Root-rot and Stem-canker Control in Faba Bean Plants by Using Some Biofertilizers Agents

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Introduction
Since the early 1990s, induction of systemic resistance by plant growth promoting rhizobacteria (PGPR) has been investigated as a possible practical way to use induced resistance in agriculture. PGPR have been tested in the green house and field for induced systemic resistance (ISR) to fungal [1,2], pathogen in various crops such as bean, carnation, cucumber, radish, tobacco, tomato and Arabidopsis. The use of plant growth promoting rhizobacteria (PGPR) isolated from cauliflower root, Pseudomonas fluorescens SP007s as biocontrol agent in protecting various plants from several diseases caused by bacteria and fungi have been reported for multiple studies [3]. Marleny et al. [4] found that plant growth promoting rhizobacteria (PGPR) are beneficial bacteria that colonize the rhizosphere and plant roots resulting in enhancement of plant growth or protection against certain plant pathogens. One practical challenge to implementing this approach is establishing beneficial microbial communities, such as plant growth promoting rhizobacteria (PGPR) to promote soil ecosystem health that contribute to suppression of plant pathogens and other pests [5,6]. Gasoni et al. [7] showed that bacteria belonging to Pseudomonas and Bacillus genera have been used as biocontrol agents. Yehia et al. [8] proved antagonistic effect of Trichoderma virdie against Fusarium solani of faba bean. Seed coating with Trichoderma virdie increased fresh and dry weight of shoots, roots and nodules number. The population densities of fungi (including Fusarium spp.,) were low in plants obtained from treated seeds. Nelsson [9] reported that Trichoderma spp., are specific biocontrol agents against fungal pathogens (from Pythium to Rhizoctonia) according to the type of antibiotic produced. Under field conditions, Ehteshamul and Ghaffar [10] observed the antagonistic rhizobia and bradirhizobia used as seed dressing or soil drench reduced infection of Rhizoctoia solani in both leguminous and no leguminous plants. Zheng and Sinclair [11] showed that Bacillus megaterium is a potential bacterial biocontrol agent against Rhizoctonia solani. Lewis and Lumsdent [12] cleared that T. harizianum and T. virdie isolates reduced damping off of different plants caused by isolate R-23 of R. solani. Jensen et al. [13] evaluated the effect of Bacillus subtils and T. harizianum alone or in combination with Captan 400 and Vitavax 200 as biocontrol treatments against the dry bean root rot pathogens. They also recorded that seed application of both biocontrol agents increased plant biomass and decreased disease severity, under greenhouse conditions. Field experiments showed that seeds treated with B. subtilis reduced bean root rot and increased yield (31%) when compared with untreated control. Maria and Joseph [14] showed that a Trichoderma harizianum strain was antagonistic; In Vitro; to Rhizoctonia solani and Verticillium dahlia and may be considered a potential biocontrol agent.
Over the last few decades, consumers demand for healthier food and governments policies focused on environmentally sustainable agricultural systems have both promoted a rapid expansion of organic farming [15,16]. Organic food production is characterized by the absence of synthetic compounds (herbicides, pesticides) [17]. Manach et al. [18] reported that nowadays, emphasis multi strains biofertilizer has already been tidied. Biofertilizers are biological preparations embodying, essentially, sufficient densities of potent strains of microorganisms, having a tangible beneficial role in filleting a proper rhizosphere for plant growth [19]. Organically grown cabbage, spinach, welch, union, green pepper generally had higher levels of flavonoids and antioxidants activity [20]. Dinitrios [21] showed that the health benefits of fruits and vegetables are largely due to the antioxidants and vitamins supported by the large number of phytochemicals, some with greater antioxidant properties. Also, Asami et al. [22] mentioned that phenolic and ascorbic acids are presented in higher levels in organic corn, strawberry and marine berry than in conventional. Dave et al. [23] found that there was quantitative increase in total phenol, total protein and major three fatty acids after treatment. Trichoderma also observed to introduce resistance capacity of Indian mustard plants. Some Trichoderma rhizosphere competent strains has been shown to have direct effects on plants, increasing their growth potential and nutrient uptake, fertilizer sufficiency, percentage and rate of seed germination and stimulation of plant stimulation of plant defenses against biotic and abiotic damage [24]. In cucurbits it has been found that ISR induction was correlated to the up-regulation of different pathogenesis related (PR) and defense related proteins (chitinases, glucanase, peroxidases and specific phytoalexins) and enzyme activities, especially phenylalanine ammonia lyase and synthesis of other phenols and related proteins [25].
The aim of this work is to study the effect of some biofertilizers and biocontrol agents either single or in combination applications on faba bean plants cultivated in soil infested with Rhizoctonia solani.
In order to isolate the causal organism of stem canker and root rot disease; infected faba bean plants were collected from different cultivated areas at Sadat City. Roots and stem bases of the obtained samples were separately washed by running tap water, surface sterilized by 70% Ethanol and then left to dry on sterilized filter papers. The samples were cut into small pieces, plated on PDA medium and incubated at 25ºC. Petri dishes were examined daily and hyphal tips were individually transferred to new PDA plates. Some other root samples were used for isolation of the outer microorganism "without surface sterilization". Obtained isolates were identified at the Agric. Botany Dept., Fac., of Agric., Min., Univ., Shibin El-kom, Egypt.

