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Growth and Yield Response of Garlic (Allium Sativum L.) to Intra-row Spacing and Variety at Selekeleka, Northern Ethiopia

Background: Garlic is an important condiment and cash crop in Ethiopia. Low yield and productivity of the crop are the major characteristics of the crop in the country. Many factors affect the productivity of the crop in the country, however poor agronomic practices, especially the use of low-yielding varieties and inappropriate spacing, are the major ones.

INTRODUCTION
Garlic (Allium sativum L.) is the second most widely produced Allium crop next to onion [1]. It is produced for fresh market, dehydrated as an ingredient for food processing, and food supplement output like dehydrated powder, essential oil, In Ethiopia, the Alliums (onion, garlic, and shallot) are important bulb crops produced for home consumption as a spice, medicinal plant, and as a source of income for farmers [7]. It grows in a wide range of climatic and soil conditions, mainly in the mid-altitudes and highlands of the country. As a cash crop, it is a source of earning foreign currency by exporting to Europe, the Middle East, Africa and USA [8]. In the Tigray region of Ethiopia, the production and marketing of high-value vegetable crops are intensifying mainly due to market-oriented government policy. Garlic is one of the highvalue vegetable crops produced in rotation with pulses during the cold season [9] (Fig. 1). The region ranks 4 th in garlic production next to Amhara, Oromia and S.N.N.P regional states of Ethiopia, covering an area of 540.97 ha with a production of 3912.826 tonnes [10]. According to Medebay Zana district office of agriculture and rural development, 2017 unpublished report, onion is one of the most important vegetable crops mostly cultivated under irrigation; during the 2017/18 cropping season, the irrigable area covered with alliums were about 226.62 hectares with a production of 10,656.25 quintals.
The average yield of garlic in Ethiopia was 9.34 t ha -1, which is far from the world average of 23.53 t ha -1 . Lack of improved and high yielding varieties, non-availability of quality seeds, low soil fertility, inappropriate agronomic practices, lack of proper pest and disease management and marketing facilities, and lack of improved post-harvest technologies are among the prominent factors for low productivity in the country [11,12]. The optimum level of any agronomic practices, such as plant population density, varies with the environment, the purpose of the crop and variety. Bulb yield and days to maturity of garlic is disposed to environmental changes resulting in variable yield due to the significant effect of genotype-by-environment interaction [13]. Lack of appropriate variety and plant population greatly affects garlic growth, yield and quality. Many authors reported a detrimental effect of lack of optimum plant population on bulb production [14 -17]. However, different garlic varieties might respond differently to different intra-row spacing, and to optimize crop yield and productivity, a full package of information is required for the specific growing system [7,8]. The use of optimum plant population density has a dual advantage. It avoids strong competition between plants for growth factors such as water, nutrients, and light. Conversely, it enables the efficient use of available cropland without wastage [9].
In the study area, most farmers cultivate alliums according to their own choice due to the absence or unavailability of standard production techniques, which contributed to the low yield of the crop [18]. The national recommended intra-row spacing is not optimum under all growing conditions as well as crop or varietal characteristics. Moreover, there are local and improved garlic varieties under cultivation in the study area. Varieties of garlic may differ in root architecture, foliage and other growth characteristics, and even a cultivar performs differently under different agro-climatic conditions [11]. Furthermore, in the study area, there is a lack of information on the growth and yield response of garlic to different intra-row spacing.
Therefore, the overall objective of the study is to evaluate the growth and yield response of garlic varieties to different intra-row spacing and to identify the optimum intra-row spacing and variety for high garlic yield in the study area.

Description of the Study Area
The experiment was conducted at Aksum University Selekleka research and demonstration center, located at Medebay Zana district, Northwestern Zone of Tigray region, during the offseason of 2018/2019. The experimental area is situated at 14° 08' 57"N latitude and 38° 17' 02" E longitudes at an altitude of 1864 m.a.s.l. The study areas belong to the cool sub-humid agro-climatic zone [19]. It receives a monomodal type of rainfall, which extends from June to September with an annual rainfall of 660 to 750 mm. The soil textural class is clay loam with a pH of 7.2. The average temperature for the past 15 years was 20.92°C with a mean maximum temperature of 30.97°C and a mean minimum of 11.4°C. The area is known for the mixed crop-livestock farming system in which cultivation of Teff (Eragrostis tef), Sorghum (Sorghum bicolor), Maize (Zea mays), Finger Millet (Eleusine coracana) and pea (Pisum sativum) are the major crops in the main season, and Garlic (Allium sativum) and onion (Allium cepa) are the leading root crops area during off-season [20].

Characteristics of Experimental Materials
Three garlic varieties, namely Bishoftu Netch and Tsedey 92, as improved varieties released by Ethiopia Institute of Agricultural Research, Debrezeit Agricultural Research Center, during 1999/2000 E.C and local garlic variety, were used for the study. The seed of the improved varieties was obtained from Aksum agricultural research center, and for the local, which is currently under production in the study area were collected from the local market. Tsedey 92 has white-colored bulbs, green leaves and takes about 110-130 days for its maturity and yields 7.5 to 8.13 t ha -1 with an average bulb weight of 41.59 g. The variety Beshoftu Netch has a white bulb color matures within 132 days, and yields 7.9 t ha -1 with a mean bulb weight is 42 g [21].

Treatments and Experimental Design
The experiment consisted of treatment combinations with four levels of intra-row spacing vis-a-vis 5cm, 7.5cm, 10cm and 12.5 cm and three garlic varieties vis-a-vis Bishoftu Netch, Tsedey92 and local variety. The experiment was set in a randomized complete block design (RCBD) with a factorial arrangement replicated three times. Bishoftu Netch and Tsedey 92 improved varieties released by Ethiopia Institute of Agricultural Research, Debre-Zeit Agricultural Research Center, during 2007. The local variety was collected from the local market in the area. For the experiment, a plot size of 3 m 2 (2 m x 1.5 m) having a distance of 0.5m, 0.3m and 1m between plots, rows and blocks, respectively, were used.

Experimental Procedure
Before planting the cloves, the experimental field was ploughed and harrowed by a tractor. Large clods were broken down to a fine tilth, and then a total of 36 plots, each with a size of 2 m x 1.5 m, were prepared, with 12 plots in each replication. On 23 rd December 2018, cured and medium to large-sized cloves of 1.50-2.50 g of the three test varieties which are not diseased, damaged, soft and discolored, were planted on well prepared raised ridges at a depth of 3 cm. Urea at a rate of 150 kg ha -1 and DAP at 200 kg ha -1 were applied as a source of nitrogen and phosphorus respectively, where DAP fertilizers were applied at planting and urea fertilizer were side dressed in a split in two applications; with half was applied two weeks after emergence and the remaining half five weeks after emergence [22]. Gap filling (re-planting) was carried out within one week to replace those seedlings which were damaged and failed after transplanting. Irrigation was supplied through the furrow irrigation method at four days interval for the 1 st month and then extended to seven days interval and stopped two weeks prior to harvesting [23]. All other management practices, including harvesting of bulbs, were carried out according to the procedure developed by [23].

