Effects of Agronomic Practices on the Severity of Sweet Basil Downy Mildew (Peronospora belbahrii)

Downy mildew (caused by Peronospora belbahrii) is a severe disease of sweet basil (Ocimum basilicum) crops around the world. We examined cultural methods for reducing the severity of sweet basil downy mildew (SBDM) under commercial conditions in greenhouses and walk-in tunnels. The effects of the orientation of walk-in tunnels, air circulation in greenhouses, plant density, and soil mulch were tested. SBDM was less severe in the tunnels that were oriented north-south than in those oriented east-west, but the yields in both types of tunnels were similar. Increased air circulation reduced SBDM severity, but did not affect yield. Gray or transparent polyethylene mulch reduced SBDM severity and, in most cases, increased yield relative to bare soil/growth medium. Yellow polyethylene mulch provided a smaller amount of control. The combination of increased air circulation and yellow polyethylene mulch provided synergistic SBDM control, whereas no synergism was observed when we combined increased air circulation with the other two types of mulch. Planting at half the usual density reduced disease severity. The reduced plant density was associated with reduced yield in the greenhouses, but not in the tunnels. All of the tested methods provided an intermediate level of SBDM control that varied among the different experiments.


Introduction
Cultural methods (i.e., agricultural practices) can play important roles in preventing or minimizing many plant diseases. Different practices can be implemented during seeding/planting, as the crop is growing or even after harvest. Cultural techniques aimed at minimizing disease include the choice of altitude at which to plant a particular crop, the means of preparing and cultivating the soil, the use of particular cultivars, the treatment of propagation material, the choice of particular planting times and depths, the exposure to air movement, the direction of plant rows, weed-management and irrigation practices, changes in plant nutrition, and general sanitation [1][2][3]. In covered crops, farmers can also consider the type of cover spread over the crop, heating, ventilation, and soil mulch [4]. Cultural methods that affect the environmental conditions inside the greenhouse and minimize the presence of water on the canopy can be effective ways of controlling diseases that are promoted by high humidity [5].
Sweet basil (Ocimum basilicum L.) is an economically important annual herb crop from the Labiatae family that is grown in polyethylene-covered structures (i.e., greenhouse structures or walk-in tunnels). Sweet basil greenhouses are common along the ridge above the Syrian-African Rift, south of the Sea of Galilee, and around and north of the Dead Sea. Crops are planted from September on, so winter and spring crops are common, but are challenged by humidity-promoted diseases [6][7][8][9].

Tunnel Orientation
The effect of tunnel orientation on SBDM could only be evaluated at Site 2. We found that SBDM was less severe in the walk-in tunnels that were oriented north-south, as opposed to east-west. The decrease in severity varied with the experiment, soil cover, and year (Table S1). In terms of the area under disease progress curve (AUDPC), the difference in SBDM severity between east-west and north-south tunnels was between 25 and 33% in the first year and was about 63% in the second year (Table 1 and Table S1). The shoot yields in the two types of walk-in tunnels were significantly similar to each other (Table 1). Thus, the north-south tunnel direction is associated with lower SBDM severity as compared with east-west-oriented tunnels.

Effect of Increased Air Circulation
The effect of increased air circulation (AC) was evaluated only in the greenhouses at Site 1. We increased the air circulation in those greenhouses by operating fans during the night. This increased air circulation reduced SBDM severity ( Table 2 and Table S2). Disease reduction reached up to 72.5% in all experiments conducted over three seasons (Table 2  and Table S2), but did not affect shoot yield (Table 2). Therefore, AC decreases SBDM.
Additionally, the results for the combination of increased air circulation with polyethylene mulch are described below.

