Seo-Jong Ho (2000) reported that soil organic matter acts as a sink and source of nutrients in the soil system because it has high nutrient holding capacity.
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IMPROVING YIELD AND DECREASING LEAF NITRATE CONTENT OF LETTUCE VIA APPLICATION OF THE BIO-ORGANIC AGRICULTURE 1
Samir G. Al-Solaimani, 1Fathy S. El-Nakhlawy, 2Fahad A. Al-Fassi, 3Frank B. Dazzo and 2&4Abu-Bakr M. Gomaa
1
Arid Land Agriculture Department, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia 2 Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia 3 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, 48824 USA 4 Agricultural Microbiology Department, National Research Centre, Cairo, Egypt ABSTRACT This field study was conducted during two successive seasons in 2014 in The Agricultural Research Station, Hada Al-Sham, King Abdulaziz University, Saudi Arabia to evaluate the ability of using bio-organic farming application to reduce the harmful nitrate content of lettuce leaves and enhance the growth and yield of lettuce in comparison with application of mineral fertilization (NPK). The results showed that lettuce yield and yield components significantly increased by treating soil with cellulose decomposing bacteria while leaf nitrate content was decreased. Lettuce fresh yield was 16,851 and 13,192 t/ha in soils treated with cellulose-decomposing bacilli vs. untreated soils, respectively. The olive by-product + barley straw or + palm tree leaves performed best as organic fertilizers in increasing fresh yield/ha and yield components and significantly decreasing the nitrate contents in lettuce leaves compared with the mineral fertilizer. Keywords: Bio-Organic agriculture, organic fertilizer, cellulose-decomposing bacteria Lettuce nitrate content, LAI. INTRODUCTION Lettuce (Lactuca sativa L.) is an annual or biennial plant grown as a leaf vegetable. Chemical fertilizer offers nutrients that are readily soluble in soil solution and therefore are immediately available to plants. Nutrient availability from organic sources is due to microbial action and improved physical condition of soil (Sarker et al., 2004). Organic sources offer more balanced nutrition to the plants, especially micro-nutrients that positively affect the number of tiller in plants (Miller, 2007). Application of organic manures plays a direct role in plant growth as a source of all necessary macro- and micro-nutrients in available forms during mineralization, thereby improving both the physical and the biological properties of the soil (Abou El-Magd et al., 2006). Seo-Jong Ho (2000) reported that soil organic matter acts as a sink and source of nutrients in the soil system because it has high nutrient holding capacity. Organic manures decompose to give humus, which plays an important role in the chemical behavior of several metals in soils through the flavonic and humic acid contentsthat have the ability to 566
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retain the metals in complex and chelate forms (Abou El-Magd et al., 2006). Organic manures also improve the water holding capacity, structure and aeration of the soil (Belay et al., 2001). Soil organic matter influences the degree of aggregation and aggregate stability and can reduce bulk density while increasing the total porosity and hydraulic conductivity of heavy clay soils (Anikwe, 2000). Ebadah et al. (2006) found that organic fertilization with the composted residue of okra or farm yard manure reduced infestation numbers with three out of the four tested blights viz. white fly, leaf miners and red spiders for composted okra residue and leaf miners, aphids and red spiders for farm yard manure. Ursinos (1986) emphasized that olive by-product contain organic compounds and K at a high level and other minerals such as N, P and Mg in noticeable amounts, and therefore its application on soil improves both physical and chemical characteristics of soil and also increases water holding capacity. He also indicated that it lowered soil pH and lead to phyto toxic effects on the annual plants. On the other hand, olive by-product applications at the rates of 20 and 30 t.ha-1 increased nutrient uptakes in faba bean and onion plants (Saime, 2011). Many researchers reported positive effects of olive by-product application to soil as an organic fertilizer for crop production in agriculture (Hermosa, 1983; Ursinos, 1986; Acunaz, 1987; Levi et al., 1989; Timur and Ozturk, 1997). Tang and Yu (1999) reported that the direction and magnitude of pH change depend on the concentration of organic anions in the