrometer method was followed to determine the sand, silt and clay percentages of soil [42]. The soil of the experimental area was deep and well-drained with a sandy loam texture (United State Department of Agriculture) comprising mean values of 64.53 % sand, 15.13 % silt and 21.37 % clay ( Table 1). The average value of the bulk density of soil was 1.56 g cm ˗3 and the average value of saturated hydraulic conductivity was 1.06 cm h ˗1 .

Climatic Condition
The climate of the study area is semi-arid and characterized by a hot summer and cold winter. The temperature of the study area varies from 7.6 • C to 45.2 • C with an average annual temperature of 24 • C. The mean annual rainfall based on a 100 year record is 790 mm. About 80% of the annual rainfall is received during the monsoon (June-September).

Soil Properties
Soil samples were collected up to a depth of 0.45 m in 0.15 m intervals. The hydrometer method was followed to determine the sand, silt and clay percentages of soil [42]. The soil of the experimental area was deep and well-drained with a sandy loam texture (United State Department of Agriculture) comprising mean values of 64.53 % sand, 15.13 % silt and 21.37 % clay ( Table 1). The average value of the bulk density of soil was 1.56 g cm −3 and the average value of saturated hydraulic conductivity was 1.06 cm h −1 . Soil fertility status is presented in Table 2. The pH values of soil range from 8.24 to 8.46 with an average of 8.36. As per soil reaction classification given by Brandy [43], the soil of the experimental field was moderately alkaline. Soil electrical conductivity (EC) varied from 0.19 to 0.24 deci Semiens per metre (dS m −1 ) with an average value of 0.21 dS m −1 . As per the salt problem classification given by Muhr 1965 [44], based on EC values, the soil of the experimental field was classified as normal. As per nutrient availability classification for available nitrogen, the upper depth (0-0.3 m) of soil fell within the medium category while the lower depth (> 0.3 m) was in a low category [45]. In the case of phosphorous and potassium availability in the soil of the experimental field, it was medium as per the limit suggested by Muhr 1965 [44].

Nursery Raising, Transplanting and Harvesting
Seeds of the Cauliflower: Brassica oleracea var. botrytis (cultivar: Indame 9803) and bitter gourd-Momordica charantia (cultivar: Indame-49) were treated with Bavistin fungicide (manufactured by Biostadt India Limited, Mumbai, India) at 2 g kg −1 of seed. Cauliflower seeds were sown in the first week of October during 2010 while bitter gourd seeds were sown in the mid of March during 2011. A hand sprayer was used to provide frequent irrigation to the germinated seedling in the nursery. During the germination stage of seedling in the nursery, no wastewater was used. Twenty-five-day-old seedlings were transplanted and the distances between plants and rows were 0.4 and 0.6 m, respectively. Harvesting of the cauliflower crop was done in February 2011 and bitter gourd in July 2011.

Crop Water Requirement
Water requirements of both the crops (cauliflower and bitter gourd) were estimated using Panman-Monteith's semi-empirical formula [46]. Weather data were collected from a nearby (40 m) automatic weather station. The actual evapotranspiration was estimated by multiplying reference evapotranspiration with crop coefficient for different months based on crop growth stages. The cauliflower was a 120 days crop and was divided into four growth stages, namely, initial: 35 days, development stage: 30 days, mid-season stage: 40 days and late-season stage: 15 days. The crop coefficients of 0.7, 0.88, 1.05 and 0.95 at initial, development, curd formation and curd maturity stages, respectively, were adopted during the crop season [47]. The same procedure was also followed for bitter gourd crop to calculate the water requirement. The value of the crop coefficient for bitter gourd crop was assumed to be 0.9 during the experimental trial. The irrigation rate was adjusted considering Agriculture 2020, 10, 291 5 of 16 the rainfall during the crop period. The irrigation efficiency of the drip system was assumed to be 90%. The total amounts of crop evapotranspiration were estimated to be 374 mm and 426 mm for cauliflower and bitter gourd, respectively. The estimated daily water requirements of both crops are shown in Figure 2.

