Abstract: Background and Objective: Roselle, Hibiscus sabdariffa L. var. sabdariffa is considered one of the most important medical plants all over the world. The application of different strains of bio fertilizers can help to control parasitic nematodes and to reduce the use of chemical nitrogen fertilizers and enhance the plant growth to decrease the production cost and environmental risk. The objective of this study was to maximize bio fertilizer and urea for controlling plant-parasitic nematode infected rosella in relation to improve growth and active constituents. Methodology: Field experiments were carried out to explore the role of three bio fertilizers: Azotobacter chroococcum; Bacillus polymyxa and Pseudomonas fluorescens singly and in combination with different doses of urea (0, 30, 60, 90 and 120 kg/feddan) in the control programs of plant parasitic nematodes infected roselle plants (Hibiscus sabdariffa L.) in relation to its growth characters under field conditions during two successive seasons. The experiment included 20 treatments, singly and combined between five levels of urea and different strains of bio fertilizers. The obtained data were subjected to analysis of variance (ANOVA) one way direction with LSD 5% using CoStat Software, Version 6.4 (2008). Results: All tested treatments significantly at 5% reduced soil and root population of the plant parasitic nematodes and increased the growth, yield and chemical constituents of roselle plants compared with untreated control. The highest reduction percentages in parasitic nematode population (84.3 and 85.9%) in the two successive seasons, respectively, occurred at the application of; A. chroococcum+60 kg urea/feddan. In addition, effectiveness of this combined gave a better enhancement in plant height (218.80 and 231.44 cm), fresh weight of whole plant (1227.22 and 1264.18 g) and seed yield (676.89 and 661.06 kg/feddan), in the two successive seasons, respectively, compared with untreated control. Conclusion: Finally, it could be concluded that, the best conformity of urea plus bio-fertilizers are A. chroococcum+60 kg urea/feddan. Which descendingly effective on parasitic nematodes reduction and roselle plant growth characters.
INTRODUCTION
Roselle, Hibiscus sabdariffa L. var. sabdariffa is considered as one of the most important medical plants of the family Malvaceae1. In addition, its drink has a laxative effect and the calyx extraction is a great therapeutic action for curing heart and nerve diseases, high blood pressure and calcified arteries2. It has been mentioned that it is beneficial for cathartic, cancer, protective, restorative, sexual stimulator, appetizer, refrigerant and anti-cough3.
The annual global loss in agriculture due to damage by plant-parasitic nematodes has been estimated as US $100 billion worldwide. Most of plant parasitic nematodes attack the roots or other parts in the soil such as bulbs and tubers and interrupt the uptake of water and nutrients by plants4. The dominant nematode genera attacking Roselle plants are Meloidogyne, Pratylenchus and Xiphinema which occurred in different numbers. One or more Meloidogyne species have been reported as pathogens of roselle. Roselle is generally susceptible to M. arenaria and M. incognita infection5,6.
Many popular fertilizers incorporate nitrogen in their composition or made specifically as a nitrogen fertilizer is necessary and critical for plant growth. The application of ammonia (NH3) has been shown to control parasitic nematodes, with exposure of J2 to ammonia at 9.3 mg plant–1 for 40 h causing 95% mortality7. Plants require nitrogen as it is a major component of chlorophyll, which is essential for the process of photosynthesis8.
It is also a major component of amino acid, which required in making proteins and that essential for the structural elements in the plant. Some soil amendments (e.g., with low C/N ratios of organic matter, in addition to NH4OH or urea) have been proposed for controlling parasitic nematodes9.
The application of different strains of bio fertilizers considered an important factors that can help to reduce the use of chemical nitrogen fertilizers and enhance the plant growth to decrease the production cost and environmental risk10,11.
The A. chroococcum; B. polymyxa and P. fluorescens strains are implicated in the control of plant parasitic nematodes, such products may be toxic to nematodes directly or it may be indirectly suppress nematode population by modifying the rhizosphere environment12,13.
Azotobacter chroococcum has been reported to inhibit hatching of juveniles of Meloidogyne incognita and its penetration in roots14. Bacillus polymyxa was previously found to have the ability, not only inhibit parasitic nematode growth but also facilitate colonization of the root and induce host resistance15. Pseudomonas fluorescens was found to exert toxic effects towards eggs and juveniles of M. javanica in vitro and contributes in reducing nematode densities and consequently root-knot disease in tomato16.
Inoculating seeds with bio fertilizers secrete some organic acid which can convert important elements from unvalued to available form through biological process17. One of the most troublesome limiting factors in increasing the cultivation lands for roselle plants in Menoufia Governorate are nematodes. Therefore, objective of this field study was reduction of plant parasitic nematodes infected Roselle plants and improvement in its growth characters.
