The current study investigates using of alkaline black liquor (ABL), a byproduct of rice straw pulping, to create a bio-active formulation that can inhibit the growth of soilborne plant pathogens. ABL was first treated to precipitate lignin, silica, and fatty acids as a calcium complex (Ca-LSF). The resulting effluent was used to digest chicken feathers under mild conditions. The resulting protein hydrolysate was added to Ca-LSF complex affording a novel formulation to be evaluated against soilborne pathogenic fungi Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, and Sclerotium rolfsii, as well as the nematode Meloidogyne incognita, the causal organisms of wilt, root rot and root knot diseases. The prepared formulation showed strong inhibitory effect against R. solaniand affected viability of M. incognita. This effect increased with increasing concentration, up to 4%, which completely inhibited fungal growth. It was also examined under field conditions, where it reduced root rot and root knot infection. Application of this formulation as seed dressing showed higher efficacy against root rot and root knot infection than its soil drench treatment, although both treatments were superior to untreated control. The presented safe, cost-effective, and eco-friendly formulation could be used to control a wide range of soilborne plant pathogens.
Article
Biopesticidal Activity for Controlling Root Rot and Root Knot Infection of Green Beans under Natural Field Conditions Utilizing Biowastes
https://doi.org/10.21203/rs.3.rs-3341786/v1
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Egypt produces a large amount of rice straw annually. Only about 20% of it is used in industries such as paper, fertilizers, ethanol production, and livestock feed. The remaining 80% is mostly discarded by burning it in the open, which creates black clouds and pollutes the air. Various efforts have been exerted to reduce the environmental pollution via utilization of agro-wastes as raw materials that are rich in bioactive components. Pulping of rice straw [1], as an example, results in the production of black liquor as a byproduct, along with the main cellulosic pulp [2].
Black liquor of rice straw mainly contains lignin, silica, hemicelluloses, and other organic compounds such as p-coumaric and ferulic acids, with known antimicrobial and antioxidant properties [3]. It’s worth mentioning that pulping rice straw under drastic conditions (high temperature and pressure) is associated with different side reactions of these components, which led to several condensed products and a dark brown color affording toxic liquor. On the other hand, the mild conditions such as in the alkaline solar pulping process leads to nontoxic black liquor that is easily refined [4–6]. This black liquor has been modified and fermented recently using a variety of methods to produce useful enzymes such as xylanase [7], Aspergillus awamori EM66 levansucrase [8], and β-glucosidase [9]. In addition, it has been used to precipitate new metal (lignin/silica/fatty acids) complexes that have been successfully used as antioxidants, fillers, and reinforcing agents in green rubber composites [6, 10–12].
Beans (Phaseolus vulgaris L.) are one of the most important food legumes in the world, grown for both local consumption and export [13]. They are a nutritious crop that is especially important for people of low income [14], as they provide a source of dietary protein as the animal protein is often lacking in their diets [15].
Plant diseases are a recurring problem in many areas. They can spread rapidly and are difficult to cure once they have taken hold. This is why most management methods focus on preventing diseases from occurring in the first place. Beans are susceptible to a number of diseases, including wilt, root rot, and leaf spot. The most common soilborne fungi that cause these diseases are Fusarium solani, Sclerotium rolfsii, and Rhizoctonia solani [16, 17]. Nematodes can also damage bean plants by feeding on their roots and reducing nutrient uptake, which can lead to root knot disease. These diseases can have a significant negative impact on plant stand and crop yield. Root rots can affect seedlings throughout the growing season, from emergence to the seedling stage. They can also infect seeds before emergence, which can lead to pre-emergence infection and the need to replant missed hills or dead plants. Root-knot nematodes (Meloidogyne spp.) are known to cause significant damage to crops all over the world. They are found in a wide range of climatic conditions and can attack a variety of crops. The damage caused by these nematodes is often severe, and can lead to significant crop losses [18–20]. The disease caused by Meloidogyne spp. is often the only, or one of the few, nematode diseases that farmers are aware of, as the nematodes' subterranean activities are not easily visible. However, the symptoms of the disease, such as stunted growth, wilting, and yellowing of leaves, are often unmistakable [21, 22].