Experimental Treatment and Setup
An experimental field plot of 25 × 30 m was utilized to test the following four treatments: PMW application using inline surface drip (T1), inline subsurface drip (T2), bioline (pressure compensating) subsurface drip (T3) and bioline surface drip (T4). The experiment followed a randomized block design (RBD). A drip irrigation system was designed for cauliflower and bitter guard crops, and consisted of a filtration system, a fertigation system, a flow control valve, pressure gauges and a flush valve. The filtration system consisted of a media filter (manufactured by Jain Irrigation Systems Ltd., Maharashtra, India) and a disc filter (Netafim Arkal Disc Filter that was manufactured by Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) with a flow capacity of 30 m 3 h ˗1 . Municipal wastewater was passed through both the filtration system before application to the experimental crop field. Lateral lines (16 mm diameter) of linear low-density polyethylene (LLDPE) were taken off from a PVC sub main line (0.06 m diameter). Lateral lines were placed at 0.6 m. Bioline and inline laterals (both the laterals were manufactured by Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) with the drip emitter spacing of 0.3 m and discharges of 2 L h −1 were used. Each lateral line was provided with a control valve to control the duration of irrigation. The experimental setup is shown in Figure 3. The bioline (Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) emitter laterals provide design flexibility and superior uniformity for wastewater disposal and soil loading. It is a low volume dripper line designed for on-site wastewater use. Inside the tubing, it has integral and evenly spaced pressure compensating drippers with the diaphragm, which allow water to flow at a constant rate and thus prevent emitter clogging [38].

Experimental Treatment and Setup
An experimental field plot of 25 × 30 m was utilized to test the following four treatments: PMW application using inline surface drip (T1), inline subsurface drip (T2), bioline (pressure compensating) subsurface drip (T3) and bioline surface drip (T4). The experiment followed a randomized block design (RBD). A drip irrigation system was designed for cauliflower and bitter guard crops, and consisted of a filtration system, a fertigation system, a flow control valve, pressure gauges and a flush valve. The filtration system consisted of a media filter (manufactured by Jain Irrigation Systems Ltd., Maharashtra, India) and a disc filter (Netafim Arkal Disc Filter that was manufactured by Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) with a flow capacity of 30 m 3 h −1 . Municipal wastewater was passed through both the filtration system before application to the experimental crop field. Lateral lines (16 mm diameter) of linear low-density polyethylene (LLDPE) were taken off from a PVC sub main line (0.06 m diameter). Lateral lines were placed at 0.6 m. Bioline and inline laterals (both the laterals were manufactured by Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) with the drip emitter spacing of 0.3 m and discharges of 2 L h −1 were used. Each lateral line was provided with a control valve to control the duration of irrigation. The experimental setup is shown in Figure 3. The bioline (Netafim Irrigation India Pvt. Ltd., Ahmedabad, India) emitter laterals provide design flexibility and superior uniformity for wastewater disposal and soil loading. It is a low volume dripper line designed for on-site wastewater use. Inside the tubing, it has integral and evenly spaced pressure compensating drippers with the diaphragm, which allow water to flow at a constant rate and thus prevent emitter clogging [38].  Table T1 and T2 are the inline laterals under surface and subsurface placement, whereas T3 and T4 are the bioline laterals under subsurface and surface placement, respectively.

Operational Procedure
Wastewater was collected from the sewage line passing nearby (approximately 500 m) the experimental field, as shown in Figure 3. The wastewater in the sewage line consisted of urban runoff, domestic wastewater and small institutional effluents. Two tanks were constructed for collecting the sewage effluent. The tank (capacity: 10 m 3 ) near the sewage line stored the sewage water for 24 h to settle the suspended particles. The second tank (capacity: 4 m 3 ) was installed near the experimental site for collecting the settled wastewater from the first tank to improve the wastewater quality by further reducing the suspended particles. Wastewater from the second tank was applied to the experimental plots through different filtration systems (sand media filter and disc filter) to prevent the chocking of the drip system. For this, a sand filter with a flow rate of 30 m 3 h −1 along with backflow mechanism and a disc filter of the same size were used. A sand filter can remove solid particles smaller than pores available in the filter by capturing the particle through both the physical and chemical mechanisms. In a disc filter, the first filtration process is done at the outer surface, which acts as a screen and collects the particles not removed by sand media filter. The second filtration process occurs inside the grooves of the disc to remove the organic materials by creating adhesion between the disc and organic materials.   Table T1 and T2 are the inline laterals under surface and subsurface placement, whereas T3 and T4 are the bioline laterals under subsurface and surface placement, respectively.