residues, the initial soil pH and the degree of residue decomposition. Microbial cellulose decomposers produce extracellular enzymes to depolymerize the larger compounds (i.e., plant polymers, cellulose, hemicellulose and lignin) to smaller fragments that are water-soluble (Hankin et al., 1976). Nitrogen fertilizers convert into nitrates that can travel easily through the soil. Because nitrate is watersoluble and can remain in groundwater for decades, the addition of more nitrogen over the years has an accumulative effect. Al-Redhaiman et al. (2003) reported that vegetables are the source of 70% of nitrate in human bodies whereas drinking water contribution is 20 %. With increased past and projections of future use of nitrogen fertilizers, this problem may increase several fold in the coming decades. Exceeding the allowable limit of nitrogen fertilization may produce considerable vegetative growth, though, many side effects could occur. High nitrate accumulation in vegetable crops is a problem when consumed by infants; it may cause the “blue baby” syndrome (Kiziloglu et al., 2007). Drinking groundwater contaminated with high nitrate levels has been linked to gastric cancer, goiter, birth malformations, and hypertension (Ogunlela et al., 2005); testicular cancer and stomach cancer. Excessive air- and water-borne nitrogen from fertilizers may cause respiratory ailments, cardiac disease, and several cancers, as well as can "inhibit crop growth, increase allergenic pollen production, and potentially affect the dynamics of several vector-borne diseases, including West Nile virus, malaria, and cholera (Kuntashula et al., 2006). The aim of this investigation was to evaluate the influence of bio-organic fertilizer application on the 567
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nitrate levels in lettuce leaves and its effect on lettuce vegetative growth, yield and other components plus some soil properties. The resultant data from the bio-organic farming were compared to corresponding data obtained from the positive control of full doses of inorganic mineral fertilizers (N, P, K). MATERIALS AND METHODS A field experiment was conducted at the Agricultural Research Station, Hada Al-Sham, King Abdulaziz University in the two successive seasons of 2014 to study the effect of different organic fertilizers in the presence and absence of cellulose-decomposing bacteria on nitrate content within leaves, fresh yield and yield components. Experimental design and treatment: Split plot design in 4 replicates was used where the main plots were with and without the two cellulose decomposing bacterial treatments The sub-plots were 7 organic fertilizers included the full dose of mineral fertilizers (N, P, K) as control treatment, cow manure, olive by-product waste, cow manure + barley straw, cow manure + palm tree leaves, olive by-product + barley straw and olive by-product + palm tree leaves. Bakr: Here provide some explanation how you obtained and processed these bioorganic fertilizers so others can duplicate the procedure. Isolation of cellulose decomposing bacilli: Serial dilution technique was used for the isolation of cellulolytic bacteria. Ten grams from each sample was added to 90 ml sterilized distilled water in 250 ml dilution bottle to give 10-1 dilution and mixed thoroughly; then further serial dilution were made up to 10-6. Aliquots of one ml from the dilutions of 10-4, 10-5 and 10-6 were transformed to sterile Petri dishes. The melted CMC agar medium (Sakthivel et al., 2010) was poured and mixed well with the added inoculum. After solidification, the inoculated Petri dishes were incubated for 24hr. at 30ºC. Bacterial colonies were purified by streaking on nutrient agar plates, incubated at 30°C for 24 hr. The pure isolated colonies were maintained on CMC agar slants at 4°C for further analysis. The organic fertilizers were applied at the rate of 20 t/ha. The organic fertilizers and cellulosedecomposing bacteria were applied in the experimental plot one month before planting to incubate and mineralize the organic fertilizer. While the NPK (20:20:20) fertilizer (control) was added weekly in equal doses (750 kg/ha). Soil analysis: Soil samples were collected from 0-30 cm and analyzed before and after harvesting according to Pansu and Gantherou (2006). Soil texture was sandy loam with sand, silt and clay contents of 71%, 22% and 7%, respectively. The chemical initial soil analysis was presented in Table (1). Table 1: Initial soil analysis of the experimental site before cultivation. pH
568
EC
Macronutrients (%)
Micronutrients (mg/kg)
O.M.