Data Collection
Data on growth and yield and attributes yield were collected from ten randomly selected plants of three rows of each plot at physiological maturity and harvesting, respectively. The total, marketable and unmarketable bulb yield was recorded from three central rows of each plot. Phenological data such as days to maturity were recorded on a plot basis at maturity.
Days to maturity was recorded by counting the number of days from planting to the time when 70% of plants in a plot fall down their foliage and neck fall. Plant height was measured from the soil surface to the tip of matured leaf using a ruler in centimeters at the time of maturity; Leaf number was determined by counting the total number of leaves produced at physiological maturity; Leaf length was measured using ruler at physiological maturity; Leaf width was recorded as the average of the width of ten randomly selected leaves measured at their widest part at physiological maturity using meter scale; Bulb length was measured using digital caliper of ten randomly selected bulbs at the time of harvesting; Bulb diameter was measured at the widest point using digital caliper at the time harvesting; Clove number was recorded by counting the number of cloves per plant at harvesting; Clove length was recorded by measuring the length of cloves using digital caliper at the time of harvesting; Clove diameter was recorded by measuring the diameter of cloves at the widest point using digital caliper at the time of harvesting; Mean bulb weight was recorded by weighting the bulbs just after curing, using digital balance; Fresh and dry biomass were recorded as the sum of the fresh and dry weights of bulbs and above ground parts respectively at the time of harvesting using digital balance where the dry biomass was dried in an oven at 70 o c for 72 hours; Marketable clove number was recorded just after topping and curing by counting of cloves having greater 1 g weight and undamaged ones according to the procedure used by Fikreyohannes (2005) [24]; Unmarketable clove numbers were recorded after topping and curing by counting the number of unmarketable cloves that are damaged and having less than 1.0 g weight [24]. Marketable bulb yield was expressed in ton per hectare and recorded from plants obtained from three rows in a plot just after curing that have a healthy and marketable bulb with a weight of 20 g -160 g [25]. Unmarketable bulb yield was recorded as the weight of bulbs that are undersized (<20 g), diseased, decayed and bulbs having the physiological disorder as described by the study [25] from three central rows after curing and expressed in ton per hectare and thus total bulb yield was recorded as the sum weight of marketable and unmarketable bulbs after curing and the yield was converted into ton per hectare. Harvest index was computed by dividing the mean fresh weight of mature marketable bulb (economic yield) to mean fresh biomass yield (biological yield) and multiplied by 100.

Data Analysis
The collected data were subjected to analysis of variance (ANOVA) using Genstat 18 th edition statistical software as per the procedure of Gomez (1984 [26]. Means were separated using Fisher's least significant difference (LSD) at a 5% probability level. Pearson's correlation was conducted to see the relationship between the recorded growth, yield and yield components.

Days to Maturity
The main effect of variety and intra-row spacing had significantly affected days to maturity of garlic while their interaction effect did not ( Table 1). Days to maturity ranges from 127.4 to 140.4 days. Tsedey 92 took a long time of 140.4 days to mature, while the local variety took shorter days of 127.4 to mature ( Table 1). The variation in maturity among the three varieties might be due to their genetic differences. Many research authors indicated that Tsedey 92 garlic variety matured late than Bishoftu Netch [27]. Intra row spacing of 12.5 cm took the longest time of 140.0 days to reach maturity, while the intra-row spacing of 5 cm reaches maturity earlier with 124.6 days ( Table 1). As the intra row spacing increases, days to maturity was delayed. The variation in maturity might be due to the fact that the growth rate is faster in closely spaced crops than widely spaced ones. This is because of high intraspecific competition for growth factors, where plants get stressed and strive to set the bulb as early as possible. Garlic planted closely mature earlier than those planted widely [28,29].

Plant Height
Plant height was tallest (68.26 cm) with Variety Tsedey 92 while it was shortest (62.02 cm) with the local variety ( Table  1). Garlic displayed great variation for plant height and other morphological characters [30]. The difference among the varieties for plant height could be attributed to their genotypic variability. The tallest plant height of 69.74 cm was obtained from the wider intra-row spacing of 12.5 cm, whereas the shortest (60.5 cm) was obtained at 5cm intra-row spacing ( Table 1). The tallest plant height at a wider intra-row spacing of garlic was reported by [31]. Similarly, the study [32 -34] reported maximum plant height at wider row spacing on the onion. This reduction in plant height at increased plant density might be due to the competition for growth factors such as soil moisture, nutrient, space and light [35,36]. Interactions of intra-row spacing and variety did not significantly (P ≤ 0.05) plant height of garlic ( Table 1).

Leaf Number
Tsedey 92 produced more leaf number of 16.53, while the local variety produced the lowest (14.01) number of leaves ( Table 1). Many scholars indicated significant varietal differences in the number of leaves produced by garlic plants [37,38]. The results might be attributed due to the genetic variation and vegetative growth differences among the studied garlic cultivars. As intra-row spacing increased from 5 cm to 12.5 cm, the number of leaves per plant increased from 13.8 to 16.90. The highest leaf number of 16.9 was obtained from 12.5cm intra-row spacing while the lowest (13.80) was recorded at 5 cm intra-row spacing ( Table 1). This statement is in conformity with that of the study [31,34,39], which also reported a reduction in the number of leaves with an increased plant density of garlic plants. This could be due to the fact that widely spaced plants produce more axillary branching than closely spaced plants that resulted in fewer leaf numbers. It might also be attributed due to the closer the spacing, the more the competition among the plants for water, soil nutrients, space, light, etc., hence making them not photosynthesizing very well. In the present study, however, interaction effects of intra-row spacing and variety did not significantly affect the number of leaves of garlic plants.

Leaf Length and Width
The results of the analysis of variance indicated that variety Tsedey 92 gave a longer leaf length of 44.33 cm while the local variety gave the shortest leaf length of 35.92 cm ( Table 1). Many research findings indicated a significant difference in leaf length among garlic varieties [38,40]. Similarly, leaf width of Tsedey 92 variety gave highest (2.24 cm) while, Bishoftu Netch gave the lowest (2.02 cm) although statistically at par with the local variety of garlic ( Table 1). In accord, the study [39,41] found a significant difference among garlic cultivars in their leaf width. This might be attributed due to inherent and genetic variation among garlic varieties in their growth habit like plant height, leaf length, leaf width, leaf number, leaf shape and vigour. There was a linear increase in leaf length of garlic from 44.89 cm to 36.44 cm with an increase in intra-row spacing from 5 cm to 12.5cm ( Table 1). The widest leaf width of 2.34 cm was obtained at intra-row spacing of 12.5 cm, while the narrowest leaf of 1.90 cm was obtained at 5 cm intra-row spacing ( Table 1). The highest leaf length of onion was obtained at wider intra-row spacing and the shortest at the narrow intra-row spacing [34,40,42]. The lowest plant densities can give wider leaf width of onion as reported by many scholars [34, 43 -45]. This might be attributed due to the fact that wider plant spacing showed less competition for resources, and as a result, leaves develop to a larger size. However, the interaction effects of intra-row spacing and variety did not significantly affect leaf length and width of garlic plants ( Table 1).

Bulb Length
There was a significant effect of variety and intra-row spacing on bulb length of garlic, but their interactions did not have any effect ( Table 2). Variety Tsedey 92 gave a maximum bulb length of 4.29 cm, although statistically at par with that of the local variety, which gave 4.22 cm. However, variety of Bishoftu Netch gave the shortest with 3.96 cm ( Table 2) [46,47]. also found a significant difference in bulb length among garlic varieties. This variation might be due to the reality that varieties can have different characteristics in their bulb shape, bulb length and bulb color.
The wider intra-row spacing of 12.5cm gave the highest bulb length of 4.49cm, while the smaller intra-row spacing of 5cm gave the minimum of 3.78 cm. Bulb length obtained at 12.5 cm intra-row spacing exceeds that obtained at 5 cm intrarow spacing by 15.81% ( Table 2). Many researchers indicated bulb length of garlic was highest at wider intra-row spacing than 5cm intra-row spacing [47,48]. The increment in bulb length at increased intra-row spacing probably might be attributed due to more nutrients, space and moisture availability so that there might be less competition for available growth resources.

