Improved Growth and Yield Response of Jew’s Mallow (Corchorus olitorius L.) Plants through Biofertilization under Semi-Arid Climate Conditions in Egypt

This study was conducted to comparatively assess the effects of fertilization typology (organic, inorganic, and biofertilization) on the growth, yield, and compositional profile of Jew’s mallow. The experiment was carried out over two growing seasons, under semi-arid climate conditions on silty loam soil. We adopted three fertilization strategies: (1) inorganic NPK fertilizer (146, 74, and 57 kg ha−1 for N, P2O5, and K2O, respectively), (2) farmyard manure (36 m3 ha−1), and (3) a biofertilizer (a set of mixed cultures of Bacillus spp., Candida spp., and Trichoderma spp. at 36 L ha−1). Treatment combinations were control (without fertilization, T1), NPK fertilizer (T2), farmyard manure (FYM, T3), biofertilizer (T4), NPK+biofertilizer (T5), and FYM+biofertilizer (T6). The T5 treatment maximized both plant and leaf biomass (up to 31.6 and 8.0 t ha−1, respectively), plant height (68.5 cm), leaf area (370 cm m−2), leaf protein content (18.7%), as well as N, P, and K concentration in leaves (2.99, 0.88, and 2.01 mg 100 g−1, respectively). The leaves’ weight incidence was lower in T5 treatment (36.7%) as compared to the unfertilized plants (T1). The results revealed that the combined application of inorganic NPK plus biofertilizer is most beneficial to increase growth, yield, and nutrient accumulation in Jew’s mallow plants.

Therefore, this study aimed to assess the bio-agronomical response of Jew's mallow to the combined soil incorporation of organic, inorganic and biofertilization, so checking the possibility to obtain a more sustainable fertilization technique for the crop.

Experimental Site
A two-year field experiment was carried out under semi-arid climate conditions on silty loam soil at the Research Farm of the College of Agriculture, Al-Azhar University, Assiut branch. The location is (27 • 12 16.67 N; 31 • 09 36.86 E) in Assiut governorate, Egypt. Table 1 shows some physical and chemical properties of the soil at the experimental site, collected at a depth of 0-30 cm and analyzed as described by [23].

Experimental Design and Treatments
Treatments were laid out using a randomized blocks design with three replications. Each plot unit included a totally flat area of 10.5 m 2 . The seeds of Jew's mallow were sprinkled on 25 March 2017 and 2 April 2018 for each season, respectively. The irrigation of experimental units was immersed-way once per 10 days, as per local custom. Weeds were removed manually at 20 and 40 days after sowing (DAS) in both growing seasons, before irrigation was affected. The treatments application comprised three fertilization types (alone or in combinations), namely an organic fertilization (farmyard manure, FYM), an inorganic NPK fertilization, and a biofertilizer. The organic fertilizer was obtained from the animal Production Farm, College of Agriculture, Al-Azhar University, Assiut, and was incorporated into the soil during plowing at the recommended dose of 36 m 3 ha −1 . Its chemical composition was reported by Silva [24] and presented here in Table 2. For the inorganic fertilization, the recommended P 2 O 5 dose of 74 kg ha −1 (as Ca super phosphate) was incorporated into the soil during plowing, while 146 kg ha −1 (as urea) and 57 kg ha −1 K 2 O (as potassium sulfate) were divided in two equal applications at 10 and 20 DAS, as commonly used for growing Jew's mallow plants, recommended by the Ministry of Agriculture [25]. The liquid biofertilizer (T.S) contains of molasse as organic material carrier of microorganisms, and a set of mixed cultures of Bacillus circulans, B. poylmyxa, B. megatherium, Candida spp., and Trichoderma spp., whose amount in terms of living cells was >0.5 × 10 9 cfu ml −1 , >2 × 10 7 cfu ml −1 , >1.5 × 10 9 cfu ml −1 , >1.5 × 10 7 cfu ml −1 and >0.5 × 10 6 cfu ml −1 , respectively. The biofertilizer was added at 36 L ha −1 with irrigation in three equal doses at 20, 30 and 40 DAS. The biofertilizer was obtained from the directorate of Agriculture in Assiut. Overall, the trials comprised an unfertilized control (T 1 ), inorganic NPK fertilization (T 2 ), farmyard manure (FYM) (T 3 ), biofertilizer (T 4 ), inorganic NPK+biofertilizer (T 5 ), and FYM+biofertilizer (T 6 ).