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7.5
dS/m 2.3
N 0.25
P
K
0.11
Ca 1.4
0.8
Mg 1.1
Na 0.12
Fe
Mn 130
160
Cu 4.2
Zn 33
(%) 0.55
Organic Fertilizer analysis: Cow manure, olive by-product manure, barley straw and palm tree leaves were analyzed for nutrient contents (N, P, K, Ca, Mg, Fe, Zn and Cu) and C/N ratio before planting the experiment and the methods for these analyses were the same as for soil (Table 2). Table 2: Chemical analysis of organic manures used in the study. N
P2O5
Manure source
K2O
Ca
Mg
Fe
(%)
Zn
Mn
Cu
(mg/kg)
OM (%)
pH
C/N ratio
Cow
2.42
1.8
1.1
1.94
0.37
110
290
322
240
61.9
6.3
14.8
Olive by-product
2.93
0.17
0.65
1.9
0.19
118
32
34.6
33.5
27.2
6.64
25.38
Barley Straw
0.581
0.073
1.49
0.43
0.08
26.8
60.21
12.13
3.12
79.2
7.1
79.07
Palm date leaves
0.192
0.555
1.494
0.35
0.18
3.9
6.9
38.5
4.86
87.7
7.8
264.9
Planting: The drip irrigation system was used under the current study. Daily irrigation interval was practiced during the growing seasons for various growth stages depending on full water requirement of the lettuce crop. The required irrigation water was calculated based on crop evapotranspiration and available water in the soil according to the Penman-Monteith equation as described by Allen et al. (2011). Lettuce seeds were cultivated in the nursery one month before transplanting using common cultural practices to grow the lettuce seedlings for two seasons. The common cultural practices were done on experimental units according to the experimental design. Recorded data: At harvest stage, 10 random guarded plants/plot were tagged and separately harvested to determine the plant height (cm), number of leaves/plant, fresh above-ground vegetative weight/plant, root fresh weight/plant, fresh vegetative yield/ha, leaf area index and nitrate content in the plant leaves according to Hotly and Potworauski (1972). Also soil organic matter (%), soil N, P and K (%) were determined after harvesting of each season according to Pansu and Gantheyrom (2006). Statistical analysis: The results of lettuce and soil characteristics were statistically analyzed according to El-Nakhlawy (2010) through the analysis of variance using the SAS program (2006). Mean separation was done using the revised LSD (RLSD) test. RESULTS AND DISCUSSION 569
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Identification of the isolated cellulose-decomposing bacilli: Two strains that having the highest cellulose decomposing activity were identified. The two strains were identified as Bacillus megatherium and Bacillus brevis according to Bergey’s Manual of Determinative Bacteriology (1994). The efficiency of the two strains in cellulose decomposition was presented in plate (1). The cellulose-decomposing bacilli were applied in a nutrient broth medium at the rate of one liter (containing 2.5x10 6 cell/ml) per plot that also received the organic fertilizers additions.
C Supernatant
C Entire culture
Sh2 Supernatant
Sh2 Entire culture
Plate 1: Cellulolytic activity of the entire cultures of C and Sh2 strains and their supernatant on 0.5% CMC basal medium incubated at 30ºC for 24 hr. What is the composition of CMC basal medium. (C, Bacillus brevis; Sh2, Bacillus megatherium). Effect of cellulose-decomposing bacilli Yield and Yield components: The statistical comparisons of the yield and yield components of lettuce with and without using the cellulose-decomposing bacteria revealed that the fresh yield/ha was higher with the biofertilizers treatment (Table 3). The fresh yield/ha increased by 1.28-foldusing these bacteria. These results are similar to those obtained by Gomaa and Magda (2007) who found that the bio-organic treatment of guar crop with 20 t/fed of farm yard 570
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manure increased yield in the presence of both bio-fertilizers isolates of Rhizobium and a soil yeast Rhodotorula. Yield components increased when inoculated with the cellulose-decomposing bacteria by the rates of 1.05, 1.13, 1.18 and 1.18 times for plant height, no. of leaves /plant, fresh weight/plant and root fresh weight/plant, respectively. Fresh yield/ha was 16.851 t/ha when inoculated with cellulose decomposing bacteria compared to 13.192 without inoculation. These results are consistent with the finding of Nabila et al. (2009) that rice cultivars responded better to the combined inoculum of Azotobacter and Rhizobium than with who mentioned strain inoculation. These results may be due to the effect of cellulose-decomposing bacteria that decomposed the cellulose of