Bulb Diameter
Tsedey 92 variety recorded the maximum bulb diameter of 4.71 cm and is at par with bulb diameter (4.62 cm) obtained with Bishoftu Netch while the minimum (4.36 cm) obtained at the local variety of garlic ( Table 2). Many researchers found a significant difference in bulb diameter among garlic varieties [49,38,46]. The possible reason for such a result could be due to variations among varieties in their bulb character, such as bulb size.
An increment in bulb diameter was observed with increasing intra-row spacing, and 12.5cm gave the highest (5.02cm) although statistically at par with that recorded at 10cm intra-row spacing. However, 5 cm intra-row spacing gave the minimum bulb diameter of 3.95cm ( Table 2). There was a bulb diameter increment of about 21.31% with an increase in intra-row spacing from 5cm to 12.5cm ( Table 2). The study [48,24] reported a maximum bulb diameter at 15 cm intra-row spacing than the narrow intra-row spacing of 10 cm. In the study, interactions of variety and intra-row spacing did not significantly influence bulb diameter of garlic ( Table 2). Note: means with the same letter within a column are not statistically significant at 5% probability level. BL = bulb length; BD = bulb diameter

Mean Bulb Weight
Mean bulb weight had significantly affected the main effect of variety and intra row spacing, while their interaction effect did not affect ( Table 2). Mean bulb weight ranged between 34.31 g to 41.58 g, where it was highest (41.58 g) with variety Tsedey 92 while it was lowest (34.31g) with the local variety ( Table 2). Garlic varieties show a significant variation in mean bulb weight [49,50]. The difference in bulb weight among garlic varieties might be accounted for the vigorous growth (production of more leaves), which helped in the synthesis of more photosynthates, increased accumulation of carbohydrates, and other metabolites, which ultimately determined the weight of bulbs per plant.
Plots planted at a wider intra-row spacing of 12.5cm gave the highest mean bulb weight of 41.39 g, while those planted at a narrower intra-row spacing of 5cm gave the lowest (34.51 g) although statistically at par with that obtained at 7.5cm intrarow spacing ( Table 2). The result showed a linear increment of bulb weight per plant as plant density per square area decreased. The study [51] reported maximum bulb weight at 20 cm intra-row spacing on Huruta shallot variety than 10 cm. Moreover, an increased mean bulb weight of onion was observed with increasing intra-row spacing from 5 cm to 10 cm [52]. Production of heavier bulbs in the wider spacing might be due to the fact that widely spaced plants experienced little or no competition for limited environmental resources compared to closely spaced plants.

Clove Number
The highest clove number per bulb of 23.74 was obtained from Tsedey 92, while the lowest (20.29) was obtained at the local variety ( Table 2). A difference in the number of cloves per bulb with different garlic varieties was reported by many authors [49,53]. The highest clove number per bulb of 24.03 was obtained from 12.5 cm intra-row spacing, while the lowest (19.96) was obtained at 5 cm intra-row spacing ( Table 2). The study [49] indicated the highest number of clove number per bulb at 20 cm intra-row spacing as compared to a plot planted with 10 cm intra-row spacing. Moreover, the highest clove number per bulb was at wider intra-row spacing [54]. These observations could be associated with garlic planted at wider spacing tend to establish better in individual bulb characteristics compared with those closely spaced plants because widely spaced plants are able to intercept more light and suffer less competition for nutrients, hence produced more yield per individual stands. Clove number per bulb of garlic did not significantly affect by the interactions of intra-row spacing and variety ( Table 2).

Clove Length and Diameter
Clove length was significantly affected by the main effect of variety and intra-row spacing as well as their interactions ( Table 3). The highest mean clove length of 3.34cm was obtained at the interactions of treatment combinations of variety Tsedey 92 and 12.5 cm intra-row spacing while the shortest clove length of 2.757 cm was obtained in the treatment combinations of local garlic variety with 5 cm intra-row spacing ( Table 3). The findings indicated that irrespective of variety, clove length was longer at wider (12.5 cm) intra-row spacing than at narrow spacing. Similarly, the study [55] confirmed an increased clove length of garlic at wider intrarow spacing.
Garlic variety significantly influenced clove diameter, and the highest clove diameter of 1.82 cm was obtained from Tsedey 92 variety, and the lowest (1.45 cm) was recorded from a local variety ( Table 4). The study [53] reported that garlic cultivars showed a significant difference in clove diameter. This might be due to variations in genetic character and genetic makeup among the evaluated garlic varieties. With regard to intra-row spacing, it has significantly affected the clove diameter of garlic with the maximum (1.74 cm) obtained at 12.5 cm intra-row spacing although at par with that obtained at 10cm intra-row spacing. However, the minimum (1.59 cm) clove diameter was obtained at 5 cm intra-row spacing, which was statistically similar to that obtained at 7.5 cm and 10 cm intra-row spacing ( Table 4). Many scholars indicated higher clove length and diameter among different varieties of garlic tested and onion at wider intra-row spacing [15,38]. However, the interaction effects of variety and intra-row spacing did not significantly affect the clove diameter of garlic.   Note: means with the same letter within a column are not statistically significant at 5% probability level.

Biomass
Main effects of variety and intra-row spacing, as well as their interactions, had significantly affected the fresh and dry biomass yield of garlic (Table 5). Variety Tsedey 92 interacted with 12.5 cm intra row spacing gave the highest fresh biomass yield of 126.43 g while local variety in combination with 5 cm intra-row spacing gave the lowest fresh biomass yield of 64.27 g although statistically at par with Bishoftu Netch in combination with 5 cm intra-row spacing which recorded 67.53 g ( Table 5). With regard to dry biomass yield of garlic observed at treatment combinations of varieties Bishoftu Netch and intra-row spacing at 10 cm gave the highest dry biomass yield of 61.6 g. However, local variety in a combination with intra-row spacing at 5 cm gave the lowest dry biomass yield of 39.0 gm ( Table 5). With all the tested garlic varieties, the fresh and dry biomass yield increased with increased intra-row spacing. The study [47] indicated lower dry biomass yield of Chelenko I and Tsedey 92 garlic varieties at a narrow intra-row spacing of 5 cm than at wider spacing. There were reports on increase in fresh and dry biomass yield of onion at wider intrarow spacing [56 -58]. This might be due to the fact that closer spacing between plants resulted in competition for resources, thus reducing the assimilation rate stored in the plant.

Marketable Clove Per Bulb
The mean square analysis revealed that the main effect of variety and intra-row spacing, as well as their interaction effects, significantly influenced marketable clove numbers per bulb ( Table 6). The highest marketable clove numbers per bulb were highest (15.73) at combinations of Tsedey 92 variety and 12.5 cm intra-row spacing, while the lowest (9.67) was obtained at combinations of local variety planted at 5 cm intrarow spacing ( Table 6). Irrespective of the tested garlic variety, marketable cloves number per bulb was lowest with decreasing intra-row spacing. Garlic varieties showed a huge variability in clove number [1]. Larger clove size at a widely spaced planting might be due to greater accumulation of assimilates, which resulted in better utilization of nutrients, moisture and light among plants and an increase in the size of cloves per bulb.

Marketable Bulb Yield
Marketable bulb yield had been significantly affected by the main effect of variety and intra-row spacing as well as their interaction effects ( Table 7). Tsedey 92 variety in combination with 5 cm intra-row spacing gave the highest marketable bulb yield of 8.05 t ha -1 while the lowest marketable bulb yield of 4.944 t ha -1 was obtained from combinations of local garlic 7DEOH 3 FRQWG variety interacted with 12.5 cm intra-row spacing ( Table 7).
In general, marketable bulb yield showed a decreasing trend with an increasing intra-row spacing in all tested garlic varieties. This variation might be due to the fact that with increased plant spacing, the population per area of land also decreases that accounts for reduced marketable bulb yield. There is an impact of plant density on marketable bulb size. With higher plant density, there is a smaller marketable bulb size [60]. Significant effect of variety and intra-row spacing combination on marketable bulb yield of garlic has also been observed [58,59]. Similarly, increasing intra-row spacing from 5 to 10 cm gave a decrease in marketable bulb yield [52]. Moreover, Pearson's correlation indicated a significant and negative relationship between marketable bulb yield of garlic with many parameters such as leaf width (-0.35*), bulb diameter (-0.53***), bulb length (-0.39***) and fresh biomass yield (-0.39*) ( Table 8).

Total Bulb Yield
There was a significant main effect of variety and intra row spacing as well as their interaction effects on the total bulb yield of garlic ( Table 7). The highest total bulb yield of 8.98 t ha -1 was obtained at a combination of variety Tsedey 92 and 5 cm intra row spacing, although statistically at par with combinations of Bishoftu Netch interacted with 5 cm intra-row spacing which gave 8.81 tha -1 . However, the lowest total bulb yield of 5.37 tha -1 was obtained from combinations of local variety interacted with 12.5 cm intra row spacing ( Table 7).
As intra-row spacing increased from 5 to 12.5 cm, total bulb yield of garlic decreased from 8.98 t ha -1 to 7.0 t ha -1 in variety Tsedey 92, and from 8.815 t ha -1 to 5.704 t ha -1 in Bishoftu Netch as well as from 8.148 tha -1 to 5.370 tha -1 in local garlic variety ( Table 7). The highest total bulb yield observed at closer spacing could be due to increased plant population. Increased bulb yield of garlic at higher plant populations is confirmed by many authors [59,60]. Moreover, total bulb yield showed a positive and significant correlation with unmarketable clove per bulb, marketable bulb yield (0.96***) and harvest index ( Table 8). Increased shallot total yield per hectare with an increase in plant density [51]. Many authors indicated that varieties could have different yield potential in different agro-ecologies due to their genetic and genetic environment interaction effects [43,58].