Operational Procedure
Wastewater was collected from the sewage line passing nearby (approximately 500 m) the experimental field, as shown in Figure 3. The wastewater in the sewage line consisted of urban runoff, domestic wastewater and small institutional effluents. Two tanks were constructed for collecting the sewage effluent. The tank (capacity: 10 m 3 ) near the sewage line stored the sewage water for 24 h to settle the suspended particles. The second tank (capacity: 4 m 3 ) was installed near the experimental site for collecting the settled wastewater from the first tank to improve the wastewater quality by further reducing the suspended particles. Wastewater from the second tank was applied to the experimental plots through different filtration systems (sand media filter and disc filter) to prevent the chocking of the drip system. For this, a sand filter with a flow rate of 30 m 3 h −1 along with backflow mechanism and a disc filter of the same size were used. A sand filter can remove solid particles smaller than pores available in the filter by capturing the particle through both the physical and chemical mechanisms. In a disc filter, the first filtration process is done at the outer surface, which acts as a screen and collects the particles not removed by sand media filter. The second filtration process occurs inside the grooves of the disc to remove the organic materials by creating adhesion between the disc and organic materials. crop season (120-130 days of life cycle). Each water sample was aseptically collected in triplicate in a sterilized, polyethylene, 1000 mL bottle. These bottles were brought to the laboratory for the microbial analysis, kept in a refrigerator at 4 • C and analyzed within a day.

Vegetable sampling
The edible parts of the vegetable samples were collected ones in the whole cropping season, i.e., during the harvesting time, in triplicates from each treatment plot. Each treatment had three replications. The freshly edible part samples were aseptically collected in sterile polyethylene bags. These polyethylene bags were brought to the laboratory for the microbial analysis and kept in a refrigerator at 4 • C.  E. coli of water samples were analysed with the help of Tryptone Blue agar with X-glucuronide plate (TBX agar) (HiMedia Laboratories Pvt. Limited, Mumbai, India) and an incubator using the unit of colony forming unit (CFU) 100 m L −1 according to standard methods [48]. Triplicate aliquots of 10, 1 and 0.1 mL of each water sample were filtered through 0.45 µm-pore-sized nitrocellulose membranes. For E. coli count, the membrane was placed onto Tryptone Blue agar with X-glucuronide plate (TBX agar) and incubated at 44 • C for 18-24 h while observing only blue-green colonies.

Soil Analysis
Escherichia coli counts were determined for soil according to standard methods [48]. One g of soil sample was aseptically added to 10 mL of sterile buffered peptone water into a sterile jar for a ten-fold dilution. The sample was mechanically shaken for 10 min and stored at room temperature for 30 min to permit bacterial cell recovery. For E. coli counts, membranes were placed onto X-glucuronide (TBX agar) plates. The plates were incubated at 44 • C for 18-24 h for E. coli count.

Crop Analysis
Escherichia coli counts were also determined for both the crops (cauliflower and bitter gourd) according to standard methods [48]. One gram of edible parts was aseptically added to 10 mL of sterile buffered peptone water into a sterile jar for a ten-fold dilution. The sample was mechanically shaken for 3 minutes and stored at room temperature for 30 min to permit bacterial cell recovery. For E. coli counts, membranes were placed onto TBX agar plates. The plates were incubated under different incubation temperatures and times at 44 • C for 18-24 h for E. coli count. To prepare the reagents, analytical grade chemicals and double glass distilled water were used.

Statistical Analysis
The data were analyzed by one-way analysis of variance (ANOVA) using pair-wise multiple comparison procedures of Tukey test by SPSS-16 software (Informer Technologies, Inc., Los Angeles, CA, USA). A p-value less than the critical level indicates there is a significant difference between the corresponding two groups. Data were represented as mean ± standard deviation.