Pot experiments
Pots (20 cm in diameter) were sterilized by immersing them in 5% Clorox for 15 min. and then left to dry in open air. Non-sterilized sandy-loam soil of Sadat City mixed thoroughly with peat moth at the rate of 1:1 were left for a week in the open air before using in this experiment.
For Rhizobium inoculation; seeds were immersed in sugar solution as an adhesive material (prepared by dissolving 20 gm. of sugar in 100 ml water) Treated seeds mixed thoroughly with the "Okadeen" biofertilizer and left for 30 min. in a shadow place for drying before cultivation.
Bacillus isolate was grown on Nutrient Broth medium for 48 hrs on a rotary shaker at 25ºC. The bacterial inoculum was applied as a soil treatment at the rate of 5 ml bacterial suspension per plant (1 10 8 cfu/ ml).
However; application of fungal isolates was carried out on Barley medium at the rate of 3% of soil weight. A disc (4 mm in diameter) from the edge of 6 days old fungal culture was added to 200 g sterilized Malt medium (75 g malt+25g soil+100 ml distilled water) and incubated for 7 days at 25ºC. The inocula were mixed with the soil at weight. The pots were watered daily for 7 days before cultivation.

Chemical analysis
Total nitrogen content: The determination of total nitrogen was carried out with Micro-Kjeldahl method. [26]. Oh point five grams of dried and finely ground shoot and root sample was taken in a Kjeldahl flask. Three g of digestion mixture (H 2 SO 4 + K 2 SO 4 ) in the ratio of 1:9 was added and followed by 20 ml of H 2 SO 4 . The sample was boiled in digestion apparatus for 1.5-2 hrs .until the contents became clear. The digested material was cooled and diluted up to 250 ml in a volumetric flask by adding distilled water. An aliquot 10 ml of it was transferred to the micro Kjeldahl distillation apparatus. It was mixed with 10 ml of 40 % NaOH and distilled in a receiver containing 10 ml of 2 % boric acid solution with methyl red as indicator. The contents of the distillate were titrated against standard sulfuric acid (N/10 H 2 SO 4 ) to light pink color end point. From the volume of acid used, percentage of nitrogen was calculated based on ammonia liberated.