Polyethylene Mulch
In greenhouses at Site 1, covering the beds with gray or transparent polyethylene decreased SBDM severity (Table 3 and Table S3). Disease severity was significantly reduced (by up to 65%) in six out of the seven evaluation comparisons (Table S3). A whole-season evaluation based on AUDPC revealed 13-64% disease reduction in the covered beds (Table 3 and Table S3). The use of polyethylene mulch significantly increased yield in five of the six greenhouse experiments (Table 3 and Table S3). Therefore, polyethylene mulch can reduce SBDM and increase yield. Three types of polyethylene mulch were compared in two growing seasons. The gray and the transparent polyethylene mulch provided more consistent and effective disease control ( Table 4). The combination of increased air circulation and polyethylene mulch was examined in a third experiment ( Figure 1, Table 5), which revealed no interaction between those treatments. Disease was significantly reduced by the increased air circulation and the gray polyethylene. The results observed for the transparent polyethylene were significantly different from those observed for the yellow polyethylene ( Figure 1, Table 5). Calculating the synergy factor (SF) of the reduction in severity provided by each of the polyethylene mulches and the air-circulation treatment revealed no synergism between increased air circulation and either the gray or the transparent polyethylene. However, there was a significant synergistic effect between the increased air circulation and the yellow mulch (SBDM severity: SF = 1.04; AUDPC: SF = 1.12).  or with extra air circulation (right). SBDM severity was evaluated on a 0-100% scale, in which 0 = healthy plants and 100% = plants completely covered by SBDM symptoms/signs. SBDM severity is presented as percentage ± SE. An analysis of these results is presented in Table 5.   Table 5. Table 5. Results from experiment M8 ( Figure 1). The effect of polyethylene mulch was also evaluated at Site 2. SBDM severity was significantly reduced in six out of eight comparisons (20-52% disease reduction) and in seven out of the eight epidemic (AUDPC) comparisons (17-51% reduction; Table 6 and Table  S6). Shoot yield was increased (8-13%) by the soil mulch in two of the three comparisons ( Table 6 and Table S6). Thus, polyethylene mulch was effective also in Site 2, where experiments were carried out in walk-in tunnel greenhouses. Table 6. Effects of polyethylene mulch on the severity of sweet basil downy mildew (SBDM) and yield of sweet basil in walk-in tunnels at Site 2a.

Plant Spacing
The effect of plant density was evaluated at both locations. We compared a density of 24-30 plants/m 2 , which is commonly used in both areas, with a reduced density of 14-15 plants/m 2 . In the greenhouses at Site 1, significant reductions in SBDM were observed in all of the reduced-density plots ( Table 7 and Table S7). The reduction in SBDM severity ranged between 32% and 68%. AUDPC was reduced by 22% to 63% (Table 7 and  Table S7). The relationship between the SBDM severity data and the AUDPC data was calculated. The relation between the severity of disease at each evaluation date and the calculated % disease reduction at the evaluation date or between the values of AUDPC and the calculated % disease reduction were assessed. A negative correlation was found between the severity of disease and % reduction in both cases, single-day disease level, or AUDPC (p < 0.01 in both cases; Table 7), pointing to the fact that under lower disease pressure, the less-dense plant spacing provides more pronounced disease suppression. In three experiments, yield was decreased by 12% to 31% by sparse planting compared to dense planting (Table 7 and Table S7). The reduced planting density, which was examined in six experiments at Site 2, resulted in a significant (19.5-52.5%) reduction in SBDM severity in six of the eight evaluations. AUDPC was reduced by 21.7% to 50.8%. The relationship between SBDM severity and disease reduction was negative (p < 0.1, Table 8). No effect on yield was recorded in the two experiments in which we measured yield (Table 8 and Table S8). Thus, in both locations, disease severity was reduced by diluted planting density, but yield was not increased.