Harvest Index
The main effect of intra row spacing significantly affected the harvest index of garlic plants, while the main effect of variety and its interaction with intra-row spacing did not ( Table  9). Intra row spacing at 5 cm gave the highest harvest index of 50.41% while, the lowest harvest index of 35.69% was obtained at the wider intra-row spacing of 12.5 ( Table 9). Higher harvest index recorded at narrow spacing's might be due to the presence of shortest leaf length, plant height and leaf diameter, which reduced above-ground biomass and resulted in a higher harvest index. On the other hand, the lowest harvest indices at the wider spacing could also be attributed to excessive vegetative growth that has a detrimental effect on the partitioning of assimilates towards the bulbs. Moreover, the lowest harvest index at a wider intra-row spacing of 12.5 might also be due to the existence of a higher number of leaves, wider leaf length and diameter at wider intra-row spacing. Similarly, harvest index positively and significantly influenced by plant height(0.63***), leaf number(0.67***), leaf length(0.54***), leaf width (0.66***), bulb diameter(0.50**), number of cloves per bulb(0.41*), mean bulb weight(0.47**), fresh bulb yield(0.88***), dry bulb yield(0.60***), marketable bulb yield(0.62***) and total bulb yield ( Table 8). Similar findings were reported for onion and shallot, respectively, in Ethiopia [52, 51]. Note: means with the same letter within a column are not statistically significant at 5% probability level.    Note: means with the same letter within a column are not statistically significant at 5% probability level, ns=non-significant

CONCLUSION
The aim of this study was to evaluate the response garlic to intra-row spacing and variety, and to identify the optimum intra-row spacing and high-yielding variety. With this notion, a field experiment was conducted to assess the effect of intrarow spacing on the growth and yield response of garlic varieties. Accordingly, the highest total bulb yield of garlic was observed at the combinations of Tsedey 92 variety and 5 cm intra-row spacing, although statistically similar with the yield obtained at combinations of Bishoftu Netch at same intra-row spacing. Marketable bulb yield was highest at the combinations of variety Tsedey 92 and 5 cm intra row spacing, and the lowest was obtained at the local variety in combination with 5 cm intra-row spacing. Therefore, it can be concluded that Tsedey 92 variety of garlic in combination with 5cm intra-row spacing showed better yield performance, and thus, it can be recommended to the study area.

HUMAN AND ANIMAL RIGHTS
No animals/humans were used for studies that are the basis of the research.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Performance Evaluation of Sesame under Regulated Deficit Irrigation Application in the Low Land of Western Gondar, Ethiopia

Sesame (Sesamum indicum L.) is the leading oil seed crop produced in Ethiopia. It is the second most important agricultural commodity for export market in the country. It is well suited as an alternative crop production system, and it has low crop water requirement with moderate resistance to soil moisture deficit. The low land of North Western Ethiopia is the major sesame producer in the country, and the entire production is from rainfed. The rainfall distribution in North Western Ethiopia is significantly varied. This significant rainfall variability hampers the productivity of sesame. Irrigation agriculture has the potential to stabilize crop production and mitigate the negative impacts of variable rainfall. This study was proposed to identify critical growth stages during which sesame is most vulnerable to soil moisture deficit and to evaluate the crop water productivity of sesame under deficit irrigation. The performance of sesame to stage-wise and uniform deficit irrigation scheduling technique was tested at Gondar Agricultural Research Center (Metema Station), Northern Western Ethiopia. Eight treatments, four stage-wise deficit, two uniform deficit, one above optimal, and one optimal irrigation applications, were evaluated during the 2017 irrigation season. The experiment was designed as a randomized complete block design with three replications. Plant phenological variables, grain yield and crop water productivity, were used for performance evaluation. The result showed that deficit irrigation can be applied both throughout and at selected growth stages except the midseason stage. Imposing deficit during the midseason gave the lowest yield indicating the severe effect of water deficit during flowering and capsule initiation stages. When deficit irrigation is induced throughout, a 25% uniform deficit irrigation can give the highest crop water productivity with no or little yield reduction as compared with optimal irrigation. Implementing deficit irrigation scheduling technique will be beneficial for sesame production. Imposing 75% deficit at the initial, development, late season growth stages or 25% deficit irrigation throughout whole seasons will improve sesame crop water productivity.

Introduction
In recent years, the world has become more complex due to an alarming growing world population and its demand for more food, water, and energy. Because of limited arable land for expanding food production, there is an increase in pressure on natural resources and ecosystem services [1][2][3]. e essential factors in food production that is per capital of cultivated land and fresh water are declining continuously as the world population increases. e natural resources over the world are limited, and excess manipulation and utilization will exaggerate resource exhaustion [4].
Fresh water and soil nutrient availability remains globally the most limiting crop growth factors [5]. Increase in threats of fresh water shortage, reduction in arable land, decline in soil fertility, and more frequent and severe drought due to climate variability/change have stimulated research into water saving strategies aiming at producing more "crop per drop" [5,6]. Crop production is the largest global consumer of fresh water, and water is a key resource in agricultural production [7]. In worldwide, agriculture accounts for 80-90% of all fresh water used by humans, and most of the water is used for the cultivation of crop [6]. Realizing the combination of high crop water productivity and improved crop yield is an important element of sustainable development.
e main limitation for irrigation development in most parts of Ethiopia is the availability of water [8]. Whenever the available fresh water supply is a limiting factor for agricultural production, deficit irrigation practices have paramount importance in improving crop water productivity [9][10][11]. Deficit irrigation application is a simple agronomic practice without extra cost and can be easily implemented as farm water saving strategy for every farming communities.
Crops recommended for deficit irrigation applications are those that are relatively drought resistant and early maturing varieties [12]. Sesame (Sesamum indicum L.) belongs to the order Tubiflorae and family Pedaliaceae and is one of the most ancient and early maturing oil crops in the world [13,14]. Sesame is commonly known as the king of oil crop because of high level oil content of the grain seed (50%-60%) [15]. It is well suited as an alternative crop production system under irrigation because it has low-level crop water requirement [16]. Ethiopia is ranked 6 th in level of sesame production with estimated 327,741 tons of production in 2013 cropping season [17]. Sesame seed is a leading crop in the country oil seed export where more than 90% of production is directed to export market [14,18]. Sesame is the second most important agricultural commodity in Ethiopia next to coffee by foreign exchange earnings in which agricultural commodities are the mainstay of Ethiopian economy [17]. e low land of North Western Ethiopia is the major sesame producing area in the country, and the entire production is from rainfed. e rainfall distribution in north western part of Ethiopia for the last 30 years was significantly varied [19]. is significant rainfall variability hampers the productivity of sesame. erefore, irrigation agriculture has immense potential to stabilize crop production and mitigate the undesirable impacts of variable or insufficient rainfall for cultivated crops [20]. Irrigation development in Ethiopian context could increase farm production, income, asset endowment, and employment opportunity; reduce the incidence of poverty at the household level; and reduce vulnerability to climate variability in the country [21,22]. Plant nutrient availability and soil moisture-related issues are the major factors causing frequent crop failure and/or hamper productivity of sesame in Ethiopian context [17]. Abasena is one of the widely cultivated sesame variety in most sesame producing areas of Ethiopia, and the productivity is improved when the production is under irrigation [17].
Recent studies in Ethiopian context support that lands under irrigation system are more stable and profitable than rainfed production. Expanding irrigation development, as supported by the Ethiopian government, is a feasible development strategy with the potential for solving food security problem in the country [23]. With high market demand, promising environmental condition, and fair favorable price for farmers, sesame has been introduced and promoted in the North Western low land of Ethiopian agricultural production system [24]. Sesame is drought tolerant and has short life cycle of 80-120 days to maturity with a good potential for crop intensification in given plot of land [25]. Given the early maturing and low-crop water requirement nature of sesame, assessing the critical growth stages where sesame is most sensitive to water stress could help in improving crop water productivity and water-related managements [25,26]. e specific objectives of the study were to identify critical growth stage/s during which sesame is most sensitive to water deficit and to evaluate the crop water productivity of sesame with induced deficit irrigation scheduling at different growth stage/s in the low land of North Western Ethiopia.

