Abstract: Background and Objective: Sugar beet is one of the cash crops grown in temperate and cold zones of the world. In Egypt, it thrives in the northern part of the country. It comprises about 30% of total sugar production. Both foliage and pulps are feed of high value for the domestic animals. Since several soil-borne fungi attack this crop causing a significant reduction in the production, the present investigation aimed to evaluate the effectiveness of selected antioxidants including GAWDA formulation along with Trichoderma fungus and organic fertilizers (compost) to induce resistance against the root rot disease along with their possible role on increasing the yield and quality. Materials and Methods: In vitro studies were carried out to select the efficient concentration of the tested antioxidants on reducing the growth of the target pathogens. In vivo studies were applied in the field plots at Mansoura University Campus during the two successful seasons of 2015 and 2016. The growth parameters as well as the root weight and their quality characters were recorded. Data were statistically analyzed by CoStat 6.3 software of analysis of variance at p<0.05 as outlined by Duncan. Results: Soaking seeds in water solution of GAWDA® formulation at a concentration of 4 g L–1 for 12 hours before planting and air dried for 1 h followed by coating them with Trichoderma harazianum (T. harazianum) before planting in soil supplemented with the composted agricultural residues significantly decreased the damping-off disease and scaled up the yield and quality. Conclusion: Results addresses a ramping up in the yield and quality of sugar beet as well as control damping-off disease when treated with friendly environmental materials.
INTRODUCTION
Sugar beet is classified as the second important sugar crop in Egypt and in many other countries after sugar cane1-3. The present world production of sugar beet reached nearly 269.714 million ton comes from about 4471580 ha with an average of 60.32 t ha–1. In Egypt, the total cultivated area of this crop reached about 211806 ha with total production of 11.046 million tons of the fresh roots and average of 52.15 t ha–1 4.
Several soil-borne fungi attack this crop causing a significant reduction in the production viz., Rhizoctonia solani (R. solani), R. crocorum, Aphanomyces cochlioides (A. cochlioides), Phoma betae (P. betae), Macrophomina phaseolina (M. phaseolina), Fusarium oxysporum f. sp., radicis-betae, Pythium aphanidermatum (P. aphanidermatum), Phytophthora drechsleri (P. drechsleri), Rhizopus stolonifer (R. stolonifer), R. arrhizus and Sclerotium rolfsii (S. rolfsii). Several fungicides have been used to control these diseases including, chlorothalonil, pencycuron, tebuconazole, azoxystrobin, trifloxystrobin and pyraclastrobin5.
Some other fungi cause post-harvest losses, in storage piles. Rhizoctonia crown and root rot caused by R. solani is one of the most damaging sugar beet pathogen. While R. solani can also cause damping-off and crown and root rot of sugar beet and other crops including beans and soybean6,7.
One of the potential tactics for management these diseases are the use of compounds of antimicrobial activities to increase the plant resistance8. Some chemical compounds i.e., salicylic acid (SA), mono and di-basic potassium phosphate (KH2PO4 and K2HPO4), hydrogen peroxide (H2O2) and Bion (BTH) have been shown to induce resistance in plants9,10.
Benhamou and Belanger11 illustrated antioxidants as a resistance inducer (SAR) of plants against pathogens and indicated that resistance more or less is associated with metabolic and structural changes inside plants. Vallad and Goodman12 suggested that SAR may be induced as a result of the stress of both biotic or abiotic elicitors, resulting in the accumulation of salicylate such as salicylic acid leading to the expression of pathogen related (PR) genes. Van Loon and Bakker13 revealed that SAR refers to a distinct signal transduction pathway that plays an important role in the ability of plants defense against plant pathogens. Recognition of plant pathogen immediately initiates a cascade of molecular signals and transcription of many genes, which eventually results in the production of defense molecules by the plant.