Data Collection
Data were collected using plant samples from 0.5 m 2 in the middle of each experimental unit. Plant height was taken from the base of the rhizome to the top of the plant using a ruler. The fresh biomass of total plants, fresh weight of leaves, and dry weight of leaves was weighed using an electronic balance (0.01 g). Fresh biomass of total plants and fresh weight of leaves were put in paper bags and transferred to a drying oven at 70 • C until constant weight to obtain the dry weight. Leaf area was estimated as described by Pandey and Singh [26], whereas leaf weight incidence, expressed on a percentage basis, was calculated by using the following Equation (1).
Leaves dressing (%) = leaves dry weight (g) plant dry weight (g) × 100 (1) Harvesting was done in the two seasons at 28-May and 5-Jun, respectively. The following soil properties were determined after harvest: cation exchange capacity (CEC) and organic-C, determined according to Clark, et al. [27]. The soil bulk density was calculated by using Equation (2).

Soil bulk density =
Dry wieght of bulk sample (g) the volume of soil core (cm3) Leaf samples from each experimental unit during two seasons were collected, the fifth leaf from the top of 20 plants after 65 DAS (the first season) and 62 DAS (second season), and washed three times with distilled water, before chemical analysis. N-content in leaves was determined using the Kjeldahl procedure according to Motsara and Roy [28]. P-content in leaves was determined by the colorimeter method (ammonium molybdate) using a JENWAY 6305 UV/Visible Spectrophotometer at 643 nm (OD643) [28]. K-content in leaves was determined photometrically using a Flame Photometer (BWB Model BWB-XP, 5 Channel) as described by Motsara and Roy [28]. Protein content in leaves (expressed on a percentage basis) was calculated as N content (%) X 6.25. Leaf chlorophyll content was determined using a mobile chlorophyll meter (SPAD-502-m Konica Minolta, Inc., Tokyo, Japan). Before taking the readings, the performance of the chlorophyll meter was calibrated according to the manufacturer' instructions. At the measurement date, 6 readings from each replicate were taken at 65 DAS (the first season) and 62 DAS (second season), using the youngest fully expanded leaves.

Statistical Analysis
All data collected were subjected to one-way analysis of variance (ANOVA) using SPSS statistical software package version 16.0 (SPSS Inc., Chicago, IL, USA). Significantly different means were separated using Duncan's multiple range test at the p ≤ 0.05 level of probability [29]. Mean values were presented as mean ± SD.

Weather Condition during the Experiment
During the first year of experiment (2017), the average mean temperature was 26.3 • C, with a gradual increase from 3 April (17.3 • C) to 15 May (29.7 • C), whereas average minimum and maximum temperatures fluctuated between 11-28.4 • C and 24.6-40.8 • C, respectively. The average relative humidity varied between 20% and 60%, with the lowest value recorded at 20 April and the highest one at 13 April ( Figure 1). During the second year of experiment (2018), the average mean temperature was 28.6 • C, with a gradual increase from 20.8 • C to 31.5 • C, whereas average minimum and maximum temperatures fluctuated between 12-28 • C and 20.8-46 • C, respectively. The average relative humidity varied between 16% and 58%, with the lowest value recorded at 6 May and the highest one at 6 April ( Figure 1).