Discussion
Cultural measures can be used to suppress SBDM. The following factors were examined in the present work: tunnel orientation, air circulation in a greenhouse, polyethylene mulch, and plant spacing. The level of disease control did not vary with the different growing seasons and intermediate-level disease control was observed in both locations. Studies of cultural means of controlling downy mildews are scarce; therefore, it is only possible to compare the currently studied means with similar methods that have been examined in other patho-systems.
Nighttime air circulation in the greenhouse was increased in an effort to reduce the amount of water on the aerial plant organs and the amount of time that water was present. Humidity-promoted pathogens such as P. belbahrii thrive in the film of water that primarily appears at night and depend on it for their reproduction and their ability to infect the plant [9,13]. Increased air circulation can reduce the presence of water at the boundary layer over the plant organs even under humid conditions [20] and can limit humiditypromoted diseases [4]. Indeed, the test carried out over three growing seasons revealed that increased air circulation at night can suppress SBDM in greenhouses. Similarly, Papas [21] concluded that B. cinerea in out-of-season tomato (Solanum lycopersicum) plants grown in unheated glasshouses in Greece can be limited by adequate indoor air circulation. Similarly, in-bed air circulation has been shown to reduce B. cinerea gray mold in lisianthus (Eustoma grandiflorum) [22].
In the sweet basil-growing regions in Israel, walk-in tunnels are traditionally oriented east-west. The tunnel orientation affects the microclimate in the tunnel, since it relates to the direction of the sun [6,9,23] and prevailing winds. Surprisingly, the north-south orientation was associated with lower SBDM severity in all of our experiments. Nevertheless, the yields from north-south and east-west tunnels were similar. These results suggest that the temperature × hours gain in the two types of tunnels are similar, but the moisture duration was different. We are not aware of similar results in other patho-systems.
Polyethylene soil cover has been suggested for the control of gray mold induced by B. cinerea [24]. A plastic cover with a white upper surface reduced the incidence of B. cinerea infection in strawberry (Fragaria × ananassa), compared to bare soil [25]. Similarly, in lisianthus, the use of a polyethylene barrier between the lower leaves and soil that prevented the lower leaves from coming into contact with the wet soil reduced the development of B. cinerea along the leaves toward the stem and prevented plant mortality [22]. The prevention of contact between the canopy and the wet soil is not relevant in the case of SBDM. However, polyethylene mulch effectively suppressed SBDM in both locations, congruent with the effects such mulch has been shown to have on B. cinerea-induced gray mold [8] and S. sclerotiorum-induced white mold [7] in sweet basil. As expected [7,8], polyethylene mulch increased shoot yield in two of three experiments.
In addition to decreasing evaporation from the growth medium, the polyethylene mulch also increases the bed temperature. Shtienberg et al. [26] showed that the polyethylene mulch causes irradiation flux across the canopy and the drying of the leaves and fruits of greenhouse tomato and cucumber (Cucumis sativus) plants. This helps to control tomato late blight (Phytophthora infestans) and downy mildew (Pseudoperonospora cubensis) in cucumber [26]. Polyethylene soil cover is associated with increased yields, thanks to the accumulation of heat in the root zone and higher soil temperatures [27]. It was suggested that passive greenhouse warming increases sweet basil's resistance to downy mildew by warming the root zone [9]. Recently, Gupta et al. [28] demonstrated that warming the root zone induces systemic resistance in plants. Indeed, warming the root zone of sweet basil under field conditions resulted in shoot resistance to the necrotrophic fungi B. cinerea and S. sclerotiorum that continued after harvest [7,8]. It may be assumed that polyethylene mulch may reduce the susceptibility of harvested leaves also to SBDM.
Reducing the density of sweet basil plants reduced SBDM, as previously demonstrated for gray mold and white mold [7,8]. Reduced plant density has also been shown to suppress disease in other patho-systems. Vieira et al. [29] reported decreased incidence of white mold and increased soybean yields when within-row densities were reduced. Lower plant density has also been shown to reduce stem gray mold in lisianthus [22]. Reducing the number of blond psyllium (Plantago ovata) seeds sown per unit area reduced the incidence of downy mildew (Peronospora alta) in that crop [30]. The reduction from 12 to 6 bean plants/m 2 decreased the severity of bean white mold (S. sclerotiorum) in one of two experiments, but did not decrease yield [31]. The severity of soybean stem canker (Diaporthe phaseolorum var. meridionalis) decreased proportionately to a decrease in plant densities [32]. In downy mildew of rose (Peronospora sparsa), reducing the density of container-grown plants had a measurable effect on the progress of downy mildew [33].
The reduction in sweet basil planting density resulted in reduced canopy volume at the beginning of the season. But, after the second harvest, the canopy was dense. Nevertheless, SBDM levels were lower in the reduced planting despite the dense canopy. The mechanism of this control could not be studied with the biotroph P. belbahrii, but our experience with B. cinerea and S. sclerotiorum pointed to reduced shoot susceptibility to pathogens [7,8]. We hypothesize that the reduction in planting density also affects the plants' susceptibility to SBDM.
We calculated the correlation between disease severity values and the intensity of disease reduction across experiments. There was a negative correlation between the disease in the denser plots and disease reduction in the plots in which plants were planted at the lower density, pointing to the fact that under conditions of lower disease pressure, increased plant spacing provides more pronounced disease suppression. Surprisingly, such a negative correlation was not found for the polyethylene mulch practice. In some experiments, we also examined the possibility of combining polyethylene mulch with reduced planting density, but that combination did not provide synergistic disease control (results not shown). When applied in combination with chemical fungicide, neither reduced plant density nor the use of polyethylene provided synergistic disease control (results not shown). As described, there was also no synergistic effect between increased air circulation and transparent or gray polyethylene mulch.