MATERIALS AND METHODS
Field experiments were carried out as Randomized Complete Block Design (RCBD); 20 treatments with three replicates for each during two successive seasons 2013 and 2014 at the experimental farm of the Faculty of Agriculture, Menoufia University, Shebin El-Kom, Egypt, to study the effect of bio and chemical fertilizers singly and in combination on plant parasitic nematodes infected roselle plants and measures growth, yield and chemical composition of Hibiscus sabdariffa L. plants.
The chemical and physical properties of the experimental soil were determined according to the method of Cottenie et al.18 as follows: pH 7.9, EC 1.73 dS m–1; 2.80% organic matter, 44.24% silt, 3.84% coarse sand, 27.40% fine sand, 23.20% clay, the texture grade was a clay loam soil. The available macronutrients were 2.30% CaCO3, 0.12% N and 0.25% P2O5.
Preparation strains of bio fertilizers: Efficient local strains of A. chroococcum, B. polymyxa and P. fluorescens were obtained kindly from Pharmaceutical Department National Research Center, Giza, Egypt. The aforementioned bacterial strains were prepared for application according to the method described by Mahfouz19. The prepared culture from each bacterial strain contained 107cell mL–1.
Experimental preparation and design: Seeds of Roselle, Hibiscus sabdariffa L. were soaked in different bacterial strains solely for 12 h, in addition, the control soaked in distilled water. Seeds were sown on 14th of April in the first and second seasons. The soil was prepared and divided into plots of 2×1.8 m with three rows at 60 cm apart and 40 cm between hills. Beside inoculation the seeds, A. chroococcum; B. polymyxa and P. fluorescens were applied (106 CFU mL–1) at a rate of 10 mL per plant after 15 days form planting20.
The experiment included 20 treatments, singly and combined between five levels of urea (46% N) and different strains of bio fertilizers (A. chroococcum; B. polymyxa; P. fluorescens plus un-inoculated seeds). Roselle plants were harvested at the first of October during maturity stage through two successive seasons (2013 and 2014).
Obtained data showed that all measurements of roselle such as: plant height (cm); number of main branches; fresh weight of whole plant (g per plant); number and fresh weight of fruits (g per plant); fresh weight of sepals (g per plant); dry weight of sepals (g per plant and kg per feddan); seed yield (g per plant and kg per feddan); fixed oil% and fixed oil yield (L. feddan).
Nematode extraction and enumeration: Each composite soil sample was carefully mixed and an aliquot of 100 cm3 was processed for nematode extraction according to methods described by Southey21, each treatment was replicated three times. An aliquant of 1 mL each of nematode suspensions were pipetted off, placed in a Hawksley counting slide and examined by using a stereomicroscope.
Nematode counts were carried out before treatment and after 1-6 months of application and the identification to generic level were based on morphology of the adult and larval forms, according to the description of Mai and Lyon22. Roots were carefully washed and the nematode galls were counted and rated as mentioned in Table 1 as well as one gram per root was stained by acid fuchsin lactophenol to counted root knot nematode stages inside the roots with the aid of a dissecting microscope.
Egg masses were assessed by staining the roots with Phloxin-B solution (0.15 g L–1 tap water) for 20 min according to Daykin and Hussey23.
Chemical analysis: Total anthocyanin content in the sepals was determined by using Du and Francis24 method. Total carbohydrate percentages in the dried leaves were determined by using the colorimetric method of DuBois et al.25. Nitrogen, phosphorus and potassium percentages were determined as follows: in the dried leaves by Kjeldahl methods, using Spectrophotometrically (Benchtop CS-800 series) and by Flame photometer (model 360, 420 series), respectively18. The pH values of sepals were determined according to Diab26. Fixed oil percentages were determined according to the AOAC 27.
Statistical analysis: The obtained data were subjected to analysis of variance (ANOVA) one way direction with LSD 5% using CoStat Software, Version 6.4 (2008).
Reduction percentages were counted according to the formula of Henderson and Tilton28 and Fleming and Retnakaran29:
| (1) |
| (2) |
RESULTS AND DISCUSSION
Effect of bio and mineral fertilizers on plant parasitic nematodes infected roselle plants
In the first season (2013): The obtained results presented in Table 2 show the effect of bio and mineral fertilizers on the average numbers of plant parasitic nematodes infected roselle plants in the first season (2013) before treatment and after 1-6 months of application under field conditions.
Statistical analysis of data in Table 2 indicated that there were significant differences among treatments at 5%. Nematode population was suppressed in the treated soils after 1-6 months in comparison with control treatment. The combined treatment of ( A. chroococcum+60 kg urea/feddan) recorded the least number of parasitic nematode (375.0 individual’s/100 g) in the 6th month (October, 2013), Azotobacter spp. is aggressive colonizers of the rhizosphere of various crop plants and has broad spectrum antagonistic activity against plant pathogens30.