Bigirimana et al.14 presented a remarkable effects of Trichoderma spp. on plants due to their direct effects on the pathogenic fungi and localized resistance in plants. Bailey and Lumsden15 revealed that the effective protection of plants against pathogen (biocontrol) exerted by Trichoderma strains is often unpredictable. The ability of Trichoderma strains as biocontrol agents is due to their high reproductive capacity, capability to survive under unfavorable conditions, effectiveness in the utilization of nutrients, ability to modify the rhizosphere, powerful aggressiveness against phytopathogenic fungi and efficiency in promoting plant growth and defense mechanisms. Li et al.16 indicated that many profitable properties of using the biocontrol agents are based on Trichoderma strains because they are living organisms and also because of their potential to survive in different environmental conditions. Until recently, the principal mechanisms for plant diseases control have been assumed to be those primarily acting upon the pathogens and included mycoparasitism, antibiosis and competition for resources and space17.
Wasternack et al.18 reported that this biocontrol fungus has two important modes of actions-direct suppression of the pathogen with production of antibiotics substances and enzymes and strong stimulation of the plants natural defence mechanism. Liu and Huany19 found that the population density of Fusarium spp. was highly decreased, when the population density of Trichoderma spp. increased in rhizosphere zone of soil treated with bio-compost.
El-Mohamdy20 showed that the bio-compost application as soil amendment were able to suppress diseases caused by R. solani and Fusarium spp. on a number of economic crops. Javaheri et al.21 studied the effects of farm yard manure and other nutrients on quality and quantity of sugar beet and resulted that 20 t ha–1 (9 t ha–1) of manure increased sugar yield by 10% with no significant effect on sugar loss in molasses. Research results show that manure could be a valuable source of nutrients for sugar beet, however, composted manure has different properties than the non-composted manure.
Cayuela et al.22 found that application of composts to soil has been proposed to control different diseases. However, not all types of compost have been shown to exert beneficial effects on plant growth and health. Loffredo et al.23 showed that a significant effect of humic fractions (HS) from soil and composts has been demonstrated on the mycelial growth and conidial germination of two formae speciales of Fusarium oxysporum) and on the growth and sclerotial formation of Sclerotinia sclerotiorum and two antagonistic Trichoderma species.
Loffredo and Senesi24 showed that the five commercial types of compost were evaluated on suppressing the root-rot pathogens (Fusarium solani, Pythium ultimum, Rhizoctonia solani and Sclerotium rolfsii) attacking sugar beet plants. El-Mohamedy et al.25 found that amendment of compost to T. harzianum accelerate its suppressive effect on controlling the plant diseases.
Al-Mughrabi26 showed that organic amendments play an important role as friendly environment and sustainable alternative approach to protect plants from the attack of the soil-borne pathogens. Soil amendments using composted agricultural wastes fortified with bio control agents is acceptable approaches in controlling a number of diseases. The use of organic agricultural wastes in this respect could be an advantageous in soil fertility, recycling of agricultural residues and provide a powerful tool for management of plant diseases. It has been reported that several composts and/or composts fortified with bio control agent used as soil amendments reduced the density of pathogen propagules and protected plants from the invasion of soil borne plant pathogens. Sabet et al.27 illustrated a significant effect of isolated bacteria and fungi from composts on a number of fungal pathogens attacking sugar beet roots.
Based on the available data, the present investigation aimed to evaluate the effectiveness of some antioxidants including GAWDA formulation along with Trichoderma fungus and organic fertilizers (compost) for inducing resistance against root rot disease of sugar beet and their possible role on increasing the production and the quality.
MATERIALS AND METHODS
This study was carried out at Laboratory of Seed Pathology, Department of Plant Pathology, Mansoura University, Egypt during three successive seasons (2015-2016).
Samples of sugar beet roots suspected to be attacked by root rot and wilt fungi were collected from different growing sugar beet areas in Dakahlia Governorates, Egypt to be used in the present study.
