Abstract: The current methods for controlling root rot and wilt disease caused by Fusarium oxysporum f. sp. radicis-cucumerinum, F. solani, Rhizoctonia solani and Macrophomina phaseolina, the most destructive fungi on cucumber are not yet rely. Salicylic Acid (SA), tartaric acid, GAWDA® formulation, compost, Trichoderma harzianum, Trichoderma hamatum, Trichoderma viride and Gliocladium virens were used in this investigation as promising methods for controlling such diseases. Results illustrated that soaking seeds in the antioxidants for 12 h before sowing followed by coating them with a combination of Trichoderma and compost, significantly reduced the disease incidence. It was also found that the triple combination of salicylic acid at 4 mM, T. harzianum and compost (2 t/fed) showed to be an intelligent tactic to enhance the resistance of cucumber plants against the root rot and wilt diseases. Moreover, this approach significantly promoted both yield and quality of the fruits.
INTRODUCTION
Cucumber (Cucumis sativus L.) is one of the oldest cultivated vegetables dating back to 5,000 years (Wehner and Guner, 2004) and classified in the Asian continent as the fourth most important vegetable crop following tomato, cabbage and onion (Tatlioglu, 1997). It ranks as the second vegetable crop in Western Europe behind tomato (Phu, 1997). The major disease contributing to yield losses is wilt disease caused by Fusarium oxysporum f. sp. radicis-cucumerinum Schlecht (Ye et al., 2004; Morsy et al., 2009; Farrag and Fatouh, 2010; Hu et al., 2010). Fusarium oxysporum, F. solani, S. rolfsii, R. solani and M. phaseolina are the commonly associated fungi with cucumber plants (Morsy et al., 2009). Since the control of such disease depends mainly on intensive application of the fungicides, risks to human health and the environment are expected. Therefore, alternative eco-friendly approaches for the control of the disease were emphasized (Mandal et al., 2009). Scaling up the Systemic Acquired Resistance (SAR) is a newly strategy to control a number of diseases in different crops (Hoitink and Boehm, 1999; Vallad et al., 2000; Nelson and Boehm, 2002). The varieties of microbes present in composts successfully induce systemic resistance in plants (Wei et al., 1991; Liu et al., 1995a, b, c). At the time being Induced Systemic Resistance (ISR) is accepted as one of the most promising methods for controling plant diseases. Some bacteria and fungi have been reported as potential candidates to induce systemic resistance in plants (Van Loon et al., 1998). Trichoderma spp. have beneficial impact on plant protection if colonized on roots, as they trigger the plant defense mechanism including the production of phytoalexins, Pathogenesis Related (PR) proteins, jasmonic acid, peroxidases and chitinases (Djonovic et al., 2006). The antioxidants increase plant growth parameters, resulting in enhancement of the yield and seed or fruit quality in a number of vegetables and field crops (Elwakil and El-Metwally, 2000; Elwakil, 2003; El-Mougy et al., 2004; Karlidag et al., 2009; Abd El-Hai et al., 2009; Zahra et al., 2010).
Therefore, the present research aimed to study the role of a number of selected antioxidants and their formulations (including Salicylic Acid (SA), tartaric acid, GAWDA® formulation, compost as well as T. harzianum, T. hamatum, T. viride, Gliocladium virens) for possible enhancement of (1) The systemic acquired resistance, (2) The control of root rot and wilt disease caused by F. oxysporum f. sp. radicis-cucumerinum, F. solani, R. solani and M. phaseolina the most aggressive fungi attacking cucumber roots, (3) The yield and quality of fruits. In this context, the increases in contents of the photosynthetic pigments and total phenols in the vegetative parts of the plants will be taken as indicators for the expected acceleration of plant defense mechanism against the attack of the damping off and wilt pathogens.
MATERIALS AND METHODS
Trials were conducted in laboratories and farm of Plant Pathology Department, Faculty of Agriculture, Mansoura University in the period from 2012-2015.
Sampling: Samples of cucumber roots presenting wilted plants grown in open fields at different areas of Dakahlia and Damietta Governorates of Egypt were collected to be used in the research.
Isolation and identification of the fungal pathogens: Roots from wilted plants washed thoroughly in tap water. Small pieces of their vascular tissues were then surface sterilized for 1 min in 0.5% sodium hypochlorite solution (NaOCl), washed again three times in sterilized distilled water and distributed on moist blotter papers containing Petri-dishes. Dishes were incubated at 22°C for 7 days in dark. The isolated fungi were identification based on the keys of morphological and cultural characteristics of the mycelium, conidiophores, conidia and colony morphology (Parmeter, 1970; Dhingra and Sinclair, 1978; Nelson et al., 1983; Booth, 1985; Burrges et al., 1988).