Performance of Laterals in Reducing E.coli in Irrigation Water
PMW was collected from different drip laterals to estimate the presence of E. coli for comparative performance evaluation of both inline and bioline laterals. The E. coli population in raw municipal wastewater was found to be 5.12 ± 50 log 10 CFU 100 mL −1 and 5.05 ± 0.43 log 10 CFU 100 mL −1 after settlement for 24 h. After passing through the filtration system and different drip laterals, the E. coli population in PMW was found to vary from 3.95 ± 0.27 log 10 to 4.73 ± 0.38 log 10 , respectively for T3 to T1 treatments (Figure 4). These values are lower than the recommended limit of E. coli population (CFU 100 mL −1 ) in wastewater used in agriculture by drip irrigation ≤10 5 [49,50], as shown in Table 3. A significant decline in E. coli population was observed in all the treatments as compared to raw and 24-h-settled wastewater (Figure 4). However, E. coli concentration was observed significantly lower in treatment T3 (3.95 ± 0.27 log 10 ) as compared to T1 and T2 (p < 0.05), while no significant difference was observed with T4.

Performance of Laterals in Reducing E.coli in Irrigation Water
PMW was collected from different drip laterals to estimate the presence of E. coli for comparative performance evaluation of both inline and bioline laterals. The E. coli population in raw municipal wastewater was found to be 5.12 ± 50 log10 CFU 100 mL ˗1 and 5.05 ± 0.43 log10 CFU 100 mL ˗1 after settlement for 24 h. After passing through the filtration system and different drip laterals, the E. coli population in PMW was found to vary from 3.95 ± 0.27 log10 to 4.73 ± 0.38 log10, respectively for T3 to T1 treatments (Figure 4). These values are lower than the recommended limit of E. coli population (CFU 100 mL ˗1 ) in wastewater used in agriculture by drip irrigation ≤10 5 [49,50], as shown in Table 3. A significant decline in E. coli population was observed in all the treatments as compared to raw and 24-h-settled wastewater (Figure 4). However, E. coli concentration was observed significantly lower in treatment T3 (3.95 ± 0.27 log10) as compared to T1 and T2 (p < 0.05), while no significant difference was observed with T4.

Yield of Vegetable Crops
The mean values of cauliflower and bitter gourd yield are presented in Figure 5. The maximum average cauliflower and bitter gourd yields (80.4 t ha −1 and 28.67 t ha −1 ) were found in treatment T3 (subsurface bioline drip laterals), while minimum average yield (57.32 t ha −1 and 22.14 t ha −1 ) was observed in T1 (surface inline drip laterals), respectively. Subsurface bioline drip resulted in significantly (p < 0.05) higher yields for both crops than the bioline surface drip. It was also found that the surface and subsurface placements of bioline drip laterals resulted in 27.66% and 25.8% higher cauliflower yields compared to surface and subsurface placement sof inline drip laterals, respectively. In the case of bitter gourd, surface and subsurface placements of bioline drip laterals resulted in 18.24% and 23.1% higher yields compared to surface and subsurface placements of inline drip laterals, respectively. Moreover, the effects of surface and subsurface inline drip lateral were not significantly different (p < 0.05) for bitter gourd yield. Similar results were also reported by Yao et al. [51], Scarpare et al. [52] and Goncalves et al. [53]. The higher yield in subsurface drip irrigation may be attributed to the better soil moisture and nutrient regime in the crop root zone due to reduced evaporation of irrigation water and minimal losses of nutrients as compared to surface drip irrigation systems.

Yield of Vegetable Crops
The mean values of cauliflower and bitter gourd yield are presented in Figures 5. The maximum average cauliflower and bitter gourd yields (80.4 t ha ˗1 and 28.67 t ha ˗1 ) were found in treatment T3 (subsurface bioline drip laterals), while minimum average yield (57.32 t ha ˗1 and 22.14 t ha ˗1 ) was observed in T1 (surface inline drip laterals), respectively. Subsurface bioline drip resulted in significantly (p < 0.05) higher yields for both crops than the bioline surface drip. It was also found that the surface and subsurface placements of bioline drip laterals resulted in 27.66% and 25.8% higher cauliflower yields compared to surface and subsurface placement sof inline drip laterals, respectively. In the case of bitter gourd, surface and subsurface placements of bioline drip laterals resulted in 18.24% and 23.1% higher yields compared to surface and subsurface placements of inline drip laterals, respectively. Moreover, the effects of surface and subsurface inline drip lateral were not significantly different (p < 0.05) for bitter gourd yield. Similar results were also reported by Yao et al. [51], Scarpare et al. [52] and Goncalves et al. [53]. The higher yield in subsurface drip irrigation may be attributed to the better soil moisture and nutrient regime in the crop root zone due to reduced evaporation of irrigation water and minimal losses of nutrients as compared to surface drip irrigation systems.