Determination of total phenolic content (TPC):
The total phenolic content (TPC) was determined by the Folin Ciocalteu method [27] using spectrophotometer (UV-200-RSLW scientific). Distilled water (3.16ml) was mixed with the 40 µl of sample, and then 200 µl of Folin Ciocalteu reagent was added. After 5 min, 600 µl of 20 % sodium carbonate solution was added and solutions were mixed again. The solution was left at room temperature for 2 hrs.
The color intensities were measured at wave length 750nm. TPC expressed as grams of Gallic acid equivalents per 100g plant.

Antioxidant capacity (DPPH Assay):
The free radical scavenging activity was estimated by 1, 12-picryl--diphenyl-hydrazyl (DPPH) assay using [28]. The reaction mixture contained 100 μl of test extracts (100-500 μg/ml) and 1 ml of methanolic solution of 0.1 mM DPPH radical. The mixture was then vigorously shaken and incubated at 37ºC for 30 min. The absorbance was measured at 517 nm using ascorbic acid (100-500 μg/ml) as positive control. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity which was calculated using the following equation: DPPH scavenging effect (%) = 100 x (Ao-A1)/(Ao) Where: Ao is the absorbance of the control reaction and A1 is the absorbance of reaction mixture containing DPPH and extract at 517 nm.
The antioxidant activity of the extract was expressed as IC50 value which is defined as the concentration (μg/ml) of extract that inhibits the formation of DPPH radicals by 50%. This was obtained from linear regression analysis.

Statistical analysis
Data were statistically analyzed by analysis of variance (ANOVA) using the Statistical Analysis System [29]. Means were separated by

Results and Discussion
Under green house and artificial inoculation condition; results present in Table 1 clear that Rhizoctonia solani caused emergence damping off of all seeds of each faba bean cultivar. However; the untreated control pots resulted 90% emerged and survived plants. These results clear that Giza 3 Mohassan faba bean cultivar are highly susceptible to R. solani.
Application of the tested rhizosphere microorganisms to the potted soil infested with R. solani showed good control to the pathogen. The best was achieved with Tricoderma harizianum where all the seeds germinated and gave 100% plant survival. Bacillus megaterium resulted 70% germination and 92.9% of them plant survival. Rhizobium leguminosarum showed the least antagonistic effect to R. solani where 65% of the seeds emerged and 69.2% of them survived. The same results were achieved when the tested three microorganisms together were applied to R. solani infested soil.
Application of the beneficial tested microorganisms gave good results in controlling R. solani. This could be due to their antagonistic effect as reported by Gasoni et al. [7], Nelsson [9], Ehteshamul-Shaque and Ghaffar [10], Zheng and Sinclair [11], Lewis and Lumsent [12], Jensen et al. [13] and Santamarina and Joseph [14]. These results also were reported by Chen et al. [1], Liu et al. [30] and Pietrse et al. [31] who showed that (PGPR) has been investigate as a possible practical way to use induced resistance to fungal pathogens in various crops. However Nelsson et al. [9] reported that Tricoderma spp., are specific biocontrol agents against Rhizoctonia solani. Table 2 indicate that there was no significant variation between the average number of leaves formed on faba bean plants up to 30 days of sowing in response to different biocontrol agents application. However, Tricoderma harizianum caused significant increase of leaves number as compared with Bacillus faba megaterium 35 days after seeding. The result was noticed for T. harizianum and Rhizobium leguminosarum 40 days after planting, where they result significantly more number of leaves than control and/or Bacillus megaterium treatment.