the organic fertilizers materials, enhancing the available macro and micro-nutrients in the soil. These would be absorbed by the plant roots and accordingly increase the accumulation of dry matter, ultimately increasing the yield components and the final fresh yield/ha. These results were also confirmed by the results of Saime (2011), Miller (2007) and Fashina et al. (2002). Leaf Area Index (LAI) As shown in Table (3) the LAI mean was significantly higher when inoculated with the cellulosedecomposing bacteria (7.121) than without inoculation. This finding is consistent with earlier studies by Gomaa et al. (2011) who found that the tallest plants, the largest leaf area (LAI) and flag leaf area were obtained when the wheat variety (Gemmiza 7) was inoculated with yeast. Nitrate Contents: The nitrate contents in lettuce leaves significantly decreased when inoculated with cellulose-decomposing bacteria (Table 3). The results agreed with those obtained by Saleh et al. (2006) who found that vines fertilized with composted municipal solid waste alone or with humic acid significantly decreased, nitrate and nitrite content in berry juice than with mineral N fertilizer (as control). Effect of organic fertilizers Yield and Yield Components: Fresh yield/ha The statistical comparison between fresh yields under the seven fertilization treatments (Table 3) showed that treatment with NPK mineral fertilizer produced the highest yields with 19.363 t/ha, followed by the organic fertilizers. The same trend was recorded by Amal et al. (2010) where combined inoculation with Azotobacter vinelandlii and Pseudomonas fluorescens surpassed the single inoculation either with a full or a double full dose of organic manure in all tested characteristics of sorghum. In comparison, the positive control of the recommended doses of NPK recorded the highest significant values at all tested plant ages. Generally no significant differences were found between the organic fertilizers in the vegetation fresh yield, except the (cow manure + palm tree leaves fertilizer) that produced the lowest yield compared with other fertilizers. The fresh 571
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yield/ha under the different organic fertilizers ranged from 14.995 t/ha to 12.569 t/ha under olive by-products waste + barley straw and cow + palm tree leaves, respectively. Plant Height: The means of plant height for the 2 successive seasons ranged from 26.11 cm to 22.55 cm under the mineral NPK and cow manure fertilizers, respectively (Table 3). Significant differences occurred between the NPK fertilizer and the organic fertilizers. Plants were taller when fertilized with the olive by-product + barley straw and olive by-product + palm tree leaves compared with other organic fertilizers. These two top-performing treatments were not significantly different from each other. The other 4 organic fertilizers did not significantly affect lettuce plant height. In earlier studies Gomaa et al. (2010) found that the bio-organic treatments increased the seed yield by 12 to 39% in comparison with the positive control. The bio-organic treatment of chicken manure +Rhizobium +Azotobacter +Rhodotorula induced the highest significant value of seed index when compared with the positive control. Number of leaves/plant: The mineral NPK fertilizers produced the highest lettuce leaves/plant with significant differences from the organic fertilizers (Table 3). [in the table] Among the organic fertilizer treatments, the olive by-product + barley straw fertilizer evoked the highest number of leaves/plant followed by olive by-product + palm tree leaves and olive by-product fertilizers. The other organic fertilizers did not affect the number of leaves/plant. Fresh weight/plant: The statistical comparisons between the means under the different fertilizers over the two seasons (Table 3) indicated that the highest fresh plant weights was produced using the mineral NPK fertilizer. The lowest fresh weight/plant was produced from the cow manure and cow manure with palm tree leaves fertilizers without significant differences between them. No significant differences were found between the other organic fertilizers on fresh weight/plant. Root fresh weight/plant: The mineral NPK fertilizer produced the highest fresh root weight/plant (Table 3). These results are in agreement with the results of Ursinos (1986), Timur and Ozturk (1997), Saime (2011), Sarker et al. (2004), Yadana et al. (2009), Miller (2007), Fashina et al. (2002) and Saeed et al. (2001).The fresh root weights/plant were not significantly different for the 6 organic fertilizers. The fresh root weight under the organic fertilizers ranged from 26.66 g to 24.58 g/plant for olive by-product + barley straw and cow manure fertilizers, respectively. The higher response obtained with the olive by-products with barley straw and with palm tree leaves may be due to the higher N contents in this olive by-product waste than the other organic fertilizers. Leaf Area Index: Statistical comparison (Table 3) showed that the highest LAI mean occurred when using the mineral NPK 572