There are a variety of methods to control Meloidogyne spp., including crop rotation, resistant varieties, and nematicides. However, no single method is completely effective, and a combination of methods is often necessary to manage these nematodes. Researchers have been working to find effective substances that can control root knot disease. As a matter of fact, chemical pesticides are often used to control plant diseases, but they can have harmful environmental and health effects. Controlling soilborne pathogens is particularly challenging because they live in the soil, which is a dynamic environment at the interface of root and soil.
Biomass-derived substances are emerging as a promising option without the dangers of using chemical pesticides, as they have been shown to be effective against nematodes and relatively safe for the environment [23].
The effectiveness of ammonia against soilborne microorganisms has been widely studied. Many ammonia-releasing compounds, such as ammonium hydroxide, ammonium phosphate, and ammonium bicarbonate, have been tested, and they all showed promising nematicidal activity in pots, with ammonium hydroxide being the most effective [24]. In addition to its toxicity to fungi, ammonia also increases soil pH and may stimulate microbial activity that can help to suppress pathogens [25]. A study by Zhao et al. [26] revealed that ammonia gas fumigation can effectively suppress Fusarium wilt, resulting in a four-fold increase in yield. Ammonium bicarbonate is another alternative fumigant that has been shown to be effective against soil-borne diseases such as southern stem rot, Sclerotinia blight of peanut [27], and Fusarium mycelia [28]. The toxicity of ammonia to susceptible organisms may be due to the passive diffusion of the non-ionized NH3 species through cell membranes. Worthy of mention are the attraction-repulsion effects of ammonia on entomopathogenic nematodes. These nematodes are parasites of insects and can be used to control insect pests. Ammonia can attract nematodes to a new host, but it can also repel them [29, 30]. The exact mechanism of this attraction-repulsion is not fully understood.
Since ammonia is supposed to be the active agent released from urea, Rodríguez-Kábana and King [31] found that urea can be used to control root knot nematodes when applied to the soil at a rate of 0.4 g/kg. However, this rate can be phytotoxic to plants. When urea is mixed with carbon-rich blackstrap molasses, the phytotoxicity is eliminated, nematode populations are reduced, and plant weight and height are increased. This suggests that adding nitrogen in the form of urea is an effective way to control root knot nematodes. Huebner et al. [32] found that soil amended with carbon rich hemicellulosic waste had more nematicidal efficacy when urea was also added to the soil.
As our program aims to develop an environmentally friendly approach to synthesize bioactive products using agro-waste instead of petrochemicals [33–36], the black liquor obtained from the solar pulping of rice straw [6] was used in the current study along with chicken feather waste to prepare and evaluate the biopesticidal activity of a new formulation and its efficiency when applied as seed dressing and soil drench to control root rot and root knot diseases of green beans in natural field conditions.
Laboratory studies
Tested material and microorganisms
Formula derived from rice straw waste were prepared at the Green Chemistry Department of the National Research Centre in Egypt. The pathogenic fungi Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, and Sclerotium rolfsii, which cause wilt and root rot diseases, as well as the culture of the pathogenic nematode Meloidogyne incognita, which causes root knot disease, were kindly provided by the Plant Pathology Department of the National Research Centre in Egypt.
Preparation of black liquor-chicken feather hydrolysate:
Black liquor from solar-pulping of rice straw (pH = 10) was prepared as previously described [6]. The tested formulation was prepared by adding 5 grams of calcium oxide to 1 liter of black liquor (pH = 10) while stirring for 1 hour. The mixture was then left overnight and was filtered to obtain 10grams of Calcium (lignin/silica/fatty acids) complex (Ca-LSF) [6]. The molasses effluent (pH = 12) was then used to digest 50 grams of chicken feathers, followed by concentration, acidification with 5% sulfuric acid till pH = 4, and filtration. After air-drying, the dried hydrolysate (75 grams) was mixed with 10 grams of Ca-LSF complex affording the tested Formulation.