Description of the Study Site.
e experiment was conducted at Gondar Agricultural Research Center (Metema Station) in North Western low land of Ethiopia from February 17 to June 7, 2017. Metema is located about 900 km northwest of Addis Ababa and 188 km West of Gondar Town (Figure 1). e site is located at 12°57′01.35″N latitude, 36°15′27.97″E longitude, and 760 meter above sea level. February to May is generally dry, and there was little rain during the experiment period ( Figure 2). e minimum and the maximum annual long-term average daily temperature ranges from 16°C to 24°C and 30°C to 40°C, respectively ( Figure 2). Average daily temperature becomes very high during the months of March to May, where it may get to as high as 43°C. e low land of North Western Ethiopia has a monomodal rainfall. e rainy months extend from June until the end of September. e soil had dark brown clay loam texture, low salinity, neutral pH, medium CEC, very low organic carbon, low total nitrogen, and low available phosphorous (Table 1).

Soil Sampling, Preparation, and Analysis.
Composite soil sample was collected using auger at soil depth 0-30 cm from the experimental field to determine selected soil physicochemical properties. However, for determination of bulk density (BD), moisture content at field capacity (FC), and permanent wilting point (PWP), undisturbed soil samples were collected using core samples from the experimental field before planting. e soil samples were analyzed following the standard laboratory procedure described by [27]. e soil moisture content at FC and PWP was measured at -1/3 and -15 bars, respectively, using the pressure plate apparatus. e available water holding capacity of the soil can be obtained by subtracting the water content at PWP from that at FC. e soil bulk density was calculated as the ratio of mass of oven-dried soil to bulk volume of soil in the core.

Experimental Design and Treatments.
e field experiment was designed as randomized complete block design with eight treatments and three replications ( Table 2). Each experimental unit had six rows. e two outer rows of each sides were used as the buffer row, while the four central rows were used for agronomic data collection. Sesame (variety Abasena) was planted 10 cm between crops and 40 cm between rows on a 2.4 × 5 m 2 plot per treatment. Two seeds were planted per hills and thinned to one plant after emergence. Frequent weeding was done manually in all experimental units when there was an invasion of weeds.
Furrows were 5 m long, closed end, and with 1% average slope of the experimental field. Nationally blanket recommended P 2 O 5 fertilizer at a rate of 46 kg ha −1 P 2 O 5 was applied at the time of planting. e source of N was urea fertilizer (46% N), and DAP (18% N) at the rate 64N kg·ha −1 was applied for all treatments uniformly with split application: half at time of planting and half 45 days after planting.
A 25 mm preplant irrigation water was applied to experimental field 5 days before planting date to break clods, to loosen the soil for better planting and to ensure that the crop germinates well and for desired crop stand establishment. Subsequent irrigations throughout the experimental period were applied as per the treatments depth. e CROPWAT 8.0 computer program which was based on the FAO Penman-Monteith method [28] was used for calculating reference crop evapotranspiration (ETo) with long-term monthly average climatic parameters. Crop water requirement (ETc) and irrigation requirement were estimated on daily basis. e crop parameters adopted to calculate crop water requirement at daily step are presented in Table 3.
ese parameters include the crop coefficient (Kc), rooting depth taken here as the sowing depth, manageable soil moisture depletion level (P), and the number of days at each growth stages. Irrigation water was applied as variable depth on fixed seven-day interval for each treatment throughout the experimentation periods. e experimental field was arranged to create favorable condition for crop growth and furrow irrigation application as per the recommendation of the agronomic requirement of sesame. e irrigation water source was from the nearby water channel. e water was then brought to the experimental field by water pump and collected in a pond for ease of controlled irrigation water application. And then the water was applied carefully to every experimental unit through narrow delivery pump to ensure application at desired level of water for each treatment and to avoid over flow of water during irrigation water application.

Data Collection and Analysis.
e following agronomic data were collected from the middle four rows of each experimental units, that is, the gross size of 2.4 × 5 m 2 . And the net (harvestable) plot was 1.6 × 5 m 2 . Plant phenological stages such as days to flowering and maturity were recorded in the plot base. Parameters such as number of capsule per plant and number of seed per capsule were recorded on plant bases. ousand seed weight (g), grain yield (kg/ha), and crop water productivity (kg ha −1 mm −1 ) were recorded in plot base. Days to flowering 50%: number of days from planting until 50% of the plant in the plot initiates flowering. Days to physiological maturity: days to physiological maturity were recorded number of days from planting until 75% of the plants in the plot bear matured capsules.
Crop water productivity (kg mm −1 ) was calculated with the following empirical formulas: where WP stands for crop water productivity (kg mm −1 ). Ya is the marketable crop yield (kg/kg ha −1 ), and ETa refers to the actual volume of water lost through evaporation and/ crop transpiration during the crop cycle (m 3 /mm) [9]. Since there is no easy way of distinguishing between transpiration by plants and evaporation from field, they are generalized under the terms of evapotranspiration [29]. e yield response factors for deficit irrigation treatments which is the ratio of relative yield reduction to relative evapotranspiration were calculated using the following equation [28,30]: where K y refers to the yield response factor, Ya stands for the actual crop yield (kg·ha −1 ), Ym stands for the maximum crop yield (kg·ha −1 ), ETa refers to actual evapotranspiration (mm), and ETm also stands for maximum evapotranspiration (ETc) in mm. e data analysis for the collected crop data was done using analysis of variance (ANOVA) following general linear model procedure of Statistical Analysis Software (SAS). Means were separated using the least significance difference (LSD) test at the 95% level of probability when there was a statistically significant difference between treatments.

Crop Water and Irrigation Requirement.
e values of the reference crop evapotranspiration (ET o ) of the experimental site were calculated using the FAO Penman-Monteith method during the cropping season on daily step, and the value varied between 4.05 mm/day in February and 6.59 mm/day in April (Table 4). e calculated ET o value of the experimental site varies from 4.05 to 6.59 mm/day, and the evapotranspiration demand ranges from moderate to high [28]. e crop coefficient (K c ) values adopted from [28] varied from 0.60 at the time of sowing to high of 1.1 during midseason (Table 3). Accordingly, the optimum irrigation amount required under no stress condition (100% ET c ) was about 548 mm (Table 3), while the minimum amount was 287 mm (50% ETc) and the maximum amount 679 mm (120% ETc).