Pathogenicity test: Pots (20 cm in diameter), filled with clay sandy soil at a rate of 2.5 kg pot–1 were inoculated with the tested fungi prepared by growing each fungal isolate in glass bottles containing sterilized sorghum grain medium (100 g of sorghum seeds mixed with sand at a ratio of 2:1 and moisted with water, transferred to conical flasks and autoclaving at 121°C for 20 min), then incubated at 25±2°C for 15 days. Soil infestation was achieved by mixing the inoculum of each fungus with the upper 5 cm layer of the soil surface and rate of 2% (w/w) for R. solani, M. phaseolina and 4% (w/w) for F. oxysporum, F. solani. To make the soils ready for seeding, they were irrigated every 2 days for a period of a week and to insure the fungi adaptation. The 6 healthy looking cucumber seeds from previously tested lot were sown in each pot. The 3 pots were used as replicates for each fungus, while 3 uninfested pots were used as a check. The treatments were irrigated as needed. All pots were kept in a net house under natural conditions while the temperature was fluctuated between 25±5°C and the humidity between 55-75%.
Effect of the tested antioxidants on the fungal growth: The following antioxidants were selected to test their possible effects on retarding the growth of the pathogenic fungi under investigation i.e., salicylic acid, tartaric acid and GAWDA® formulation (Tri-Sodium Orthophosphate 1 mM+Tartaric acid 2 mM+Hydroxyquinoline 1 mM+Calcium Chloride 6 mM+Magnesium Chloride 5 mM+Calcium Borate 5 mM). The antioxidants salicylic acid, tartaric acid and GAWDA® formulation were dissolved in distilled water to obtain the desired concentrations of (2, 4, 6 and 8 mM), (5, 10, 15 and 20 mM), (1, 2, 3 and 4 g L–1), respectively. Disks of each fungal growth (0.6 cm diameter) from 7 days old cultures were transferred onto centers of PDA plates supplemented with different concentrations of the selected antioxidants. The possible inhibitory effects of these antioxidants on the tested fungi were recorded. The 3 replicates were used to present one treatment. All cultures were incubated at 25±2°C for 7 days in dark. Linear growth of each fungus was measured and recorded.
Trichoderma spp.: Trichoderma harzianum, T. hamatum, T. viride and G. virens obtained from the Department of Plant Pathology, Faculty of Agriculture, Mansoura University Egypt were used in this investigation. Seeds were coated with a formulation consists of one sp of Trichoderma spp. (at a rate of 1 g/20 g seeds) and Acacia gum at a concentration of (2 g/10 mL water), air dried for 2 h. At this stage, seeds were ready for sawing.
Antagonistic effects of Trichoderma spp. on the selected pathogens: The possible antagonistic competency of T. harzianum, T. hamatum, T. viride and G. virens to inhibit the growth of the tested fungi were investigated using dual culture technique (Baker and Cook, 1974). Discs of 0.6 cm taken from the growing edge of 7 days old cultures of F. oxysporum, F. solani, R. solani and M. phaseolina were placed at a distances of 1 cm from the edge of Sterilized Petri dishes containing sterilized PDA. At the inverse side of the medium, 0.6 cm diameter disc of 5 days old culture of T. harzianum, T. hamatum, T. viride and G. virens singly placed at a distances of 1 cm from the edges. The PDA containing petri dishes were inoculated at their centers with 0.6 cm diameter discs of F. oxysporum, F. solani, R. solani and M. Phaseolina to be used as checks. All plates were incubated at 25±2°C under 12 h in dark for 8 days. The inward linear growth (distance between the center of the disc and the edge of the colony) was measured after 2, 4, 6 and 8 interval days and the interaction between the 2 antagonized fungi was recorded either in the form of inhibition zone or the over growth of T. harzianum, T. hamatum, T. viride and G. virens on F. oxysporum, F. solani, R. solani and M. phaseolina as described by Desai et al. (2002).
Source of seeds: Seeds of cucumber cv. Beta Alpha Hybrid (produced by Seminis Co, USA) were used in the research.