Effect on Fungal Linear Growth
The prepared formulation was evaluated against the growth of the above-mentioned soilborne pathogenic fungi at different concentrations (1%, 2%, 3%, and 4%). A certain weight of the formulation was added to autoclaved melted potato dextrose agar (PDA) medium and carefully mixed by gentle swirling to ensure that the concentration was evenly distributed before pouring into 9 cm diameter Petri dishes. After the agar solidified, a 5 mm disk was cut from the edge of an actively growing colony of each tested fungus and placed individually in the center of each Petri dish. A separate PDA plate without the formulation powder was used as the control treatment. All Petri dishes were incubated at 25 ± 1°C, and the radial growth of the fungi was observed daily until the colonies completely covered the medium in the control set. The diameter of the mycelial growth was then measured in millimeters. Three replications were used for all treatments, and the experiments were repeated three times. The reduction percentage in fungal growth was calculated relative to its growth in the control treatment using the following formula:
Where dc is mean colony diameter in control sets and dt is mean colony diameter in treatment sets.
Effect on total fungal count in agricultural soil
Randomized different cultivated soil samples were collected from a natural heavily infested field with soilborne pathogens located at Researches and Production Station, National Research Centre, Beheira governorate, Egypt. The samples were mixed together and then divided into two subsamples of 1 kilogram each. One subsample was amended with 5% of formulation powder, while the other subsample was left untreated. Both subsamples were water irrigated to field holding capacity and left overnight at room temperature. The total fungal counts in the two prepared soil samples were then determined using the plate count technique, as described by Alexander [37]. This procedure was repeated three times to ensure the reliability of the results.
Effect on the viability of Meloidogyne incognita J2 phase
The efficacy of the tested formulation on the total number of J2 (second stage juveniles) of the nematode Meloidogyne incognita was evaluated using the plate count technique with the aid of a microscope, as described by Ashoub and Amara [38]. The tested formulation was investigated at three concentrations (1%, 3%, and 5%) using distilled water. A certain number (100 larvae) of live moving larvae were placed into Petri dishes (9 cm in diameter). The prepared solutions of the tested formulation were then poured individually into these plates. A blank solution, without any formulation powder, was used as the control treatment. Five replicates were used for each treatment. All plates were incubated at room temperature (22–27°C) and examined microscopically after 24, 48, and 72 hours. The percentage of mortality or unmoved larvae was calculated using the following formula:
At the end of the experiment, after 72 hours, the larvae were transferred to distilled water and left for 24 hours. They were then examined, and the final number of dead larvae in each treatment was calculated. The recovered larvae, which had detached themselves from the formulation powder, were not included in the final count.
Field experiment
All plant experiments were conducted in accordance with relevant institutional, national, and international guidelines and legislation.
The field experiments were conducted for two consecutive growing seasons (September 2017 and March 2018) at the Researches and Production Station of the National Research Centre in the Nobaria region of Beheira governorate, Egypt. The experiment was conducted to evaluate the efficacy of formulation powder as a seed dressing at a rate of 3 grams per kilogram of seeds and soil drench treatments for controlling the incidence of root rot and root knot diseases in green beans. The experimental field consisted of plots measuring 2 meters by 15 meters, each with 10 rows (1.5 meters between rows, 2 meters long each, and 8 holes per row). At the same field, green bean seeds Giza 4 cv. were sown in all treatments at the rate of two seeds per hole. Green bean seeds were coated with the formulation powder at a rate of 5 grams per kilogram of seeds before sowing. The formulation powder was applied to the soil as a soil drench at rates of 10, 20, and 40 kilograms per feddan (4200 square meters). These treatments were incorporated into the same cultivated row site on top of the top 20 centimeters of soil surface. Green bean seeds Giza 4 cv. were also sown in all treatments at the rate of two seeds per hole.
Monitoring and scouting for root rot disease incidence was conducted weekly in all cultivated plots. After 3 weeks of seedling emergence, the percentage of root rot and root knot incidence was calculated. The percentage of root rot disease incidence was estimated as the mean of all replicates using the following equation:
Similarly, the root-knot nematode was recorded in each treatment. Random plants that showed a yellowish appearance were selected for nematode infection. The tops of the plants were cut off and the roots were gently washed to remove any adhering soil. The roots were stained with hot acid fuchsin-lactophenol, cleared with lactophenol, and the number of nematode galls and egg masses were counted [39]. The centrifugal-flotation technique [40] was used to extract nematode juveniles from soil (500 grams of soil per plant). The perineal patterns of isolated Meloidogyne females were prepared for identification to the species level [39]. Five replicates were used for each treatment, and untreated plots were also included. All plots were conducted in a completely randomized block design. The traditional agricultural practices, such as soil plowing, fertilization, and irrigation, were followed at all experimental plots. The field experiments were conducted at the same field for two consecutive growing seasons, and then the average of the root rot and root knot diseases incidence, as well as the number of J2 per 500 grams of soil, the number of galls in roots (5 grams), and the number of egg masses in roots (5 grams) for the two seasons were calculated.