The highest reduction percentages of the nematode in the soil were recorded in the combined treatments of (A. chroococcum+60 kg urea/feddan) followed by (B. polymyxa+90 kg urea/feddan) and (P. fluorescens+30 kg urea/feddan) with (84.3, 73.0 and 59.1%), respectively, descendingly as shown in Table 3.
Some species of Azotobacter and Bacillus are reported to induce systemic resistance in plants against invading pathogens and antagonists to root-knot nematodes31.
Such findings could lead us to make some restrictions in choosing the suitable fertilizers level to, fit with our requirements. The main tool in this respect must be the behavior of soil fertilizer under different environmental conditions.
| Table 1: | Rating scale levels of resistance or susceptible by gall numbers |
| Source: Southey21 | |
| Table 2: | Effect of mineral urea levels and bio-fertilizers on the population density of plant parasitic nematodes infected roselle plants under field conditions (2013) |
Second season (2014): Data presented in Table 4 showed that the side effect of applied treatments on the population density of plant parasitic nematodes infected Roselle plants under field conditions.
| Table 3: | Reduction percentages of plant parasitic nematodes infected roselle plants as affected by bio and mineral levels fertilizers under field conditions (2013) |
Statistical analysis indicated that all treatments significantly at 5% suppressed nematode population in the soil treated after 1-6 months in comparison with control treatment. The combined treatment of (A. chroococcum+60 kg urea/feddan) recorded the least population density of parasitic nematode (239.5 ind’s /100 g) in the 6th month (October, 2014).
Followed by that found in combined treatments of (B. polymyxa and P. fluorescens+90 kg urea/feddan) for each, resulted nematode population density of (597.0 and 829.0 ind’s/100 g), respectively.
The application of ammonia (NH3) has been shown to control nematodes, with exposure of J2 to ammonia at 9.3 mg per l for 40 h causing 95% mortality7. Some soil amendments (e.g., with low C/N ratios of organic matter, in addition to NH4OH or urea) have been proposed for controlling nematodes9.
Results in Table 5 showed that, the highest reduction percentages of the nematode in the soil, occurred in the combined treatment of A. chroococcum+60 kg urea/feddan followed by B. polymyxa+90 kg urea/feddan and P. fluorescens+30 kg urea/feddan with 85.9, 67.8 and 58.8% respectively, descendingly.
It is very interesting to know that, volatile compounds, fatty acids, hydrogen sulfide, enzymes, hormones, alcohol and phenolic compounds are among the bacterial products implicated in the control of plant parasitic nematodes13.
Such products may be toxic to nematodes directly or it may be indirectly suppress nematode population by modifying the rhizosphere environment 12.
This study results are in agreements with those obtained by Wen et al.32, who found that urea+bacteria to be effective against RKN and ammonia may act by disrupting cell membranes.
| Table 4: | Effect of mineral urea levels and bio-fertilizers on the population density of plant parasitic nematodes infected roselle plants under field conditions (2014) |
All plant treatments decreased the gall index and egg-masses production (Table 6). The combined treatment of A. chroococcum+60 kg urea/feddan recorded the highest decrease percentage in root gall index (-80 and -80%) and egg masses production (-86.0 and -88.8%) in the two seasons, respectively.
| Table 5: | Reduction percentages of plant parasitic nematodes infected roselle plants as affected by bio and mineral levels fertilizers under field conditions (2014) |
Our results are in agreements with those obtained by Li et al.33 and Anwar-ul-Haq et al.34.
Statistical analysis showed that, there are significant differences at 5% among all of experimental factors between each of months; bio and mineral fertilizers; population density of plant parasitic nematodes and the interactions within the experimental factors.
Vegetative growth characteristics
Effect of nitrogen application: The obtained data in Table 7 indicated that, the application of urea levels had a significant at 5% effected on vegetative growth characters i.e., plant height, number of main branches as well as fresh weight of whole plant which reached to optimum values as a results of using 90 kg/feddan during the two seasons (2013 and 2014).
These results could be explained by using nitrogen in building protein molecules and enhancing cell division and cell elongation which was reflected in promoting the growth of lateral buds and producing more branches, fresh weight and increasing the length of the plant and was similar to those obtained by Sakr et al.35 on rosella plants.
Effect of bio-fertilizers application: The three bio-fertilizers gave significantly at 5% increased in growth characters of roselle plants (Table 7). The A. chroococcum produced the best results followed by B. polymyxa and P. fluorescens, during the two successive seasons, respectively. This effectiveness may be due to producing adequate amounts of IAA, gibberellins, cytokinins and vitamins which improve roots capacity, length, hair branching and mineral up take from the soil36. This study results stated the same treat with those obtained by Aly et al.37 on dill plants.