Weather Condition during the Experiment
During the first year of experiment (2017), the average mean temperature was 26.3 °C, with a gradual increase from 3 April (17.3 °C) to 15 May (29.7 °C), whereas average minimum and maximum temperatures fluctuated between 11-28.4 °C and 24.6-40.8 °C, respectively. The average relative humidity varied between 20% and 60%, with the lowest value recorded at 20 April and the highest one at 13 April ( Figure 1). During the second year of experiment (2018), the average mean temperature was 28.6 °C, with a gradual increase from 20.8 °C to 31.5 °C, whereas average minimum and maximum temperatures fluctuated between 12-28 °C and 20.8-46 °C, respectively. The average relative humidity varied between 16% and 58%, with the lowest value recorded at 6 May and the highest one at 6 April ( Figure 1).  Tables 3-5 show the effect of organic, inorganic, and biofertilizers supplementations on the growth and yield of Jew's mallow plants. The results showed no significant difference in the plants height under T2, T3, T5, and T6, but they were higher than those under the other treatments in the  Tables 3-5 show the effect of organic, inorganic, and biofertilizers supplementations on the growth and yield of Jew's mallow plants. The results showed no significant difference in the plants height under T 2 , T 3 , T 5 , and T 6 , but they were higher than those under the other treatments in the mean of both growing seasons (Table 3). There were statistically significant differences between the treatments, where the maximum fresh plants weight, leaves fresh weight, plant dry weight, leaves dry weight, and leaves area (3.16 kg.m −2 , 797.88 g.m −2 , 646.79 g.m −2 , 223.35 g.m −2 , and 369.5 cm 2 .m −2 , respectively) were showed by the plants treated with NPK with biofertilizers (T 5 ), and that of without fertilization, T 1 treatment gave the lowest values (Tables 3 and 4) in the mean of both growing seasons.

Growth Variables
The highest leaves weight incidence was observed in plants treated with biofertilizer (T 4 ) having non-significant difference among FYM+biofertilizer (T 6 ), NPK fertilizer (T 2 ), and T 1 during first growing season, while in second growing season the plants treated with T 1 showed significant highest values (Table 5). There was no significant difference in dry matter content of plants under all the treatments except T 1 , but T 4 was higher than other treatments in the first season, while in the second season, there was no significant difference in dry matter contents of plants in T 1, T 4 , T 5 , and T 6 treatments, but T 4 was highest compared to other treatments in the mean of both growing seasons.

Compositional Variables
As shown in Tables 3-5, the accumulation of protein in Jew's mallow plants in both seasons was the significantly highest under NPK with biofertilizers (T 5 ). Data presented in Table 6 shows that the average accumulation of N, P, and K in leaves were under T 5 higher than other treatments in both growing seasons, while T 1 gave the least N, P, and K accumulation.  Values are means of three replicates; different letters in the same column indicate significant differences according to Duncan's multiple range test at p ≤ 0.05. Where without fertilization (T 1 ), NPK fertilizer (T 2 ), farmyard manure (T 3 ), biofertilizer (T 4 ), NPK+biofertilizer (T 5 ), and Farmyard manure +biofertilizer (T 6 ).  Values are means of three replicates; different letters in the same column indicate significant differences according to Duncan's multiple range test at p ≤ 0.05. Where without fertilization (T 1 ), NPK fertilizer (T 2 ), farmyard manure (T 3 ), biofertilizer (T 4 ), NPK+biofertilizer (T 5 ), and Farmyard manure +biofertilizer (T 6 ).
The average leaf chlorophyll content the first season was higher than in the second season in all variants, where the highest value of leaf chlorophyll content 41.21 mg g −1 was obtained in T 5 , while the lowest one (29.47 mg g −1 ) was recorded in T 1 (Figure 2). Agronomy 2020, 10, x FOR PEER REVIEW 9 of 14 The average leaf chlorophyll content the first season was higher than in the second season in all variants, where the highest value of leaf chlorophyll content 41.21 mg g −1 was obtained in T5, while the lowest one (29.47 mg g −1 ) was recorded in T1 ( Figure 2).

Soil Properties at the End of the Experimental Period
The result of soil properties after harvesting Jew's mallow plant showed that the soil was variably influenced by the different treatments. The average values for the soil organic-C (%) contents were influenced by the individual treatments. The highest value for soil organic-C, as shown in Figure 3a, was observed in T3 in both of seasons and in T6 in the 2018 season, while the lowest values were recorded in T1 and T2. As shown in Figure 3b, the application of organic manure with biofertilizers significantly enhanced the CEC value. The highest average values CEC were noticed in T6 (17.98 cmol kg −1 ), and T3 (17.93 cmol kg −1 ) which were statistically undifferentiated. The lowest value (15.72 and 16.01cmol kg −1 ) were obtained in control (T1) and NPK fertilizer (T2), respectively. The treatment effects on the average soil bulk density for two seasons are presented in Figure 3c. These treatments (T3-T6) had positive and significant effects on soil bulk density. The bulk density was reduced in the T3-T6 treatments (1.39 g cm −3 , on average) and showed statistically lower values than obtained in the control (1.46 g cm −3 ).