Statistical analysis
The data were analyzed using IBM SPSS software version 14.0. Analysis of variance (ANOVA) was performed to determine the significance of the differences between the means. The mean values were compared using Duncan's multiple range test at a significance level of P < 0.05.
Chemical composition:
The chemical composition of chicken feather hydrolysate (table 1) shows that it contains a significant amount of nitrogenous compounds (about 50%), such as amino acids and peptides, which are derived from the digested feather ketenes. These nitrogenous compounds are released and degraded slowly affording ammonia, which makes them beneficial for plant growth. The hydrolysate also contains about 39% of hemicelluloses/cellulose, which can serve as a carbon source for plants and help to reduce the phytotoxicity. Additionally, the hemicelluloses/cellulose can be biodegraded under field conditions, releasing active chemicals such as furfural [41], with beneficial effects on plant growth.
The chicken feather hydrolysate can be further enriched with calcium (lignin/silica/ fatty acids) complex (formulation 1). This complex contains lignin with known antioxidant, and antibacterial properties [6, 10]. Therefore, such Ca-LSF enriched feather hydrolysate has the potential to be used as a biofertilizer, and biopesticide.
Table (1): Chemical composition of the black liquor pH (10)-chicken feather hydrolysate
|
Component |
% on air dried basis (as it is) |
|---|---|
|
Crude protein |
49.67 |
|
Cell wall constituents |
|
|
NDF |
40.42 |
|
ADF |
8.54 |
|
Lignin |
1.12 |
|
Hemicellulose |
31.88 |
|
Cellulose |
7.42 |
Effect on fungal linear growth
The effectiveness of the prepared formulation against the growth of some soilborne pathogenic fungi was evaluated under in vitro conditions (Fig. 1). Table 2 shows that it began to affect the growth of the tested fungi at a concentration of 3%, with the highest growth reduction was 72.4–82.6% at a concentration of 4%.
Table (2): Linear growth of some fungi in response to different concentrations of the tested formulation in vitro
|
Concentration (%) |
|
||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
Tested formulate |
Growth (mm) of tested fungi |
||||||||||||||||||||||||||||||
|
F. oxysporum |
F. solani |
R. solani |
S. rolsii |
||||||||||||||||||||||||||||
|
1.0 |
2.0 |
3.0 |
4.0 |
1.0 |
2.0 |
3.0 |
4.0 |
1.0 |
2.0 |
3.0 |
4.0 |
1.0 |
2.0 |
3.0 |
4.0 |
||||||||||||||||
|
1 |
90.0 ± 0.1g |
90.0 ± 0.1g |
86.8 ± 0.1g |
82.6 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
80.4 ± 1.9f |
78.6 ± 1.9e |
90.0 ± 0.1g |
90.0 ± 0.1g |
80.4 ± 1.9f |
78.4 ± 1.9e |
90.0 ± 0.1g |
88.8 ± 1.9f |
78.2 ± 1.9e |
72.4 ± 1.9e |
|||||||||||||||
|
Control |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
90.0 ± 0.1g |
|||||||||||||||
Means ± standard deviations within a column followed by the same letter are not significantly different at P < 0.05
Effect on total fungal count in agricultural soil
The inhibitory effect of the tested formulation was assayed in cultivated soil sample treated with 5% formulation. Then, the total fungal counts per gram of dry soil was calculated. Table 3 shows that the total fungal counts in the treated soil (12 x106 cfu/g) were drastically reduced by 64.7% compared to the untreated soil (34x106 cfu/g). This reduction could be attributed to the release of ammonium throughout the soil particles when the formulation was incorporated into the soil. The fungicidal and nematicidal effects of ammonium have been reported previously. Ammonium was found to inhibit the growth and spore germination of soilborne fungi [42]. It also exhibited a nematicidal effect against root knot nematodes [24].