Materials and Methods
Experiments were carried out at two experimental stations (Sites 1 and 2, described in detail below) under semi-commercial conditions during the years 2013-2015. Sweet basil cv. Peri [34] plants were used in all of these experiments. Plugs were prepared in a commercial nursery (Hishtil, Ashkelon, Israel) and transplanted 3 to 4 weeks after seeding. Each plug contained 3 to 5 plants, but the plugs are usually referred to as plants. "Peri" is susceptible to P. belbahrii [9]. The experiments were carried out in greenhouses (Site 1) and in walk-in tunnels (Site 2). Downy mildew epidemics occurred naturally at the field sites, following the placement of infected basil plants next to the plots as described below.

Inoculation with P. belbahrii and Disease Evaluation
Spores of P. belbahrii were harvested in water by washing sporulating leaves of sweet basil plants that were kept in an experimental greenhouse at the Volcani Center, Agricultural Research Organization, Israel. The suspension was then filtered through cheesecloth. The concentration of spores was determined using a hemocytometer and a light microscope, and adjusted to 1 × 10 3 cells ml −1 . Potted sweet basil plants were inoculated by spraying with a spore suspension (5 mL plant −1 ), incubated at high RH (>95%) in the dark in a growth chamber at 22 ± 1 • C for 12 h and then incubated in a greenhouse chamber at 22 ± 2 • C for 1 week, and incubated at high RH (>95%) in the dark in a growth chamber at 22 ± 1 • C for 12 h and then incubated in a greenhouse chamber at 22 ± 2 • C for symptom development [9]. The potted sweet basil plants subjected to this artificial inoculation served as a source of inoculum to ensure even inoculum loads across the greenhouses and walk-in tunnels. The plants were placed at the borders of each plot.
The evaluation of the severity of sweet basil downy mildew (SBDM) in the plots included all plants except those along the 1 m edges of each plot. The severity of SBDM was determined periodically in all plants of each plot in each experiment on a scale of 0 to 100, in which 0 = all plants visually healthy, 10 = 10% of the leaf area in the plot covered by typical downy mildew symptoms of chlorosis and/or dry necrotic lesions or P. belbahrii spores on the undersides of the leaves, and 100 = all leaves on all plants in the plot show typical downy mildew symptoms/signs [9].