Soil Properties at the End of the Experimental Period
The result of soil properties after harvesting Jew's mallow plant showed that the soil was variably influenced by the different treatments. The average values for the soil organic-C (%) contents were influenced by the individual treatments. The highest value for soil organic-C, as shown in Figure 3a, was observed in T 3 in both of seasons and in T 6 in the 2018 season, while the lowest values were recorded in T 1 and T 2 . As shown in Figure 3b, the application of organic manure with biofertilizers significantly enhanced the CEC value. The highest average values CEC were noticed in T 6 (17.98 cmol kg −1 ), and T 3 (17.93 cmol kg −1 ) which were statistically undifferentiated. The lowest value (15.72 and 16.01 cmol kg −1 ) were obtained in control (T 1 ) and NPK fertilizer (T 2 ), respectively. The treatment effects on the average soil bulk density for two seasons are presented in Figure 3c. These treatments (T 3 -T 6 ) had positive and significant effects on soil bulk density. The bulk density was reduced in the T 3 -T 6 treatments (1.39 g cm −3 , on average) and showed statistically lower values than obtained in the control (1.46 g cm −3 ).

Growth Variables
The positive effects of NPK with biofertilizers (T5) on growth variables may have been due to the efficiency of the microorganism in the biofertilizer in immobilizing N for a longer time in the form of NH4 + , which helped in the nutrient uptake by the plant [30]. According to Alori and Babalola [31], a biofertilizer is a living organisms that is added into the soil as inoculant that helps to provide certain nutrients for crop growth. Furthermore, these positive effects may be related to the increased

Growth Variables
The positive effects of NPK with biofertilizers (T 5 ) on growth variables may have been due to the efficiency of the microorganism in the biofertilizer in immobilizing N for a longer time in the form of NH 4 + , which helped in the nutrient uptake by the plant [30]. According to Alori and Babalola [31], a biofertilizer is a living organisms that is added into the soil as inoculant that helps to provide certain nutrients for crop growth. Furthermore, these positive effects may be related to the increased availability of nutrients provided by mineral fertilization, which also served as an energy source for the microbial community [32]. Similar to our findings, Al-Zabee and Al-Maliki [32] reported that the combination of mycorrhizal fungi, algae, and yeast with a higher rate of chemical fertilization (120 kg N, 60 kg P, and 200 kg K per hectare) was beneficial to soil microbial metabolism and potato yield. Besides, Asmamaw, et al. [33] reported that the application of dry cyanobacterial biofertilizer could serve as an auxiliary N source to inorganic fertilizer for pepper, maize, and kale production.
It was also noted that the use of biofertilizers in combination with chemical N fertilizers increased growth, productivity, and chemical compositions of the dill plant (Anethum graveolens L.) compared to the untreated control, where the highest values of plant growth were recorded when biofertilizer was used in combination to 97.6 kg.ha −1 N [34]. Observations have also shown that the most effective treatment for growth characteristics of barley cultivars (Giza-128 and Giza-129) under newly reclaimed sandy soil was 178.57 kg N ha −1 + Yeast [35]. Moreover, Sen, et al. [36]  The co-application of biofertilizers like Azospirillum and Phosphobacteria spp. and inorganic fertilizers had a significant effect on the growth variables of cucumber (Cucumis sativus L.) [37]. Application of biofertilizer at 300-400 kg ha −1 dose combined with inorganic fertilizer at 75% of crop requirement dose was the best combination for increasing NPK nutrient uptake for rice crop and weight of milled dry rice. Marlina, et al. [38] recommended the use of dry cyanobacterial biofertilizer which serve as a supplementary N source in place of inorganic fertilizer for rice production in inception soil of lowland swamp area.