Impact of the antioxidants on seed germination: Groups of 10 g of cucumber seeds were separately soaked in one of the following antioxidants for 12 h. Salicylic acid at (2, 4, 6 and 8 mM), tartaric acid at concentration (5, 10, 15 and 20 mM) and GAWDA® at (1, 2, 3 and 4 g L–1). After soaking seeds of each treatment were seeded onto moist blotter paper in 9 cm diameter Petri dishes while 3 replicates presented one treatment. The check was prepared by soaking healthy seeds in a tap water. All plates were incubated at 25±2°C for 7 days in dark, then the number of germinated seeds were rated to assess the impact of the antioxidants on seed germination. The best concentration of the tested antioxidants was determined and recorded.
Possible impact of the used antioxidants on Trichoderma spp.: Disks of 0.6 cm diameter from 5 days old cultures of each candidate (T. harzianum, T. hamatum, T. viride) were set onto centers of PDA plates each supplemented with 4 mM salicylic acid or 10 mM Tartaric acid or 1 g L–1 GAWDA® and incubated in dark at 25±2°C up to 7 days. Three replicates were used per each treatment. The check was prepared by placing each fungus on PDA free from the antioxidants. At the point in which Trichoderma cover the whole surface of the check treatments, colony diameters were measured and the possible percentage of growth retardation was calculated.
Field trails: Field trails were carried out at the experimental farm of Plant Pathology Department, Faculty of Agriculture, Mansoura University, to compare the in vitro results versus the in vivo studies. Split-Split Plot design of 6 replicates was applied. The compost were allocated in the main plots, antioxidant sub plots and Trichoderma sub plots. Cucumber seeds cv. Beta alpha hybrid were sown in ridges of 100 cm apart in hills spaced 35 cm apart on one side of the ridge.
| Table 1: | Chemical analysis of the used compost |
Compost: Commercial compost produced by the Egyptian Company for Solid Waste Recycling (ECARU) at the rate of 2 t/fed was used in this research. The composition of the compost is shown in Table 1. A daily observation for damping-off and wilt disease were recorded.
Forty days beyond seed sowing, a number of physiological characters were determined. In blades of third leaves of the growing plants, total phenols were measured following the method of Maliak and Singh (1980). Chlorophyll a, b and carotenoid were detected according to the method of Mackinney (1941). Ascorbic acid content (vitamin C) was detected following the method of (AOAC., 1970). Total soluble solids in fruits were detected by using a refractometer (ATAGO-MANUAL, Japan) (AOAC., 1970), fruit firmness was measured in 6 fruits from each sample employing a hand penetrometer and the measurements were taken on the 2 cheeks at the center of each fruit using 0.654 cm diameter head of penetrometer (Gullimex FT327, Italy), acidity in cucumber juice was measured following the method developed by Rangana (1979). Fruit moisture was calculated by taking a fixed weight, cut it to few slices, distribute them in opened Petri dishes for a partial dryness followed by incubation for 3 days at 70°C to insure a complete dryness. The difference between the 2 weights was recorded. Total carbohydrates in the fruit was measured following the method described by Sadasivam and Manickam (1996). At maturity stage, the following characters were recorded: yield (g)/plant, number of fruits/plant, fruit length and fruit diameter.
Statistical analysis: The obtained data were statistically analyzed using COSTAT (2005) software of analysis of variance (Gomez and Gomez, 1984). The means were compared using Least Significant Difference (LSD) at p = 0.05 as outlined by Duncan (1995).
RESULTS AND DISCUSSION
Results in Table 2 illustrate the degrees of variance in damping-off severity caused by F. oxysporum, F. solani, R. solani and M. phaseolina isolates. F. oxysporum diseased 78% of the total number of tested plants, F. solani 89%, R. solani 94% and M. phaseolina 78% when compare with the check.
Results in Table 3 show that salicylic acid at concentrations of 2, 4, 6, 8 mM, tartaric acid at (5, 10, 15, 20 mM) and GAWDA® formulation at (1, 2, 3 and 4 g L–1) significantly reduced the linear growth of F. oxysporum, F. solani, R. solani and M. phaseolina isolated from cucumber roots. It was also noticed that the reduction in the linear growth was correlated to the increase in concentration of the tested antioxidants.
The effect of T. viride, T. hamatum, T. harzianum and G. virens on the mycelial growth of F. oxysporum, F. solani, R. solani and M. phaseolina is shown in Table 4.
| Table 2: | Pathogenicity of the isolated root rot and wilt fungi in cucumber and percentage of their severity |
Trichoderma harzianum presented the highest antagonistic effect (100%) on the mycelial growth of R. solani and M. phaseolina while least inhibition percentage was shown from T. viride on the linear growth of R. solani (76.8%).