Table (3): Effect of the tested formulation at different concentrations on the growth of some pathogenic soilborne fungi
|
Soil sample |
Total fungal count (cfu/g) dry soil |
Reduction (%) in fungal counts |
|---|---|---|
|
Treated soil sample with formulation powder |
12x106 |
64.7 |
|
Untreated soil sample |
34x106 |
-- |
Effect on the viability of Meloidogyne incognita J2
The effect of the tested formulation on the viability of nematode Meloidogyne incognita J2 was evaluated under in vitro conditions (Fig. 2). Table 4 shows that the number of dead nematode larvae increased with increasing concentration of the formulation and with prolonged exposure time. The highest percentage of dead nematode larvae was observed at 5% of formulation, which recorded 73%, 98%, and 100% mortality after 24 hours, 48 hours, and 72 hours, respectively. In contrast, no mortality was observed in the control group (sterilized distilled water).
Table (4): Effect of the tested formulation at different concentrations on the viability of J 2 nematode Meloidogyne incognita
|
Concentrations (%) |
Exposure time |
||||||
|---|---|---|---|---|---|---|---|
|
24 hrs |
48 hrs |
72 hrs |
|||||
|
Live |
Dead |
Live |
Dead |
Live |
Dead |
||
|
The tested formulation |
1.0 |
65 |
35 |
2 |
96 |
0 |
100 |
|
3.0 |
43 |
57 |
2 |
98 |
0 |
100 |
|
|
5.0 |
27 |
73 |
4 |
98 |
0 |
100 |
|
|
Control |
100 |
0 |
100 |
0 |
100 |
0 |
|
The pesticidal activity of the tested formulation can be attributed mainly to the presence of polyphenolic compounds from hydrolysable lignin residues [43,44]. These compounds synergize with the protein hydrolysate peptides and amino acids, which slowly degrade and release the known bioactive ammonia under field conditions [45, 46]. This is in addition to the presence of calcium salts of natural hydroxyl acids derivatives present in the black liquor of rice straw and those of the amino acids [47]. These compounds, along with hemicelluloses as a carbon source, contribute to the pesticidal activity of the presented formulation [48].
Field experiment
Under field conditions, the presented formulation was evaluated as a safe and effective alternative to chemical pesticides for controlling root rot disease in green beans. The formulation was applied as a seed dressing (5 g/kg seeds) or a soil drench at different rates (10, 20, and 40 kg/feddan).Visual scouting and monitoring throughout the study period revealed that all of the treatments were effective in reducing the incidence of root rot disease. Data in Table (5) and Figure (3) show that the highest percentage of root rot incidence was 18.8%, which was reduced by 16.8% with the 10 kg/feddan soil drench treatment. The 20 kg/feddan and 40 kg/feddan soil drench treatments reduced the incidence of root rot disease by 30.5% and 44.2%, respectively. Moreover, the seed dressing treatment with the rate of 5g/Kg seeds was the most effective, with a disease incidence of only 10.3%, which is a reduction of 54.4% over the untreated control.