Compositional Variables
The data in Tables 5 and 6 indicated differences in the average proportion of protein, N, P, and K content in leaves among treatments. The NPK with biofertilizers (T 5 ) treatment significantly increased these variables in both seasons. The results presented by Hellal, Mahfouz and Hassan [34] showed that the highest NPK-accumulation were recorded after the combination of biofertilizer with 476.2 kg ha −1 ammonium sulphate (20.5% N) in the Dill plant. The pronounced positive effect on protein, N, P, and K in leaves resulting from T 5 addition may be attributed to the increased uptake of N by plants, and thus, the biosynthesis of protein was increased. Moreover, Tisdale et al. [39] reported that the addition of N in combination with adequate P tended to increase K-uptake by plants. They also showed that K concentration may be high in the NH 4 + -nourished plants as it is adsorbed by soil colloids, so it does not get leached from the soil. This gave the plant a greater chance of taking up N, and thus some nutrients, to build the dry matter. Also, data in Figure 2 for leaf chlorophyll content supported the results of Hellal, Mahfouz and Hassan [34], where it was observed that the highest values of chlorophyll content were recorded where biofertilizer was used in combination to 97.6 kg ha −1 N in the dill plant. Moreover, Sen, Choudhuri, Chatterjee and Jana [36] reported that the combination of 100% of the recommended dose of inorganic fertilizers with biofertilizer increased the leaf chlorophyll content (13.18 mg.g −1 ) in cumin black (Nigella sativa L.) in the eastern Himalayan region of West Bengal. Moreover, Youssef, et al. [40] reported that the combined application of organic manures and biofertilizer (EM) had a synergistic effect on the total chlorophyll content of plants.

Soil Properties at the End of the Experimental Period
The combined application of the biofertilizer with the organic or the inorganic fertilizer was beneficial for the physical and chemical properties of soil and were important for the quality and productivity of the soil. The application of organic fertilizer in T 3 and its combination with biofertilizer in T 6 increased the soil organic-C content of the soil at the end of experimental period by 91.25 % and 68.75%, respectively, over the control treatment (without fertilization T 1 ). This organic fertilizer in the soil can increase the soil organic-C due to higher soil organic matter added from organic fertilizer. This serves as nutrient sources for plants and improves physical, chemical, and biological properties of the soil through improved structure and stable aggregates. This is because organic matrices are a natural chelating material with high moisture retention capacity [41,42]. These results are in agreement with Nesgea, et al. [43], who reported that the application of organic fertilizer increased the soil organic-C content after harvest by up to 65%.
As for the cation exchange capacity of the soil, our results showed that the application of organic fertilizer with biofertilizer increased in CEC of the soil after crop harvest, which were statistically undifferentiated with T 3 and T 5 . The increased CEC might be attributed to the addition of organic fertilizer with the biofertilizer, which might have helped in releasing more nutrients into the soil. This could be an indication of increased exchange sites on the surface of the soil colloids. In line with this result, Tana and Woldesenbet [44] reported that CEC significantly increased with increasing organic fertilizer (15 ton ha −1 ) with inorganic fertilizer.
The data in Figure 3c indicated that there were no differences between treatments (T 3 , T 4 , T 5 , and T 6 ), but the highest average reduction in soil bulk density was recorded in farmyard manure +biofertilizer (T 6 ). Soil bulk density was reduced after the combined use of organic manure with biofertilizer (T 6 ) compared to soil amended with only inorganic fertilizer (T 3 ). This could be due to improved soil aggregation as a result of decreased soil bulk density. Several studies have shown that the appropriate addition of combined biofertilizers, inorganic and organic, improved soil porosity and decreased its bulk density. Our results are in harmony with Khan, et al. [45] who reported that organic fertilizer improved soil organic matter content and decreased soil bulk density.

Conclusions
Biofertilizers play a significant role in improving soil structure, and inorganic fertilizers are important due to their ability to provide essential nutrients, resulting in the better growth and productivity of crops. The results of the present study, conducted on a silty loam soil and in semi-arid climate conditions, revealed that the Jew's mallow plants treated with the combined application of biofertilizer and NPK fertilizer showed maximum growth and productivity among all other treatments. Although the current study unfolded the performance ability of a neglected crop under the application of different kinds of fertilizers, there is a further need to understand the molecular mechanism behind it and to improve the fertilization techniques and material according to the need for crops.