Shoot Weight
In selected experiments, shoots longer than 15 cm were harvested and weighed three to five times during the growing season, as detailed below. The yield was collected separately for each plot, sorted for quality, and calculated per m 2 bed. The cumulative yield figures for the various harvests were calculated and those figures are presented.

Site 1-Eden Experimental Station
Experiments were conducted at the Eden Experimental Station (32 • 46 79 N, 35 • 48 88 E; 120 m below mean sea level) at the Emek Hamaayanot Research and Development Center. The regional climate is Mediterranean, semiarid with winter rains and a dry, hot summer. At this site, experiments were carried out in two 400 m 2 greenhouses. The structures were covered with 150 µm-thick Sunsaver Clear IR AV polyethylene (Ginegar Plastic Products, Kibutz Ginegar, Israel). The greenhouses were aerated during the day and closed during the night (18:00 to 07:00). At night, the greenhouses were heated to 12 • C to prevent physiological damage to the leaves. There were five bays in each greenhouse and the bays were separated with 1.8 m-high transparent polyethylene.
The potting material was tuff (volcanic gravel; 3 to 6 mm particles) placed in plastic containers that were 1 m wide × 15 cm deep × 20 m long (Mapal, Mevo Hama, Israel). Plants were irrigated daily according to local extension service recommendations, allowing 30% drainage, and fertigated proportionally with 5-3-8 N-P-K fertilizer at a rate of 2 L/1000 L water. The nutrient concentrations were therefore 8.6, 1.0, and 4.0 mM N, P, and K, respectively. Fertigation was performed using a 17 mm drip-irrigation pipe with a 1 L/h dripper embedded in the pipe every 20 cm. Plots were 5 m long each, containing 108-125 plants/plot at the higher plant density (24-25 plants/m 2 ) mentioned below.
Experiments were carried out over three consecutive growing seasons, with planting dates of 9 September (fall 2013), 24 February (spring 2014), and 19 January (winter 2015). Treatments consisted of different cultural methods, as detailed below and in Table 9. Plots consisted of one bed (1 m wide and 4.5 to 5.0 m long) and there were 4-8 plot replicates.

Cultural Methods Applied at Site 1
Air circulation: Four fans (60 cm diam., Adirom Heating and Ventilation Engineering Ltd., Ashkelon, Israel) were installed 2 m above the beds, facing the canopy of plants that were planted at the area of one third of a greenhouse bay toward the north or south edge of the greenhouse. The fans were operated once every hour for 15 min from 19:00 until 08:00. Beds were covered with gray polyethylene mulch and the planting density was 24-25 plants/m 2 .
Planting density: Sweet basil plants were planted at two densities: 24-30 plants/m 2 , as is customary in the area, and 14-15 plants/m 2 . The higher planting density was also used in experiments in which planting density was not a tested parameter. The beds were left bare or covered with gray or transparent polyethylene (Table 9).
Polyethylene mulch: The beds were either left uncovered (bare growing medium) or covered with sheets of polyethylene. Several types of polyethylene were examined: (1) transparent 30 µm-thick Sunsaver Clear IR polyethylene (Ginegar), (2) gray-black 30 µm-thick Mulch-More polyethylene (Ginegar) with the gray-colored side visible and the black-colored side facing the ground, and (3) yellow-brown 30 µm-thick Mulch-More polyethylene (Ginegar) with the yellow side visible and the brown side facing the ground. The plant density was 24-25 plants/m 2 (Table 9).