E. coli Incident in Cauliflower Curd and Bitter Gourd Fruit
The E. coli concentrations in both the vegetable crops, i.e., cauliflower curd and bitter gourd, irrigated with PMW using bioline and inline emitters, were also examined in this study ( Figure 6). The results showed that the higher concentration of E. coli was found in T1 (surface placement inline drip lateral) treatment for both the crops than the subsurface placement drip laterals. Similar findings were reported by Sacks and Bernstein [54], who studied the effects of surface and subsurface drip irrigation with reclaimed wastewater on E.coli contamination in melon crop and found a significantly lower count of the bacteria in melon irrigated with the subsurface placement of lateral as compared to surface laterals. In cauliflower curd, the E. coli concentration was found to be 35 ± 2.66 CFU g −1 , whereas a lower count was observed in the bitter gourd fruit (25 ± 1.72 CFU g −1 ) ( Figure 6). Further, in bitter gourd fruit, E. coli population was significantly (p < 0.05) lower as compared to cauliflower curd for both inline and bioline laterals. Among all the treatments, E. coli

E. coli Incident in Cauliflower Curd and Bitter Gourd Fruit
The E. coli concentrations in both the vegetable crops, i.e., cauliflower curd and bitter gourd, irrigated with PMW using bioline and inline emitters, were also examined in this study ( Figure 6). The results showed that the higher concentration of E. coli was found in T1 (surface placement inline drip lateral) treatment for both the crops than the subsurface placement drip laterals. Similar findings were reported by Sacks and Bernstein [54], who studied the effects of surface and subsurface drip irrigation with reclaimed wastewater on E.coli contamination in melon crop and found a significantly lower count of the bacteria in melon irrigated with the subsurface placement of lateral as compared to surface laterals. In cauliflower curd, the E. coli concentration was found to be 35 ± 2.66 CFU g −1 , whereas a lower count was observed in the bitter gourd fruit (25 ± 1.72 CFU g −1 ) ( Figure 6). Further, in bitter gourd fruit, E. coli population was significantly (p < 0.05) lower as compared to cauliflower curd for both inline and bioline laterals. Among all the treatments, E. coli concentration was significantly lower in treatment T3 (subsurface placement drip laterals) for both crops. Lonigro et al. [11] found similar results of E. coli concentration in different crops, such as tomato (35 CFU g −1 ) and lettuce (45 CFU g −1 ). Shock et al [55] also found a significant reduction in microbial contamination of onion crop irrigated with wastewater by subsurface drip irrigation. Song et al. [56] and Allende and Monaghan [57] also found a reduction in microbial contamination in lettuce crop using subsurface drip irrigation. The results of the present study also revealed that there were reductions of 7.59% and 8.13% in E. coli counts in the bioline drip lateral as compared to the inline drip lateral for cauliflower and bitter gourd crops, respectively. This shows that the bioline drippers with sand and disc filters can reduce microorganism concentrations in wastewater significantly.
concentration was significantly lower in treatment T3 (subsurface placement drip laterals) for both crops. Lonigro et al. [11] found similar results of E. coli concentration in different crops, such as tomato (35 CFU g −1 ) and lettuce (45 CFU g −1 ). Shock et al [55] also found a significant reduction in microbial contamination of onion crop irrigated with wastewater by subsurface drip irrigation. Song et al. [56] and Allende and Monaghan [57] also found a reduction in microbial contamination in lettuce crop using subsurface drip irrigation. The results of the present study also revealed that there were reductions of 7.59% and 8.13% in E. coli counts in the bioline drip lateral as compared to the inline drip lateral for cauliflower and bitter gourd crops, respectively. This shows that the bioline drippers with sand and disc filters can reduce microorganism concentrations in wastewater significantly.