Isolation: Diseased roots washed thoroughly in tap water followed by cutting small pieces, surface sterilized for 1 min in NaOCl (0.5%), re-washed three-times in distilled sterilized water and distributed on petri-dishes containing 3 layers of moist blotter papers. Plates were incubated for 7 days at 22°C in the dark. The grown fungi were identified depending on their habit characters, colony pigment, size and shape of conidia and other morphological structures described by Gilman28, Parmeter29, Dhingra et al.30, Nelson et al.31, Booth32 and Burgess et al.33.
Pathogenicity: About 20 cm diameter pots, filled with sandy-clay soil (1:1) at a rate of 3 kg/pot, were inculcated with the tested fungi. Each fungus was grown in glass bottles containing sterilized moisted sorghum grain and incubated at 25±2°C for 15 days. Infestation was accomplished by mixing the inoculum with the upper 5 cm layer of the soil at a rate of 2% (w/w) for Rhizoctonia solani, Sclerotium bataticola and 4% (w/w) for Fusarium monliforme, Fusarium solani. Soil was irrigated 2 days intervally up to 8 days. Five healthy seeds from previously tested were sown in each pot. Three pots were used as lot replicate for each fungus, while three un-infested pots were used as a check. Pots were maintained under natural conditions. Observation for damping-off was recorded during the life span of the plants.
Antioxidant: The following antioxidants i.e., Salicylic acid, Tartaric acid and GAWDA® formulation Patent No. 23798, the Academy of Science and Technology of Egypt (Tri-sodium orthophosphate 1 mM+tartaric acid 2 mM+hydroxyquinoline 1 mM+calcium chloride 6 mM+magnesium chloride 5 mM+calcium borate 5 mM) were tested for their effect on the growth of the tested pathogenic fungi.
Compost: The Egyptian company for Solid Waste Recycling, Talkha, Dakahlia, Egypt kindly provided samples of its production used at the rate of 2 tons/fed was used in this study.
Tricohderma spp.: Tricohderma harzianum and Tricohderma viride were obtained from Plant Pathology Department, Faculty of Agriculture, Mansoura University, Egypt.
Effect of antioxidants on the fungal growth: The following antioxidants; Salicylic acid, tartaric acid and GAWDA® formulation each was dissolved in distilled water while concentrations of (2, 4, 6 and 8 mM), (5, 10, 15 and 20 mM), (1, 2, 3 and 4 g L–1) were used respectively. Disks presented 7 days old cultures were transferred onto the centers of PDA plates supplemented with the above antioxidants. The inhibitory effects were measured and recorded. Three replicates were used to present one treatment. All cultures were incubated for 7 days at 25±2°C in the dark. Linear growth of each fungus was recorded.
Effect of Trichoderma spp. on the fungal pathogens: The inhibition rate of T. harzianum and T. viride on the growth of F. solani, F. monliforme, R. solani and S. bataticola were investigated using the dual culture technique34 while the interaction was recorded as described by Desai et al.35.
| Table 1: | Designed combinations |
Effect of antioxidants on Trichoderma spp.: Disks from 5 days old cultures of T. harzianum and T. viride were transferred onto the center of PDA plates appended with 4 mM salicylic acid or 10 mM tartaric acid or 4 g L–1 GAWDA®. Cultures were incubated in dark for 7 days at 25±2°C. Three-replicates were used per each treatment. The inhibitory effect of the Trichoderma on pathogenic fungi were observed and recorded.
Formulation of Trichoderma spp.: After seeds soaking with selected antioxidants for 12 h, seeds were coated with one species of Trichoderma (1 g/20 g seeds) plus Acacia gum (1 g/100 mL water), then air dried.
Efficacy of antioxidants and GAWDA formulation as well as Trichoderma and the organic fertilizer (compost) on the sugar beet growth: Field plots located at the Campus of Mansoura University were used for the in vivo studies. The experiment was carried out according to Split-Split Plot design of six replicates. Compost was presented the main plots while the antioxidants were presented in the sub plots, Trichoderma sub-sub plots. Sugar beet seeds cv. Tenor were grown in ridges of 20 cm apart in hills spaced 60 cm apart on one side of the ridge.