Site 2-Zohar Experimental Station
This research station is located in the Sedom area south of the Dead Sea and is part of the Northern Arava Research and Development Center. It is located at 30 • 94 656.2 N, 35 • 40 341.7 E at 354 m below mean sea level. The weather at the Zohar Station is arid. In the winter, rain is rare and the mean daytime temperature is 22 • C. The summers are dry and hot, with an average daily temperature of 33 • C. The work at the Zohar Experimental Station was carried out in 10 walk-in tunnels. Each tunnel was 40 m long and 5 m wide (200 m 2 ). The structures were covered with 100 µm-thick Sunsaver Clear IR AV polyethylene (Ginegar Plastic Products, Ginegar, Israel). The front and back openings of each tunnel were covered with 50-mesh netting. Five round aeration openings (50 cm diam.) were cut along the length of the tunnels and covered with 50-mesh netting. Onemeter-wide sandy soil beds were planted with 30 plants/m 2 , unless otherwise noted. Plants were irrigated with local brackish water (4 decisiemens per meter), according to the local extension service recommendations, and fertigated with 1.0 L/1000 L 8-2-4 N-P-K fertilizer. Nutrient concentrations were therefore 6.9, 0.33, and 1.0 mM N, P, and K, respectively. Fertigation was performed using a 17 mm drip-irrigation pipe with a 1.2 L/h dripper embedded in the pipe every 20 cm. Each plot consisted of two beds that were each 9 m long, unless otherwise mentioned.
Experiments were carried out over two consecutive growing seasons, with planting dates of 19 February (spring 2014 season) and 11 November 2014 (winter 2015 season). Treatments consisted of different cultural methods, as detailed below and in Table 10. Plots

Cultural Methods Applied at Site 2
Tunnel direction: Walk-in tunnels were oriented north-south or east-west with either bare soil or with transparent polyethylene mulch. The planting density in the plots was 30 plants/m 2 (Table 10).
Planting density: Sweet basil plants were planted at two densities: 30 plants/m 2 , as is the common local practice, or 15 plants/m 2 . The higher planting density was also used in experiments in which the planting density was not a tested parameter. The soil was covered with transparent polyethylene (Table 10).
Polyethylene soil mulch: The beds were either left uncovered (bare growing medium) or covered with transparent polyethylene (30 µm-thick Sunsaver Clear IR polyethylene; Ginegar). The beds were planted with 30 plants/m 2 in tunnels oriented either north-south or east-west (Table 10).

Experimental Design and Statistical Analysis
Treatments in each year and each field experiment were replicated 4-10 times. Replicates of each treatment were arranged randomly. Disease severity was evaluated in each plot (replicate). Area under the disease severity progress curve (AUDPC) values were also calculated. Data in percentages were arcsine-transformed before further analysis. Disease severity (%) and AUDPC (% × days) data were analyzed using ANOVA and Tukey's HSD test. Standard errors (SE) of the means were calculated and disease levels were statistically separated following a one-way analysis of variance. Treatments in experiments with combined two-treatment factors were statistically separated following a two-way analysis of variance. Statistical analyses were performed using JMP 5.0 software (SAS Institute, Cary, NC, USA).
Disease reduction was calculated as follows: % disease reduction = 100 − 100 × (disease severity TT /disease severity control ). (1) The combined effect of the control measures used was estimated using the Abbott formula [35,36]. The expected disease reduction (control efficacy) and the combined suppressive activity were calculated as: CE exp = a + b − a × b/100 and SF = CE obs /CE exp , where a = disease reduction due to one measure when applied alone, b = disease reduction due to the other measure when applied alone, CE exp = expected control efficacy of the combined treatment if the two measures act additively, CE obs = observed disease reduction for the combined treatment, and SF = the synergy factor achieved by the combined treatment.
When SF = 1, the interaction between the control measures is additive. When SF < 1, the interaction is antagonistic, and when SF > 1, the interaction is synergistic [26,35,36]. The same formula was used to calculate SF in the context of yield.

Conclusions
Increased air circulation, reduced plant density, polyethylene mulch, and the northsouth orientation of walk-in tunnels moderately reduced SBDM under commercial conditions. These practices can contribute to efforts to reduce the dependence on chemical fungicides in sweet basil crops that commercially, because of demands of minimized chemical residues, can tolerate only limited use of such chemicals at application times that are temporally far from harvest.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.