Variation of E. coli Incident with Respect to Plant Height in Bitter Gourd Fruit
The variation of E. coli counts with the location of fruits for bitter gourd was also assessed in this study. Notably, no study has been reported in the literature dealing with bitter gourd irrigated with municipal wastewater using a drip irrigation system in combination with bioline drippers. The results show that within the same plant of bitter gourd, a significant reduction in E.coli concentration was observed between the top fruit (1m above the ground level, 10.8 ± 1.06 CFU g −1 ) and fruit near the surface (near the ground level 25.2 ± 2.26 CFU g −1 ), as shown in Figure 7. According to EFSA [58], direct contact of vegetable crops to microbial contaminated irrigation water should be avoided. The average E. coli concentrations in the fruits of bitter gourd at the top and bottom of all the treatments were found to be 7.8 ± 0.58 CFU g −1 and 22.5 ± 2.05 CFU g −1 , respectively. The lowest concentration of E. coli was found in T3 treatment at the top and bottom both with values varying from 6.8 ± 0.53 CFU g −1 to 18.9 ± 2.04 CFU g −1 , respectively.

Variation of E. coli Incident with Respect to Plant Height in Bitter Gourd Fruit
The variation of E. coli counts with the location of fruits for bitter gourd was also assessed in this study. Notably, no study has been reported in the literature dealing with bitter gourd irrigated with municipal wastewater using a drip irrigation system in combination with bioline drippers. The results show that within the same plant of bitter gourd, a significant reduction in E.coli concentration was observed between the top fruit (1m above the ground level, 10.8 ± 1.06 CFU g −1 ) and fruit near the surface (near the ground level 25.2 ± 2.26 CFU g −1 ), as shown in Figure 7. According to EFSA [58], direct contact of vegetable crops to microbial contaminated irrigation water should be avoided. The average E. coli concentrations in the fruits of bitter gourd at the top and bottom of all the treatments were found to be 7.8 ± 0.58 CFU g −1 and 22.5 ± 2.05 CFU g −1 , respectively. The lowest concentration of E. coli was found in T3 treatment at the top and bottom both with values varying from 6.8 ± 0.53 CFU g −1 to 18.9 ± 2.04 CFU g −1 , respectively.

E. coli Incident in Soil Profile
The results of E.coli concentration in the soil after harvesting of crops under different treatments are presented in Figure 8a,b. Results revealed that the highest concentration of E. coli (470 ± 70.5 and 410 ± 36.9 CFU g −1 ) was found in T1 treatment in 0-0.15 m soil depth after harvesting of crops, while the minimum was observed in T3 (154 ± 13.86 and 95 ± 14.25 CFU g −1 ) treatment. At 0.15-0.3 m, the highest concentration of E. coli was observed in T2 (407 ± 40.7 and 402 ± 52.26 CFU g −1 ) followed by T3 (369 ± 55.35 and 368 ± 29.44 CFU g −1 ). The reason for this pattern of distribution of E. coli population is attributed to the greater incidence of the bacteria in proximity to the drip lateral position (at surface and 0.2 m). It was also observed that, in cauliflower harvested soil, the E. coli concentration was higher than in the bitter gourd harvested soil, as shown in Figure 8a,b. That may have been due to a lower temperature and higher moisture content in the soil because the cauliflower was grown in the winter season, whereas bitter gourd was grown in summer. This result is also supported by the findings that the survival rate of E. coli is higher in the winter season than in summer [59][60][61].