Experimental design: The design made for the use of each component and their combination to assess their effect on retarding the severity of the pathogens under field condition was shown in Table 1.
Assessment of the photosynthetic pigments in the leaves: The third upper parts of a number of sugar beet leaves collected from plants were used to determine their contents of the photosynthetic pigments according to the method described by Mackinney36.
Total phenols: Fresh leaves of sugar beet plants were collected to determine their contents of the total phenols using Foline-ciocalteau reagent37.
Plant growth characters: After 200 days of sowing date, the following characters were measured:
| • | Plant weight (g), root weight (g), root length (cm), root diameter (cm), shoot weight (g), shoot length (cm), number of leaves/plant |
Total soluble solids (TSS) and sucrose content: The TSS was measured in juice of fresh roots by using hand refractometer (Hycle groupe lifasa bio 21320 Pouuilly by Auxxois-Fransa). Sucrose percent was determined by using polarimetric on lead acetate extract of fresh macerated roots according to the method of Carruthers and Oldfield38 and Fatouh39.
Statistical analysis: Obtained data were statistically analyzed according to CoStat 6.31140 of analysis of variance41 at p<0.05 as outlined by Duncan42.
RESULTS
Pathogenicity test: Data in Table 2 show that the degree of variance in damping-off percentage caused by the selected fungi was 42% in case of F. monliforme, 34% in case of F. solani, R. solani (60%) and S. bataticola (46%).
In vitro effect of the selected antioxidants on the growth of the isolated fungi: Results in Table 3 presented salicylic acid at a concentration of 2, 4, 6, 8 mM, tartaric acid at (5, 10, 15, 20 mM) and GAWDA at (1, 2, 3 and 4 g L–1) significantly reduces the linear growth of F. monliforme, F. solani, R. solani and S. bataticola.
Interaction between Trichoderma spp. and the isolated fungi: Tricohderma viride and T. harzinum retarded the mycelial growth of F. monliforme, F. solani, R. solani and S. bataticola as shown in Table 4. T. harzinum recorded the highest antagonistic effect (100%) on the mycelial growth of both R. solani and S. bataticola only.
| Table 2: | Pathogenicity of damping-off pathogens isolated from sugar beet roots |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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| Table 3: | Effect of selected antioxidants on the linear growth of the pathogenic fungi attacking sugar beet plants |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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| Table 4: | Interaction between Trichoderma spp. and the isolated fungi |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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| Table 5: | Effect of the selected antioxidants on the mycelial growth of Trichoderma spp. |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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Effect of the tested antioxidants on Trichoderma spp.: The results presented in Table 5 revealed that salicylic acid at 4 mM, tartaric acid at 10 mM or GAWDA formulation at 4 g L–1 show a significant effect on the linear growth of Trichoderma spp.
| Table 6: | Effect of the selected formulations of the antioxidants, Trichoderma and compost on the damping-off percentage of sugar beet |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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Effect of the combination of antioxidants, Trichoderma and compost on the damping-off percentage: The combination consists of GAWDA formulation at 4 mM and T. harzianum before sowing in soil amended with organic fertilizer (compost) has a significantly effects on decreasing the incidence of damping-off as shown in Table 6.