Phenological Variability and Plant
Height. Deficit irrigation water application showed significant difference (p < 0.05) on plant height in sesame crop which was recorded at the time of physiological maturity (Table 5). e maximum plant height (182 cm) was observed under optimum irrigation water application throughout the growth season (UO), and the lowest plant height was also recorded (158 cm) on experimental plots which received 50% deficit irrigation throughout the growth season. Similar research findings on sesame have been reported in [31,32]. Water is important to build plant tissue and carries out biochemical and physiological activities within the plant body to express the development.
Crop phenological data collected from the field study showed that days to 50% flowering were significantly affected (p < 0.01) by different levels of irrigation treatments and stage-wise deficit irrigation water application (Table 5). International Journal of Agronomy e 50% deficit application throughout the growing season (0.5 × ETc) and deficit irrigation water application during the midseason stage (DM) took the shortest number of days (52) to bear flowers, while experimental plots provided with uniform optimal irrigation (UO) through the whole growth season took the longest number of days (61) from planting to 50% plants in a plot to bear flowers. Deficit irrigation water application during the midseason stage could reduce number of days from planting to 50% flowering.
Statically significant (p < 0.05) variability in the days to maturity was observed (Table 5) among irrigation treatments. Meanwhile, experimental plots which received 50% deficit throughout the whole growth season also mature 10 days in advance compared with maturity date of optimally treated experimental units. For both days to 50% flowering and days to maturity, the variability among the stage-wise deficit irrigation application treatments was not statistically significant (p < 0.05). However, significant variability in days to 50% flowering for stage-wise deficit irrigation has been observed when deficit has been induced at the development stage. is may be explained by the fact that the crop was able to recover from the stress (deficit irrigation induced at early crop growth stages) when optimum irrigation amount was applied during the rest of growth stages. e effect of different deficit irrigation water applications resulted in a statistically significant (p < 0.05) variability in the number of capsules per plant in the sesame field experiment (Table 5). e maximum number of capsules (122) per plant was observed under optimal irrigation water application treatment (UO), and the minimum number of capsules (67 and 71.1) per plant was obtained under deficit 75% during midseason stage (DM) and 50% deficit irrigation application throughout the whole growth season, respectively. ese results are in accordance with the findings of Nadeem et al. [33] who reported that number of capsules per plant in sesame plants have been significantly affected by different levels of soil moisture regimes. e variability among different irrigation water application treatments was statistically significant in the number of seeds per capsule on sesame plants (p < 0.05). e maximum number of seeds (74.4) per capsule was obtained under the optimum irrigation water application throughout, while the lowest number of seeds (57.7) per capsule was obtained when 50% water deficit is induced throughout the whole growth season. Among the stage-wise deficit irrigation water application treatments, 75% deficit applied during the midseason (DM) stage resulted in a statistically lower number of (58.3) seeds per capsule. Flower abortion due to stress at the midseason growth stage might be an explanation why the number of capsules per plant for sesame is substantially reduced when deficit irrigation is induced at the midseason growth stage [32].

Yield and Crop Water Productivity.
e different deficit irrigation treatments affected the average grain yield of sesame significantly (p < 0.01) ( Table 6). For uniform deficit irrigation water application treatments throughout the whole growth season, the lowest yield (670 kg·ha −1 ) was obtained when 50% deficit irrigation was induced throughout the whole growth season, while the highest yield (1840 kg·ha −1 ) was obtained under optimum irrigation (UO). e average grain yield sesame reduction was not statistically significant among stage-wise deficit irrigation application treatments at the initial, development, and late season growing stages. But when the stage-wise deficit irrigation was induced at flowering and pod-bearing stage (deficit during the midseason stage), significantly lower yield (58.6%) was recorded compared with the optimal irrigation (UO). e grain yield reductions of sesame with stage-wise deficit irrigation when compared with uniform optimal irrigation application are as follows: when the deficit was induced at the initial stage, the yield reduction was (4.0%); when the deficit was induced at the development stage, the yield reduction was (13.3%); and when the deficit was induced at the late season stage, the yield reduction was (8.9%).  International Journal of Agronomy e significant impact of the deficit application at the midseason stage on sesame grain yield was in line with other findings which indicate that water deficit that occurs at the reproductive stage specially during flowering and capsule formation stage affects grain yield more severely [32,34].
Crop water productivity (CWP) of sesame varied from 2.041 kg·ha −1 mm −1 under deficit irrigation application at the midseason stage (DM) to 4.281 kg·ha −1 mm −1 when 75% deficit was applied throughout the growth seasons (Table 6). Deficit irrigation application at the flowering/grain feeling (DM) stage resulted in the lowest crop water productivity among the whole stage-wise deficit irrigation treatments which were even less than the optimal irrigation water application (UO). Hence, inducing deficit irrigation water application during the midseason period (DM) could not be valuable for both yield improvement and for the enhancement of crop water productivity. is result highlights that, in stage-wise deficit irrigation application, inducing deficit irrigation at flowering and capsule bearing stage for sesame crop yield reduction is amplified. It is therefore important to consider the growth stage of the crop when we want to plan stagewise deficit irrigation. Accordingly, in planning deficit irrigation scheduling practice, deficit should be mild at the midseason growth stage to reduce grain yield reduction for sesame. e finding in [35] reported that the grain yield of maize is severely affected by soil moisture stress at anthesis/early reproductive stages ultimately affecting crop water productivity. Another finding related to stage-wise deficit irrigation on mung bean by Ambachew et al. [36] also noted that application of adequate water during flowering and pod development was the most significant factor in deficit irrigation scheduling.

Yield Response Factor to Water
Deficit. From our field study, the yield response factor to each growth stage except  G. mean, grand mean; LSD, least significant difference; NS, no significantly different at probability level of 5%; CV, coefficient of variation. Means with same letter in each column are not significantly different at probability level of 5% using the LSD test. 6 International Journal of Agronomy water deficit at the development stage in stage-wise deficit irrigation scheduling was less than one ( Figure 3). Yield response factor (K y ) reflects the complex relationship between production and water use by the crop. is relationship could reflect a remarkable validity and allowable procedure to quantify the effect of water deficit on economical yield. erefore, relative yield reduction was less than the relative evapotranspiration deficit when deficit irrigation scheduling was induced at initial, development, and late development stages [30]. Among all deficit irrigation scheduling treatments, the highest K y value (1.9) was obtained when the deficit was induced at the midseason stage. is reflects the sensitivity of the crop to water deficit with proportionally larger yield reduction when deficit was implemented at this stage. e midseason stage in sesame was found to be very sensitive to water stress, while water deficit induced during the late growth stage has limited impact on yield. Inducing deficit irrigation at early stages (at initial or at development stage) and then the crop could get enough time for recovery, provided that optimal irrigation is applied to the rest of the growth stages. Variability in the yield response factor (K y ) might be observed under different irrigation practices, such as cultivation method, stage of the crop deficit irrigation induced, and extent of deficit irrigation induced [37].

Conclusion and Recommendation
Sesame is commonly grown as a rainfed crop in north western part of Ethiopia, but its productivity significantly improved when the crop is cultivated under irrigation. e field experiment revealed that crop phenology, yield component, grain yield, and crop water productivity of sesame significantly affected deficit irrigation treatments at different growth stages and throughout the growth season. Deficit irrigation scheduling can be applied for sesame production. But in stage-wise deficit irrigation, applying deficit at the midseason stage will not be beneficial because substantial yield reduction has been observed with induced deficit irrigation at this growth stage. With 30% irrigation water deficit (with stage-wise deficit irrigation) at the midseason stage, 58.6% grain yield reduction has been recorded. In deficit irrigation scheduling through the whole growth season, mild deficit is more beneficial in sesame production. When deficit irrigation is imposed with 25% uniform deficit throughout, the crop water productivity of sesame will improve significantly with no or reasonable crop yield reduction. e productivity of sesame will enhance by implementing irrigation production practice, and the crop is worthy for deficit irrigation scheduling with due emphasis on the growth stage when the deficit is stage-wise deficit irrigation. Sesame is found to be moderately sensitive to drought and with a potential for deficit irrigation scheduling.
is study is one-year single location experiment; furthermore, similar studies are recommended for validating this finding and to go for further possible growth stage combinations under deficit irrigation scheduling to refine crop water productivity of sesame in the district.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.  G. mean, grand mean; LSD, least significant difference; NS, not significantly different at probability level of 5%; CPW, crop water productivity; CV, coefficient of variation. Means with same letter in each column are not significantly different at probability level of 5% using the LSD test.