Effect of interaction: Interaction occurred within most of combined treatments caused significant differences at 5% level in all vegetative characters during both seasons under study, reached the maxima with application of the combined treatment of A. chroococcum+60 kg urea/feddan.
| Table 6: | Decrease of gall index and egg-masses production on roselle as influenced by treatments application in the two seasons (2013 and 2014) |
Its values were varied within both seasons, with higher levels in 2014 than that found in 2013 seasons. Such findings may be affected by different environmental factors 38. Results obtained are in harmony with that found by Mohamed et al.39 on sweet basil plants.
Yield parameters and oil production
Effect of nitrogen application: Data recorded in Table 8 and 9 showed that, all urea levels significantly at 5% increased number of fruits, fresh weight of fruits (g per plant), fresh weight of sepals (g per plant), dry weights of sepals (g per plant and kg per feddan), seed yield (g per plant and kg per feddan), fixed oil% and fixed oil yield (L per feddan). Level of 90 kg urea per feddan was the best treatment and this may be due to the direct effect of fertilization on increasing the photosynthetic activity and different metabolic processes which encourage the yield of plants. These results are in agreements with those obtained by Khalil and Yousef40 on roselle plants.
Effect of bio-fertilizers application: Bacterial strain of A. chroococcum was the best treatment which significantly at 5% increased both total yield parameters and oil production, while P. fluorescens caused the higher value of fixed oil% in both seasons.
This may be due to that bio-fertilizers improve physical, chemical, microbiological characteristics of the soil and increase its hold capacity which led to increase the yield41. These results are in harmony of that found by Patel et al.42 on Plumbago zeylanica.
Effect of interaction: All combined treatments caused significantly at 5% increased of total yield parameters and oil production, reached the maximum by application A. chroococcum+60 kg urea/feddan in both seasons, followed by the application of 90 kg urea/feddan plus of each A. chroococcum and P. fluorescens which resulted the highest fixed oil yield and fixed oil percentage in both seasons, respectively. Similar results were found by Mohamed et al.38 on dill plants.
Quality of sepals and chemical composition
Effect of nitrogen application: The obtained results show that, all urea levels increased the quality of sepals including anthocyanin content and pH values which arrived to the greatest values by added urea at 60 and 90 kg/feddan in both seasons, respectively (Table 10).
| Table 7: | Effect of mineral urea, bio-fertilizers and their interaction on vegetative characters of roselle plants during the seasons (2013 and 2014) |
| U: Urea, B: Biofertilizers, UB: Interaction | |
Total carbohydrate N and K (%) increased by using different levels of urea except phosphors percentage which increased at 30 kg urea/feddan and then decreased gradually by increasing urea level up to 120 kg/feddan. These results are in accordance with those found by Abo-Baker and Mostafa43 on roselle plants.
Effect of bio-fertilizers application: The highest mean value in this trail were obtained by using B. polymyxa in the two seasons except anthocyanin content which reached its maxima by using P. fluorescens. Similar results were obtained by Sakr et al.35 on roselle plants.
Effect of interaction: High increments in total anthocyanin content, pH values in roselle leaves by the application of P. fluorescens+90 kg urea/feddan and B. polymyxa+60 kg urea/feddan in both seasons, respectively (Table 10).
| Table 8: | Effect of mineral, bio-fertilizers and their interactions on roselle fruits during two seasons (2013 and 2014) |
| U: Urea, B: Biofertilizers, UB: Interaction | |
| Table 9: | Effect of mineral urea, bio-fertilizers and their interactions on seed yield, fixed oil and fixed oil yield of roselle plants during the two seasons (2013 and 2014) |
| U: Urea, B: Biofertilizers, UB: Interaction | |
| Table 10: | Effect of mineral urea, bio-fertilizers and their interactions on chemical composition of roselle plants during the two seasons (2013 and 2014) |
| U: Urea, B: Biofertilizers, UB: Interaction | |
While the combined treatments of B. polymyxa+each of 30, 60, 90 and 120 kg urea/feddan produced the highest value of P (%), total carbohydrate, K (%) and maximum N (%), respectively, in the two successive seasons. These results conformity with those obtained by Abo-Baker and Mostafa43 on roselle plants.
CONCLUSION
Finally, it could be concluded that the best and optimum combined treatments among bio-fertilizers and urea levels occurred by application (A. chroococcum+60 kg urea/feddan). This application gave the highest plant parasitic nematodes reduction percentages and consequently flourished all vegetative growth characteristics; yield parameters and chemical composition of roselle plants through both seasons (2013 and 2014).
SIGNIFICANCE STATEMENTS
This study discovers that the use of Azotobacter chroococcum as bio-fertilizer combined with urea in roselle plantation gave adequate control of plant parasitic nematodes as well as improved growth characters.