Effect of the combination of antioxidants, Trichoderma and compost on the content of the photosynthetic pigments and total phenols in leaves: The combination consists of GAWDA formulation at 4 mM and T. harzianum before sowing in soil amended with organic fertilizer (compost) significantly increased the content of chlorophyll A to record (2.592 mg g–1 fresh weight), chlorophyll B (1.719 mg g–1 fresh weight), total chlorophyll (4.311 mg g–1 fresh weight) and carotenoid (0.116 mg g–1 fresh weight). The total phenols recorded (943 mg/100 g fresh weight) as shown in Table 7.
| Table 7: | Effect of the combination of antioxidants, Trichoderma and compost on the content of the photosynthetic pigments and the total phenols in the leaves of healthy sugar beet plants |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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Effect of the combination of antioxidants, Trichoderma and compost on the growth parameters of healthy sugar beet plants: The combination of the compost, GAWDA formulation at 4 mM and T. harzianum significantly increased the plant weight to record (2619 g), plant height (328 cm), root weight (1405 g), root length (124 cm), shoot weight (1213 g), shoot length (205 cm), root diameter (20.3 cm) and leave numbers (96 leaves) (Table 8).
Effect of the selected formulations on the content of the total sugar and TSS in healthy sugar beet plants: The combination of the compost, GAWDA formulation at 4 mM and T. harzianum significantly increased the content of total sugar to record (23.66%) and TSS (28.08%) (Table 9).
DISCUSSION
The results illustrate obvious incidence of F. moniliforme, F. solani, R. solani and S. bataticola in wilted roots of sugar beet plants grown in different areas at Dakahlia Governorate of Egypt.
A modern method for controlling diseases attacking sugar beet roots caused by F. moniliforme, F. solani, R. solani and S. bataticola was applied as a friendly environmental method of fungal control. The application of compost, antioxidant i.e.: Salicylic acid, Tartaric acid, GAWDA® formulation and T. harzianum and T. viridae individually or combinations accelerate the resistance of sugar beet plants against F. moniliforme, F. solani, R. solani and S. bataticola.
| Table 8: | Effect of the selected formulations of the antioxidants, Trichoderma and compost on growth parameters in healthy sugar beet plants |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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The in vitro studies showed that salicylic acid, tartaric acid, GAWDA® formulation and antagonistic fungi; T. harzianum and T. viridae in the presence of the selected compost inhibit damping-off pathogens. These results were in support with the finding of Abdel-Monaim43, Ismail44 and Elwakil and El-Metwally45.
Salicylic acid at 2, 4, 6 and 8 mM, tartaric acid at 5, 10, 15 and 20 mM and GAWDA® formulation at 1, 2, 3 and 4 g L–1 significantly decreased the linear growth of F. monliforme, F. solani, R. solani and S. bataticola. Moreover, the presented results are also in support with the finding of Galal et al.46, Shahda47, Shabana et al.48 and Abd El-Hai et al.49.
Giridhar and Reddy50 presented the antioxidants as a friendly green chemicals which inhibit of functions of several enzymes by oxidized compounds, dissolved membrane lipids and interfere with membrane functions including transport of nutrients and interferes with proteins, RNA and DNA synthesis.
It was also, obvious that the best concentration for soaking seeds in salicylic acid was 4 mM, tartaric acid 10 mM and GAWDA® one g L–1 and this was in a harmony with those obtained by El-Mougy et al.51.
The application of selected antioxidants increases the sugar beet growth parameters. This increase may attribute to the role of antioxidants in stimulating of the physiological processes and reflecting an improvement in the vegetative growth followed by active translocation of the photo assimilation. In this respect, antioxidants might also increase enzyme activates such as α-amylase and nitrate reductase, which accelerates the sugar translocation from the leaves to developing fruit52.
| Table 9: | Effect of selected formulations of antioxidants, Trichoderma and compost on the content of the total sugar and TSS in healthy sugar beet plants |
*Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p<0.05 |
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CONCLUSION
This research highlights that the designed formulation of GAWDA at 4 mM and T. harzianum used for treating sugar beet seeds before sawing in soil supplemented with compost at a rate of (2 ton/fed) is an innovated method to overcome the incidence of damping off disease of sugar beet plants and significantly improve the yield and quality of the harvested roots.
SIGNIFICANCE STATEMENT
This data may help the researchers to direct their attention to use green chemicals as safe alternative to the toxic chemicals used in the agriculture regime, subsequently produce healthy food and keep the environment and soil clean.