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fertilizer (7.691), significantly followed by the (olive by-product + barley straw) and olive by-product + palm straw fertilizers without significant difference between these two fertilizers, while the lowest LAI occurred with the cow manure fertilizer (Table 3). These results may be due to the high amount of available N, P and K in the mineral NPK, reflected in the high number of leaves/plant accordingly increased
the plant leaf area. As for the organic
fertilizers effect on the LAI, the olive by-products with barley straw or palm trees contained high and available macro and micronutrients, beside high organic matter in the soil compared with other organic fertilizers accordingly the leaf area increased and reflected into LAI. These results are similar to the results of Levi et al. (1989), Timur and Ozturk (1997), Obi et al. (2005) and Sridhar and Adeoye (2003). Nitrate content: The statistical comparison of the means (Table 3) showed that organic fertilizers significantly decreased nitrate content in lettuce leaves. The highest nitrate content was found in leaves fertilized with mineral NPK (3880 mg/kg F.W.) and the lowest nitrate content occurred using the organic fertilizers of olive by-product + barley straw or + palm tree leaves. These results are likely due to the positive effect that organic fertilizers with their low N contents can have in reducing the nitrate content in lettuce leaves compared with mineral NPK fertilizer. These results are in agreement with those obtained by Saleh et al. (2006). Table 3: Means of plant height (cm), no. of leaves/plant, fresh weight/plant, root fresh weight, fresh yield/ha, leaf area index (LAI) and leaf nitrate contents (mg/kg fresh wt.) and soil organic matter as average of the 2 successive seasons. Treatments
Plant height (cm)
leaves (no/plant)
With Without
23.95a* 22.73b
33.48a 29.69b
F1 (control) F2 F3 F4 F5 F6 F7
26.11a 22.55c 22.77c 22.73c 22.72c 23.28b 23.04b
40.40a 29.55d 30.07c 29.74d 29.43d 31.34b 30.59c
Fresh Root fresh weight weight (g/plant) (g/plant) Cellulose decomposing bacteria 323.97a 28.61a 274.66b 24.22b Organic fertilizers 357.27a 32.00a 283.49c 24.58b 293.38b 25.73b 289.26b 25.02b 276.28c 24.98b 303.52b 26.66b 292.00b 25.99b
Fresh yield (t/ha)
LAI
Leaf nitrate content (mg/kg)
16.851a 13.192b
7.121a 6.255b
2214b 2790a
19.363a 14.067bc 14.995b 14.435bc 12.569c 15.006b 14.72b
7.691a 5.716d 6.739c 6.517c 6.139c 7.117b 6.996b
3880a 2755b 2530c 2164d 2184d 1932e 2066de
*Means followed by the same letter (s) are not significantly different according to RLSD at p ≤ 0.05. *F1: NPK fertilizer (20:20:20), F2: Cow manure, F3: Olive by-product waste, F4: Cow manure + Barley straw, F5: Cow manure + Palm tree leaves, F6: Olive by-product + Barley straw and F7: Olive by-product + Palm tree leaves.
Conclusion The present study revealed that inoculation of lettuce with cellulose-decomposing bacteria significantly increased its yield and yield components. Also, organic fertilizer treatments significantly decreased the lettuce nitrate content in contrast to the NPK mineral fertilizer that significantly increased it. Olive by-product + barley straw or + palm tree leaves fertilizers provided the best performance in increasing the lettuce yield, yield 573
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components, LAI and soil organic matter. Also, cellulose-decomposing bacteria and organic fertilizers significantly decreased the lettuce nitrate content compared with non-using cellulose decomposing bacteria and without organic fertilizers. These finding support the recommendation to use these latter organic fertilizers to increase lettuce production and decrease leaf nitrate content. Acknowledgment The project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah under the grant number 27/34/RG. The authors, therefore, acknowledge with thanks DSR technical and financial support.
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