E. coli Incident in Soil Profile
The results of E.coli concentration in the soil after harvesting of crops under different treatments are presented in Figure 8a,b. Results revealed that the highest concentration of E. coli (470 ± 70.5 and 410 ± 36.9 CFU g −1 ) was found in T1 treatment in 0-0.15 m soil depth after harvesting of crops, while the minimum was observed in T3 (154 ± 13.86 and 95 ± 14.25 CFU g −1 ) treatment. At 0.15-0.3 m, the highest concentration of E. coli was observed in T2 (407 ± 40.7 and 402 ± 52.26 CFU g −1 ) followed by T3 (369 ± 55.35 and 368 ± 29.44 CFU g −1 ). The reason for this pattern of distribution of E. coli population is attributed to the greater incidence of the bacteria in proximity to the drip lateral position (at surface and 0.2 m). It was also observed that, in cauliflower harvested soil, the E. coli concentration was higher than in the bitter gourd harvested soil, as shown in Figure 8a,b. That may have been due to a lower temperature and higher moisture content in the soil because the cauliflower was grown in the winter season, whereas bitter gourd was grown in summer. This result is also supported by the findings that the survival rate of E. coli is higher in the winter season than in summer [59][60][61].
Among all the treatments, E. coli concentration in soil was significantly lower in T3 (subsurface bioline drip laterals) after harvesting of both crops. Sacks and Bernstein [54] also studied the effect of surface and subsurface drip irrigation in the melon crop on the incidence of E. coli in the soil profile and found significant differences in E. coli concentrations in soil between the surface (higher) and subsurface drip placements of laterals. The concentration of E. coli in T3 was also significantly different than in treatment T2 and T4 in cauliflower (p < 0.05) and bitter gourd (p < 0.05) harvested soil, respectively.
The placement of laterals significantly influenced the population of E. coli, increasing the number of E. coli at the respective depths. The increased population of E. coli in wastewater was mainly due to the domestic sewage discharges which mix up with the wastewater sources at several points. Additionally, the soil reaction being alkaline in nature, the E. coli survived with much ease compared to soil with acidic reaction [62]. Wastewater derived from domestic sources is laden with E. coli contamination and the intensity of contamination increases with the increase in the percentage of wastewater used for irrigation [63]. Since the majority of the wastewater is used for irrigation of vegetable crops in the peri-urban localities which have a chance to be consumed raw, wastewater irrigation increases the chance of contamination and diseases unless proper measures are taken. In the present study, the placement of laterals played a significant role in the reduction of coliform contamination with a lower population at the soil surface due to subsurface placement of laterals. Our results are similar to those of Li and Wen [64] and Wen et al [65], who reported that E. coli concentration was observed in the proximity of emitters and that subsurface placement of drip laterals can avoid the contamination of the soil surface layer. Hence, subsurface drip irrigation could help to reduce E. coli contamination in crops and use the wastewater more efficiently. Similar observations have also been made by Balkhair [63]. This shows that the bioline subsurface drippers with sand and disc filters are efficient in reducing the microorganism concentrations of wastewater used with drip irrigation systems. Among all the treatments, E. coli concentration in soil was significantly lower in T3 (subsurface bioline drip laterals) after harvesting of both crops. Sacks and Bernstein [54] also studied the effect of surface and subsurface drip irrigation in the melon crop on the incidence of E. coli in the soil profile and found significant differences in E. coli concentrations in soil between the surface (higher) and subsurface drip placements of laterals. The concentration of E. coli in T3 was also significantly different than in treatment T2 and T4 in cauliflower (p < 0.05) and bitter gourd (p < 0.05) harvested

Conclusions
The treated or partially treated wastewater is now becoming an important source of irrigation for the sustainable use of limited freshwater resources, owing to its potential economic and environmental benefits. Though wastewater has already been used in agriculture for decades, still, it is challenging to follow the guidelines worldwide along with old irrigation practices, particularly in developing countries like India, where the adoption of these guidelines and new techniques to increase the benefits of wastewater reuse along with a reduction in human health risks has not yet reached a larger scale.
The results of this study show that the E. coli concentration in irrigation water and in vegetable fruits can be reduced significantly by using inline and bioline drip laterals. As far as the microbial quality of irrigation water is concerned, a combination of the subsurface drip having a bioline lateral was found to yield lower concentrations of E. coli as compared to the combination of the surface drip having an inline drip lateral. Similarly, among all the treatments, the E. coli concentration was significantly lower in the case of subsurface drip irrigation with bioline laterals (T3) for both crops, i.e., cauliflower curd and bitter gourd. Bitter gourd crop was less contaminated with E. coli as compared to cauliflower. It was also observed that within the same plant of bitter gourd, top fruits were much safer for use as compared to surface fruits because of the low population of E. coli. Results also revealed that E. coli variation in the soil profile was in the proximity of the emitter. Overall, it is concluded that the wastewater applied through the drip irrigation system with pressure compensating drip laterals coupled with sand and disc filters would be a better option for reducing the microbial population in irrigation water, which leads to lowering the E.coli concentration in the soil profile and in vegetable crops. Further, the bitter gourd crop is a better option for irrigating with wastewater as compared to cauliflower.
Author Contributions: Experiments were conducted by D.S. and N.P. The manuscript was written by D.S., N.S., S.P., A.G.-T. and P.K.S. Statistical analysis was performed by N.S. and S.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.