On the other hand, soaking cucumber seeds in salicylic acid at concentrations of (2, 4, 6, 8 mM), tartaric acid at concentrations of (5, 10, 15, 20 mM) as well as GAWDA® formulation at concentrations (1, 2, 3 and 4 g L–1) for 12 h significantly increased the percentage of seed germination (Table 5).
The results also show that the combination of T. viride, T. hamatum, T. harzianum and G. virens and salicylic acid at 4 mM, tartaric acid 10 mM or GAWDA® formulation 1 g L–1 did not show any adverse effect on the linear growth of Trichoderma spp. used in this investigation (Table 6 and Fig. 1).
The triple combination consists of compost, Trichoderma and antioxidants significantly reduced the percentage of damping-off syndrome. Precisely the combinations of (compost, salicylic acid and T. viride or T. hamatum or T. harzianum or G. virens) or (compost, tartaric acid and T. harzianum or G. virens) as well as (compost, GAWDA® formulation plus T. harzianum or G. virens) were highly effective on reducing the incidence of damping-off as shown in Table 7.
| Table 3: | Effect of different concentrations of antioxidants on the linear growth of the tested pathogenic fungi attacking cucumber plants |
| *Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p = 0.05 | |
| Table 4: | In vitro effect of Trichoderma spp. on the linear growth of the tested fungi |
| *Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p = 0.05 | |
The impact of compost, Trichoderma and antioxidants solely or in their combinations on the content of photosynthetic pigments and the total phenols in leaves of cucumber plants revealed that the triple combination of compost, salicylic acid at 4 mM and T. harzianum presented a significant increase in the content of chlorophyll A (1.905 mg g–1), chlorophyll B (0.964 mg g–1), total chlorophyll (2.869 mg g–1) and carotenoid (0.296 mg g–1) as well as the total phenols to present a rate of (153.3 mg g–1 fresh weight) (Table 8).
It was also found that the combination of compost and salicylic acid at 4 mM in the presence of T. harzianum present a significant increase in the content of total carbohydrate (51.2%), ascorbic acid 45.4% (16.00 mg/100 g fresh weight). Firmness 60% (16 lb/in2). Moisture was reduced by 1.56% and the total soluble solids by 32% as shown in Table 9. The acidity reduced by 42.8% to record 1.12% when compare with the check (Table 9).
This triple combination also showed a significant increase in the yield 66.6%, as the number of fruits/plant increased by 220%, fruit length by 61% and the fruit diameter was reduced by 40% as shown in Table 10.
In this study 4 fungal isolates i.e.,: F. oxysporum f. sp. radicis-cucumerinum, F. solani, R. solani and M. phaseolina obtained from wilted roots of cucumber plants grown in fields at different areas in Dakahlia and Damietta Governorates.
| Table 5: | Effect of different concentrations of salicylic acid, tartaric acid and the formulation of GAWDA® on germination of cucumber seeds in vitro |
| *Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p = 0.05 | |
| Table 6: | Effect of the tested antioxidants on the linear 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 | |
| Fig. 1(a-d): | Effect of the tested antioxidants on the linear growth of Trichoderma spp (a) Trichoderma viride, (b) Trichoderma hamatum, (c) Gliocladium virens and (d) Trichoderma harzianum, 1: Check, 2: Salicylic acid (4 mM), 3: GAWDA® (1 g L–1) and 4: Tartaric acid (10 mM) |
| Table 7: | Effect of the triple combinations of the antioxidants, Trichoderma and compost on the percentage of damping-off of cucumber seedlings under field conditions |
| *Means followed by different letter(s) in the column are significantly different according to Duncan's multiple range test at p = 0.05 | |
These fungi were purified and identified based on the identification keys while the pathogenicity tests revealed that all tested isolates have potential to attack cucumber roots cv. Beta alpha hybrid and cause a syndrome of damping-off, root rot and wilt, revealing a reduction in the number of healthy plants. These results are similar to those obtained by Morsy et al. (2009) and El-Mougy et al. (2012) who reported that F. oxysporum, F. solani, S. rolfsii, R. solani and M. phaseolina were associated with cucumber damping-off and Root Rot diseases.