Conflicts of Interest
International Journal of Agronomy 7


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Effects of Peel Extract from Citrus reticulata and Hesperidin, A Citrus Flavonoid, on Macrophage Cell Line

1. Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia 2. Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia 3. Army Medical Center, Jl. Mayjen Soetoyo, Cililitan, East Jakarta, Jakarta 13640, Indonesia 4. Department of Histology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Jl. Farmako, Sekip Utara, Yogyakarta 55281, Indonesia 5. Faculty of Science and Technology, Universitas Aisyiah Yogyakarta, Jl. Ring Road Barat 63, Gamping, Sleman, Yogyakarta 55292, Indonesia

INTRODUCTION
Citrus plants (family: Rutaceae) have attracted researchers' attention due to their biological activities since the early 18 th century (Manthey et al., 2001). Their phytochemical healthpromoting properties are mainly based on the anti-oxidant activities of flavonoid compounds that contribute to the cardiovascular disease and cancer prevention, anti-inflammatory, antiviral, and antimicrobial properties of citrus (Barreca et al., 2017;Benavente-Garcia et al., 1997). Citrus species contain a variety of flavonoids, namely flavonones, 261 flavons, and flavonols (Benavente-García et al., 1997). Hesperidin, a flavonone glycoside, is a major flavonoid compound in Citrus reticulata (reviewed in Barreca et al., 2017). The first description of hesperidin by Lebreton in 1828 marked the beginning of researchers' vast interest in citrus flavonoids (reviewed in Manthey et al., 2001). As a fruit crop that is abundantly cultivated and consumed across the globe, citrus fruits generate a large amount of waste every year (Sharma et al., 2017). Citrus peels are one of the solid citrus waste products that need to be managed. Since 2007, the Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada (UGM), Indonesia has been exploring the extract of citrus peels in order to reveal its chemopreventive properties and, at the same time, to increase the utility of citrus waste (reviewed in Meiyanto et al., 2012). One of the studies was focused on the ethanolic extract of Citrus reticulata, commonly known as tangerine or mandarin, which has been shown to contain flavonoids (Armandari, 2010;Meiyanto et al., 2011) (Table I). In addition, hesperidin, as one of the main compounds, has been studied (Table I).
In vitro, it induces cell proliferation and angiogenesis in MCF-7 breast or WiDr colon cancer cells at concentrations of 10−1500μg/mL Puspita et al., 2008;Yunas et al., 2007) but can suppress MCF-7 cell
In vivo, 150-600μg Citrus extract shows antiangiogenic activities (Chrisnanto et al., 2008) and suppresses epithelial breast and hepatic cell proliferation in rats with chemically induced cancer at 750-1500mg/kg BW (Supriyati et al., 2008;Meiyanto et al., 2011). In the same manner as Citrus extract, hesperidin also displays differing properties depending on its concentration and the cell type. Hesperidin (5-200μM) increases cell viability in MCF-7 and WiDr cells but decreases cell viability more effectively in combination with an anticancer agent than the anticancer agent alone (Gilang et al., 2012;Hermawan et al., 2010). In the other cancer cell lines, T47D breast, HeLa cervix, and MCF-7 with HER2 over-expression (MCF-7 /HER2), hesperidin exhibits an IC50 value varying from 11 to 200μM (Febriansah et al., 2014;Kusharyanti et al., 2011;Setiawati et al., 2011). The effect of Citrus extract on MCF-7/HER2 is contrary to the estrogenic effect seen in ovariectomized mice (Adelina et al., 2015). In summary, Citrus extract and hesperidin may act differently depending on the concentration and the cell type. The dual or biphasic effect of these compounds on cell proliferation will influence whether the expected effect on cells is antiproliferative or proliferative. Therefore, the precise action of Citrus extract and citrus flavonoids need to be elucidated in various cell types. Besides the exploration of its anticancer potency, citrus and citrus flavonoids have also been studied for their anti-inflammatory activities (Manthey et al., 2001). Our group's work has been focused mainly on cancer cells, not on normal or inflammation-related cells (Table I). To our knowledge, no study has yet addressed the dual effect of citrus extract or citrus flavonoids on cell proliferation. Hence, an investigation of Citrus extract and citrus flavonoids on normal or inflammatory-related cells, i.e., macrophages, is important, especially considering the probability of their contradictory concentration-dependent effect.
In this study, we evaluate the effect of ethanolic extract from Citrus reticulata peel (Citrus extract) and hesperidin at various concentrations on the modulation of cell proliferation in the RAW 264.7 macrophage cell line. We reveal that both Citrus extract and hesperidin show a biphasic effect on RAW 264.7 macrophage cell viability. Additionally, Citrus extract at a moderate tested concentration can induce the expression of interleukin-10 (IL-10), an anti-inflammatory cytokine.

Preparation of Citrus extract
Citrus reticulata fruits were obtained from Kalisoro, Tawangmangu, Central Java, Indonesia in September and were identified in the Laboratory of Pharmacognosy, Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada (UGM). Healthy, green, mature but unripe fruits were washed, and the peels were collected and air dried without direct sunlight. Dried peels were powdered and macerated in 70% ethanol (Merck, Darmstadt, Germany) (10L for 1kg powder) for 5 days as previously described . The ethanolic fraction was separated and evaporated in a rotary vacuum evaporator until a brown viscous extract was obtained (yield was 3.21% (b/b)) (Armandari, 2010). The obtained extract was then identified by thin layer chromatography to detect flavonoid components as previously described . After citrobroric treatments, positive spots indicating flavonoids were developed under 245 and 366nm UV lights (Armandari, 2010). This extract is heretofore referred to as "Citrus extract."

Cell culture
The murine monocyte macrophage RAW 264.7 cell line was a gift from Prof. Tatsuo Takeya (Nara Institute of Science and Technology, Japan). Cells were cultured in Minimum Essential Media (Gibco, USA) supplemented with 10% (v/v) Newborn Calf Serum (NBCS) (Gibco, USA) and 2% (v/v) penicillin-streptomycin (Gibco, USA) in a 37ᵒC, 5% CO2 incubator and grown to confluence in 75cm 3 tissue culture flasks. After they reached 80% confluence, cells were scraped and used for experiments.

Cell viability assay
Cells (5 × 10 3 cells/well) were plated in 96well plates and cultured in a complete medium for 48h. The medium was then replaced with a medium containing various concentrations of Citrus extract or hesperidin and incubated for 24h. Cell viability was assessed by the MTT method as previously described (Ikawati et al., 2018) and carried out in at least triplicate for each experiment. Briefly, the absorbance at 595nm of diluted formazan after addition of 0.5mg/mL 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, Germany) in phosphate-buffered saline (PBS) followed by stopper solutions (10% SDS (Merck, Germany) in 0.1NHCl (Merck, Germany)) were measured in a microplate reader (Bio-Rad, Japan). The detailed step-by-step protocol is described in Armandari (2010). Untreated cells served as a control, while wells without cells served as a blank. The percent cell viability and the IC50 value were calculated as (absorbance of treated cells − absorbance of blank)/(absorbance of control − absorbance of blank) × 100% and by linear regression analysis as follows: cell viability (%, y axis) vs log concentration (μg/mL or μM, x axis) (Ikawati et al., 2018), including all tested dosage of the concentration series). Data are presented as the mean of two or three measurements per condition per experiment.

Immunostaining
Immunocytochemistry with an anti-IL-10 mouse monoclonal antibody (Dako) was carried out by the avidin-biotin complex method as previously described . Cells (1 x 10 5 cells/well) were plated on coverslips in a 24well plate until they reached 80% confluence. Cells were then incubated with Citrus extract at the designated concentrations for 15h. After removal of the medium, cells were washed with cold PBS, and then were fixed in cold methanol for 10min at -20ᵒC. The endogenous peroxidase activity was blocked with hydrogen peroxide, and nonspecific sites were blocked with normal goat serum (Novocastra), for 10min each at room temperature, followed by incubation in the primary antibody, anti-IL-10 (1:50), overnight at 4ᵒC. After washing with PBS, cells were incubated in IgG biotinylated universal secondary antibody (Novocastra) for 10min. Following washing, cells were incubated with streptavidin-peroxidase complex reagent (Novocastra) for 10min and then incubated with 3,3-diaminobenzinidine (DAB) substrate solution (Novocastra) for 2-10min to visualize the bound biotin. Cover slips were washed in distilled water and counterstained with Mayer's hematoxylin (Dako) for 1-3min. Cells were dehydrated in ethanol, cleared in xylene, and cover slips were mounted with a mounting medium. Protein expression was observed qualitatively with a light microscope (Olympus). A stained cover slip without primary antibodies served as a control.

Statistical analysis
Data are presented as mean ± standard deviation (SD) and were analyzed for significance using the Student's t-test. Values of p <0.05 were considered to indicate significance.