Table (5): Efficacy of seed dressing and soil drench application of the tested formulation on root-rot incidence of green bean for two successive seasons under natural field conditions
|
Treatment |
Average root rot incidence (%)* |
Reduction in root rot incidence (%) |
|
|---|---|---|---|
|
Soil drench |
10Kg/feddan |
18.8 ± 2.1b |
16.8 |
|
20Kg/Feddan |
15.7 ± 1.9c |
30.5 |
|
|
40Kg/feddan |
12.6 ± 1.1d |
44.2 |
|
|
Seed dressing |
5g/Kg seeds |
10.3 ± 2.1e |
54.4 |
|
Untreated control |
22.6 ± 0.8a |
||
| * Average records for the successive cultivation seasons | |||
Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05
Furthermore, the applied treatments also reduced nematode infection, the number of soil-J2 nematode Meloidogyne incognita, the number of galls, and the number of egg masses in the roots of green bean plants, compared to the untreated control (Table 6). Plant infection by nematode showed descending order related to the rate of the applied formulation as soil drench. The percentage of plant infection by nematodes decreased in the following order:
-
13.3% at 10 kg/feddan of soil drench with formulation powder
-
8.3% at 20 kg/feddan of soil drench with formulation powder
-
5.0% at 40 kg/feddan of soil drench with formulation powder
It is interesting to note that seed dressing with the presented formulation powder resulted in remarkable protective effect against root knot incidence, as well as the number of M. incognita-J2 nematodes, the number of root galls, and the number of egg masses, when compared with soil drench and control treatments. The seed dressing treatment resulted in a root knot incidence of only 4.1%, which is a reduction of 88.2% compared to the soil drench treatments, which had a range of 5.0–13.3% disease incidence (Fig. 4). A similar trend was observed for the other measurements: the number of J2 nematodes per 500 grams of soil, the number of galls in 5 grams of roots, and the number of egg masses in 5 grams of roots.
Table (6): Effect of formulation seed and soil treatments on root knot infection, number of Meloidogyne incognita juveniles in soil, galls and egg masses on roots of green bean for two growing seasons under field conditions*
|
Treatment |
Plants infected with M. incognita (%) |
Number of J2 per 500 gm soil** |
No. galls in roots (5g) |
No. egg-masses in roots (5g) |
|
|---|---|---|---|---|---|
|
Soil drench |
10Kg/feddan |
13.3 ± 1.0b |
900 ± 150.0b |
52 ± 4.0b |
14 ± 3.0b |
|
20Kg/Feddan |
8.3 ± 1.6c |
720 ± 100.0c |
46 ± 2.0c |
10 ± 1.8c |
|
|
40Kg/feddan |
5.0 ± 1.2d |
252 ± 15.0d |
34 ± 3.0d |
8 ± 0.6d |
|
|
Seed dressing |
5g/Kg seeds |
4.1 ± 0.4de |
132 ± 12.5e |
12 ± 2.0e |
5 ± 1.2e |
|
Untreated control |
35.0 ± 2.0 a |
1045 ± 55.0a |
87 ± 11.0a |
17 ± 1.6 a |
|
| * Average records for the two successive cultivation seasons | |||||
| **Imitational nos. was 467/500 gm soil | |||||
Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05
This is likely because the ammonia released around the seed bed and later through the plant root system and the rhizosphere area, which provides more protection to the plant roots from soilborne pathogens. In the case of soil drench, a similar effect was observed.
The importance of the current study is to explore the potential of pesticidal activity of rice straw black liquor/chicken feather hydrolysate to be used as an alternative in a protective formulation that can inhibit harmful soilborne plant pathogens and prevent them from causing damage. Based on the above mentioned results, it seems that the action of soil microorganisms in decomposing of organic contents of our tested formula namely: lignin, hemicelluloses, phenol, volatile and fixed fatty acids, along with amino acids and peptides are expected to produce a wide range of nematicidal agents that have also significant fungicidal and nematicidal activity in the soil. This led to a reduction in the causal pathogens of root rot and the root knot nematode Meloidogyne incognita, as well as its galling and egg masses. This suggests that the presented formulation powder can protect plants from pathogens invasion and prevent them from completing their life cycle inside plant roots.
Apparently, the formulation applied in this study also helped to maintain soil conditions that are favorable for root development and provided protection against soilborne pathogens. It also possesses beneficial ingredients for plant growth and increasing natural enemies.
Overall, the formulation powder showed promising results against root rot pathogenic fungi, root knot nematodes, and their harmful effects on green bean plants. It could be a useful alternative to petrochemical derived pesticides for controlling a wide range of soilborne plant pathogens and suggests an ecofriendly useful utilization of wastes instead of its bad disposing.
Acknowledgement
The authors are grateful to the Administration Board of the Researches and Production Station, National Research Centre, Beheira governorate for providing the facilities that made this work possible.
Author contributions
All authors contributed to the study conception &design, Material preparation, data collection and analysis. The first draft of the manuscript was written by all authors and they approved the final manuscript.
Competing interests
The authors have declared that no competing interests.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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No competing interests reported.