The results also perform the role of the tested antioxidants i.e.,: Salicylic Acid (SA), tartaric acid, GAWDA® formulation as well as the antagonistic fungi T. viride, T. hamatum, T. harzianum, G. virens as potential triple combination for suppressing root rot and wilt pathogens of cucumber. The results are also in agreement with the finding of Abdel-Monaim (2013) who reported that salicylic acid and T. viride either individually or in combination inhibited the growth of pathogenic fungi attacking roots of faba bean. Ismail (2006) found that tartaric acid at 10 mM reduced linear growth of fungi causing damping-off, root rot, wilt in sesame (Sesamum indicum L). El-Metwally (2005) reported that GAWDA® formulation reduced the linear growth of the common seed borne fungi on Peanut. Hassan et al. (2014) reported that T. harzianum significantly reduced the linear growth of pathogenic fungi causing root rot and wilt diseases in Roselle. Mosaad (2008) reported that T. harzianum, G. virens reduced the radial growth of pathogenic of cucumber. Verma et al. (2007) reported that Trichoderma spp. is widely used as antagonistic fungal against several pests and enhance the plant growth. All these results support the finding of the authors.
However, the results presented here revealed that the suggested and tested triple combination of T. viride, T. hamatum, T. harzianum and G. virens and salicylic acid (4 mM), tartaric acid (10 mM) and GAWDA® formulation 1g L–1 did not present significant differences in their effect on linear growth of the root rot and wilt fungi of cucumber when compared with the solely uses of each one. Accelerate seed germination was also found when soaking seed in one of the tested antioxidants (4 mM of salicylic acid, 10 mM of tartaric acid or 1 g L–1 of GAWDA formulation). These results are also in a harmony with those obtained by El-Mougy et al. (2004) who reported that no significant reduction in seed germination was observed when lupine seeds were treated with SA and ASA up to 2 and 3 g kg–1, respectively.
The in vivo results insured that the triple combination of compost+salicylic acid 4 mM+T. harzianum has a significant impact on damping-off of cucumber seedlings under the natural field condition, significant increase in the content of the photosynthic pigment and the total phenols in the cucumber plants. Also improved fruit quality including total carbohydrates, ascorbic acid, firmness, moisture, TSS and acidity %. A significant increase in the yield (g)/plant, number of fruits/ plant, fruit length and fruit diameter were significant. In conclusion it is recommended to use a triple designed formulation of Salicylic Acid (SA) at 4 mM and T. harzianum for treating seeds following by sawing in soil supplemented with compost at a rate of (2 t/fed).
| Table 8: | Effect of the triple combinations of the antioxidants, Trichoderma and compost on the content of the photosynthetic pigments and the total phenols in the upper leaves of cucumber plants |
| *Means followed by different letter(s) in the column are significantly different according to Duncan's multiple range test at p = 0.05 | |
| Table 9: | Effect of the triple combinations of the antioxidants, Trichoderma and compost on the fruit contents of total carbohydrates, ascorbic acid, firmness, moisture, total soluble solids and acidity |
| *Means followed by different letter(s) in the column are significantly different according to Duncan’s multiple range test at p = 0.05, TSS: Total soluble solid | |
| Table 10: | Effect of different combinations of the antioxidants, Trichoderma and compost on Yield (g)/plant, number of fruits/plant, fruit length and fruit diameter |
| *Means followed by different letter(s) in the column are significantly different according to Duncan's multiple range test at p = 0.05 | |
This tactic is an intelligent method to overcome the incidence of root rot and wilt disease of cucumber and improve its yield and quality of the fruits. It is also recommended for the growers to follow it for increasing their income and market competition. These results are also in agreement with the finding of Abdel-Kader et al. (2013) who reported that combined treatments (compost+T. harzianum+ thyme or compost+T. harzianum+lemongrass) were effective in controlling peanut crown rot disease under field conditions and reduced the disease incidence in both pre and post-emergence growth, respectively. Abo-Elyousr et al. (2014) reported that under greenhouse and field conditions, T. harzianum (isolates No. 1 and 2) and compost individually or in combination were effective in controlling Rhizoctonia root rot disease and showed a suppressive effect on severity of the disease and increased the yield of soybean. On the other hand, Elwan and El-Hamahmy (2009) found that spraying salicylic acid at a rate of (10‾6 and 10‾4 M) was a promising compound to control pepper diseases. This application at a low concentration (10‾6 M) positively increased the foliage fresh and dry weight, fruit number, average fruit weight, fruit yield, vitamin C, carotenoids content, cuticle thickness of fruit pericarp and translocation of sugars from leaves to fruits.