Results shown in
The same table also showed that faba bean plant height has been affected by soil infestation with Rhizoctonia solani, even in the presence of the significant higher plants. These results have been shown as early as 20 days and stayed up to 40 days of sowing. Generally, application of either Tricoderma harizianum or Rhizobium leguminosarum to the soil infected with R. solani showed the best plant growth. While application of Bacillus megaterium and/ or the three biocontrol agents resulted the worst results of plant height. Table 3 clear that flowers number of faba bean plants didn't affect by various beneficial biocontrol agents. There were no significant differences between the averages numbers of flowers emerge on the plants of various treatments up to 45 days after seeding. It is of interesting to notice that individual treatment with Rhizoctonia solani resulted complete pre-emergence damping-off. The same table showed that average number of nodules significantly decreased in response to the soil infested with R. solani even in the presence of biocontrol agent(s). This was noticed in comparison with the non-infested control soil. However, number of Rhizobium leguminosarum nodules was the worst when the three microorganisms were applied to the soil infested with R. solani. This could be due to the antagonistic effect(s) of the tested microorganisms to R. solani as mentioned before. On the other hand, the best number of nodules was achieved when Bacillus megaterium applied to the infested soil. Table 4 indicate that the average total length of faba bean plants severely affected with soil infestation with Rhizoctonia solani. Control plants, sown in sterilized soil, gave the best plant length (58.0cm) after 45 days from seeding. Complete death was obtained when Giza 3 Mohassan faba bean cv. was seeded in R. solani infested pots. Significant reduction of total plant length was noticed in all treatments contained R. solani an one or all of the beneficial soil **ND = Not determined due to total damping-off seedlings Shoot system fresh weight of Giza 3 Mohassan cv. Plants was significantly less than control in response to soil infestation with Rhizoctonia solani, in most cases ( Table 5). The most fresh weight reduction of shoots was achieved with application of the three microorganisms to the infested soil with R. solani 4.5/8.8%). However, the competitive saprophytic ability could minimized the antagonistic role of each tested biocontrol agents itself [32]. Bacillus megaterium application caused (50.5%) reduction in fresh weight. Both mentioned treatments also resulted significant reduction in roots fresh weight (6/12.4 and 5.8/12.4% respectively). Results shown in the same table nearly clear the same response of plants dry weight as found in fresh weight. These results are also reported by Yehia et al. [8].

Results in
Results present in Table 6 clear that application of Rhizobium leguminosarum to faba bean plants sharply increased nitrogen content in both of plant roots and shoots. This may be due to its antagonistic effects against Rhizoctonia solani. Tricoderma harizianum resulted 4.7% more nitrogen level when compare with the untreated control plants, while Bacillus megaterium gave the same result as the control. Application of the three microorganisms to the soil gave 23.7% more total nitrogen content of the whole plant than control. The same results were obtained with the protein content of the roots an shoots of plants. R. leguminosarum gave 156.2% more protein content followed by the application of the three microorganisms which increased the protein content by 148.12% while the application with T. harizianum gave 29.3% and B. megaterium gave the same result as the control. These results are in agreement with Dave et al. [23].
The results above clear the aggressiveness of tested Rhizoctonia    This pathogen caused complete pre-emergence dampping off all faba bean seedlings. It also affected the growth characters of those survive in the presence of the biocontrol agents, which has a good role in this investigations as reported by Marleny et al. [4] and Shoresh et al. [24] who showed that PGPR and Tricoderma spp., are beneficial microorganisms resulting in enhancement of plant growth against certain plant pathogens. Figure 1 show that the application of the three microorganisms gave highest amount of poly phenolic content (153.1%) followed by the treatment of Tricoderma harizianum (137.3%) and Bacillus megaterium (137.3%) in the roots of faba bean plants.
The results shown in Figure 2 illustrate that the phenolic content in the shoots are similar in the roots. Table 7 show that applicaton of Rhizobium leguminosarum + Bacillus megaterium to faba bean plants gave the best levels of antioxidants in shoots ( 90.89%) while application each of Tricoderma harizianum and Bacillus megaterium alone gave the same level(90.58%) of antioxidants in shoots Rhizobium leguminosarum gave 85.7%. The same results were obtained in roots (33.06, 31.01, 28.76 and 26.71%) for Rhizobium leguminosarum + Bacillus megaterium, Tricoderma harizianum, Bacillus megaterium and Rhizobium leguminosarum respectively. This result is in agreement with Ren et al. [20].