Higher, but not lower, concentrations of Citrus extract decrease RAW 264.7 cell viability
To examine the inhibition or induction of cell proliferation, RAW 264.7 cells were treated with a series of concentrations of Citrus extract, in the range from 1 to 1,000μg/mL, for 24h. As expected, lower concentrations of Citrus extract did not affect cell morphology. The cell morphology at lower concentrations (i.e., 10μg/mL and 50μg/mL) appeared the same as that of control cells ( Figure 1A). Moreover, the cell density increased after treatment with 50μg/mL extract. In contrast, at higher concentrations, starting at 500μg/mL, more cells appeared rounder and less flattened out in the well. These morphological observations imply a biphasic effect of Citrus extract.
After 24h of incubation with Citrus extract, cell viability was assayed by the MTT method. The higher concentrations of Citrus extract (500μg/mL, 750μg/mL, and 1000μg/mL) were able to decrease cell viability significantly (64%, 46%, and 36%, respectively, compared with 100% in the control without Citrus extract treatment) ( Figure 1B). The cell viability in the presence of 250μg/mL Citrus extract was 96%, while lower concentrations increased the cell viability, though not significantly, ranging from 108% to 138%. The highest cell viability was given by the lowest tested concentration, 1μg/mL. Thus, Citrus extract demonstrated a biphasic effect on RAW 264.6 macrophage cell proliferation.

Biphasic effects of hesperidin on RAW 264.7 cell viability
Hesperidin is one of the citrus flavonoids, a major compound in Citrus extract or citrus in general. Therefore, a similar cell viability assay was also carried out for hesperidin to determine 264 Volume 30 Issue 4 (2019) whether this compound would exert a similar biphasic effect to the extract. A series of concentrations varying from 1μM to 500μM (equal to 0.6μg/mL−305.3μg/mL) was used (Figure 2). The lowest tested concentration did not significantly affect cell viability. However, at 5−100μM, hesperidin increased cell viability significantly (116%−136% compared with 100% in the control without hesperidin treatment). At higher concentrations, hesperidin decreased cell  Effects of hesperidin on cell viability in RAW 264.7 macrophage cells. Cells were treated with a concentration series of hesperidin, incubated for 24h, and then assayed by MTT assay in triplicate. The graph of hesperidin treatment demonstrated a biphasic effect on cell viability. The experiments were carried out three times, and the graph represents means ± SD (n=3). Statistical significance was determined by means of the Student's t-test. Hash tags indicate significant increase ( # ; p<0.05) on cell viability compared with the control. Asterisks indicate a significant decrease (***; p<0.001 ****; p<0.0001) in cell viability compared with the control. viability by as much as 61% and 10% at 250μM and 500μM, respectively. These data confirmed the biphasic effect of Citrus extract on cell proliferation, and that effect was likely caused by its citrus flavonoid content.
Citrus extract has a lower IC50 value compared with hesperidin in RAW 264.7 cells IC50 values were calculated based on linear regression equations derived from the graph of concentration versus cell viability (Figure 3). The IC50 of Citrus extract and hesperidin was 756 g/mL and 203μg/mL (332μM), respectively, as expected ranging at the high tested concentration. The IC50 of Citrus extract was 3.7 times higher than that of hesperidin. This is plausible because Citrus extract may contain other compounds. Nevertheless, at the given IC50, studies on the utilization of Citrus extract rather than hesperidin may yield additional advantages, especially if the accessibility of a pure compound is limited. Figure 3. The IC50 value of Citrus extract or hesperidin in RAW264.7 macrophage cells. Cells were treated with a concentration series of Citrus extract (A) or hesperidin (B) for 24h and then assayed by the MTT method. Graphs of concentration versus percentage cell viability are presented as indicated. Points in A and B are presented as the mean of two or three experiments, respectively. The IC50 value was calculated by linear regression analysis as stated on each graph.

Citrus extract can induce IL-10 expression
Treatment with 250μg/mL Citrus extract induced expression of IL-10, an anti-inflammatory cytokine, indicated by a brownish color in the cytoplasmic area compared with control IgG (Figure 4, rightmost panel versus leftmost panel). However, at a concentration of 25μg/mL, Citrus extract did not affect IL-10 expression and appeared similar to cells without Citrus extract treatment (0μg/mL). Higher concentrations should be tested further to confirm whether the modulation of IL-10 expression caused by Citrus extract is also ensuing the biphasic trend. Figure 4. Effects of Citrus extract treatment on the expression of interleukin-10 (IL-10). Cells were incubated with Citrus extract at the indicated concentrations for 15h. Cells were immunostained for IL-10 (brown) and counterstained with Mayer's hematoxylin to visualize nuclei (blue). Staining without primary antibody served as a control (leftmost panel). Scale bar = 50μm.
Our findings exhibit that the ethanolic extract of Citrus reticulata (Citrus extract) and hesperidin, a citrus flavonoid, display a biphasic effect on the proliferation of a monocyte macrophage cell line, indicated by the cell viability parameter ( Figure 1B and 2). More importantly, the concentration of Citrus extract required to increase RAW 26.7 cell viability varied from 1μg/mL to 250μg/mL ( Figure 1B) compared with 10-1,500μg/mL in cancer cell lines ( Yunas et al., 2007;Ardiani et al., 2008;Puspita et al., 2008). Meanwhile, hesperidin at low concentrations (5−100μM) can increase cell viability (Figure 2), similarly in cancer cell lines (5-100 or 200μM) (Hermawan et al., 2010;Gilang et al., 2012). The IC50 value of hesperidin in the monocyte 266 Volume 30 Issue 4 (2019) macrophage cell line is 1.7-30 times higher than in cancer cell lines ( Figure 3B) (Kusharyanti et al., 2011;Setiawati et al., 2011;Febriansah et al., 2014). Taken together, the use of Citrus extract or hesperidin should carefully deliberate the concentration or dosage and the cell type.
Macrophages express interleukin-10 (IL-10) and usually triggered by inflammatory stimuli (Chung et al., 2007). Since citrus flavonoids has been studied extensively for their antiinflammatory activity (Manthey et al., 2001), it is plausible to observe IL-10 expression under Citrus extract or hesperidin treatments. Due to our limitations (Figure 4). At 250μg/mL, Citrus extract can induce IL-10 expression, but not at lower concentrations. In the future study, higher concentrations of Citrus extract should be tested to confirm whether the modulation of IL-10 expression caused by Citrus extract also follows the pattern of a dual effect. Furthermore, the expression of cyclooxygenase-2 (COX-2), one of the key enzymes in inflammation, was also tested. However, there was no COX-2 detected (data not shown). An inflammatory stimulus such as lipopolysaccharide (LPS) should be administered to the cells, similarly to the ones that have been reported previously (Kang et al., 2011;Sakata et al., 2003).
At lower concentrations, Citrus extract was able to maintain cell viability independently of the expression of the anti-inflammatory cytokine IL-10. Meanwhile, hesperidin at a lower concentration was able to promote cell proliferation. Therefore, a low concentration of Citrus extract and hesperidin is possibly useful for treating inflammatory diseases. On the other hand, application of a low concentration of Citrus extract or hesperidin should be avoided for cancer cells because it induces cell proliferation as previously reported in a colon cancer cell line by Ardiani et al. (2008). Alternatively, at higher concentrations, Citrus extract or hesperidin is beneficial to cancer cells due to their cytotoxic effects. Recently, genistein, another flavonoid found in soybean, a widely known potential chemopreventive agent, has been reported to demonstrate a biphasic mechanism on CHO-K1 cells: at low concentrations, it induces senescence and apoptosis in combination with estrogen, and at high concentrations, it modulates the cell cycle (Jenie et al., 2019). Hence, an investigation of the dose-dependent mechanism of Citrus extract and hesperidin should be carried out further.

CONCLUSION
Citrus extract and hesperidin exerted a biphasic effect on a macrophage cell line. The future development of Citrus extract as a co-chemotherapeutic, anticancer, or immunemodulatory agent should include careful consideration of its biphasic effect on each cell type.

ACKNOWLEDGMENT
This study was partly funded by "Hibah Penelitian Berkualitas Prima" (Prime Quality Research Grant) Faculty of Pharmacy UGM 2009 (granted to MI). The authors wish to state their contributions as follow: IA performed experiments for the Citrus extract; AK and YE performed cell viability assays for hesperidin; MI and IA wrote the manuscript and prepared the figures; MI oversaw all aspects of the study. All authors have read and approved the manuscript.