2.1. Plant resources were utilized in the experiment:
2.1.1. Faba bean seeds source:
Giza 716 seeds were obtained from the Legume Crop Research Department, Field Crops Research Institute, Agricultural Research Center (ARC), Giza, Egypt.
2.1.2. Biological antagonists:
Trichoderma album, T. atrovirde, T. hamatum and T. harzianum (30x106 spore/ml) were four different biocontrol agents that was added at the rate of 1 Lit./50 Lit. water and were graciously donated by Biological Control Production Unit Central Lab. of Organic Agriculture, CLOA; ARC.
2.1.3. Biocide preparations:
There are some biocide formulations were used as a comparison with other treatments as follow:-
- Blight Stop was recommended as biocide preparation which contain wild fungal isolate (Trichoderma harzianum 30x106 spore/ml) that is added at the rate of 1 Lit./50 Lit. water and were kindly provided by Biological Control Production Unit Central Lab. of Organic Agriculture, CLOA; ARC.
- Bio Zeid 25% WP, which was provided by kz, was suggested as a biocide preparation. It contains the wild fungus isolate "T. album 2.5% (w/w)" and is added at a rate of 250g/100 L water.
2.2. Chocolate spot fungal pathogen isolation and identification:
Leaf samples of faba beans infested naturally were collected from a field in Nubaria City, EL-Behira Governorate, Egypt. The infected leaves were collected, cut into small pieces, and surface disinfected with 3% sodium hypochlorite for 2 minutes before being repeatedly washed in disinfected distilled water. The pieces were dried between a pair of sheets of sterilized filter paper to get rid of excessive distilled water before plating on faba bean leaf extract agar medium "FBLA" as suggested by Hanouike and Hasanain (1986). Three slices were placed in each Petri dish, and they were incubated at 20 °C for 12 days. according to Sinclair and Dhingra (2019), the hyphal tip approach was used to purify the isolated fungus.
2.3. Antagonistic effect of different antagonists against Botrytis fabae:
The antagonists suspensions were added to warm sterilized PDA medium at a 10% concentration and placed into Petri dishes (10 ml/plate) before solidification. A disc (5 mmØ) of B. fabae, taken from the periphery of a mycelium that was 7 days old on the same medium, was put in the center of each plate after it had solidified. Plates containing media without antagonists and inoculated only with B. fabae served as control treatment. Three plates were used for each treatment. Inoculated plates were incubated at 22±2 °C. The experiment was terminated when mycelial mats covered the surface in the control treatment, all plates were examined and the percentage reduction in mycelial growth of the fungus was calculated using the formula suggested by Ahmed (2005) and Ahmed (2013) as following:
Where: G1: pathogenic fungus growth in the control only, G2: growth of the pathogen against the tested antagonists
2.4. Assessments of Botrytis fabae isolates under greenhouse conditions:
In order to produce a high quantity of spores, Botrytis fabae isolates from Nubaria City were propagated on "FBLA" medium for 12 days at 22 °C with a photoperiod of 12 h light/12 h dark as described by (Haggag et al., 2006). Five seeds of faba bean cv. Giza 716 were planted in pots (30 cm in diameter, three replicates of each cultivar). In order to preserve high relative humidity, the established plants were sprayed with the appropriate isolate's spore suspension 45 days after sowing and covered with polyethylene bags for 24 hours (Barakat et al., 2014). In this regard, biological control treatments were sprayed at the suggested concentrations five days after the pathogen was artificially inoculated. The following parameters were taken into account after the experiment:
a. Disease incidence:
Ahmed (2005) and El-Shennawy, 2011 used the following formula to calculate the percentages of disease incidence and survived plants in each treatment.
b. Disease severity (DS %):
The disease severity of chocolate spot was determined after two weeks from inoculation when the initial symptoms appeared using the 0–9 scale described by Ding et al. (1993), where 0= no visible leaf infection, 1= less than 10% infection, 2= less than 20% infection, 3= less than 30% infection 4= less than 40% infection, 5= less than 50% infection, 6= less than 60% infection, 7= less than 70% infection, 8= less than 80% infection and 9= infection more than 80% of the foliage. Disease severity scores were converted to percentage severity index (DSI) for analysis using the following formula developed by (Kora et al., 2017).
Where n = Number of plants in each category; v = Numerical values of symptoms category; N = Total number of plants; 9 = maximum numerical value of symptom category
2.5. Field trials:
Field trials were carried out during two winter growing seasons (2021/22 and 2022/23) to estimate the efficiency of different antagonists for controlling chocolate spot of faba bean plants in naturally infested farm have history of high infestation with Botrytis fabae which located at Nubaria city, EL-Behira Governorate, Egypt. Soil texture is sandy loamy. Nile water is available in this area with drip irrigation system. Three replicated plots for each biological treatment in addition to a control were used in a complete randomized block design for all of the trials, and the experimental plot's surface area was 24.75 m2, comprised of 3 ridges (5m×1.5 m width) with about 50 cm apart. Each row was sown with 25 seeds of faba bean cv. Giza 716 on 15th October of both seasons (Zewdineh et al., 2022). All faba bean cultivars received the same organic fertilizers and irrigation regime. The faba bean plants were sprayed after 45 days from sowing at the beginning of the flowering stage, the second one was 60 days after sowing in the end of flowering stage with suspensions of the four bioagent isolates— Trichoderma album, T. atrovirde, T. hamatum and T. harzianum (30x106 spore/ml), as well as with the two commercial biocide products, including Blight Stop and Bio Zeid at the recommended doses as mentioned above. Super film as a surfactant and sticker material, was mixed with each treatment prior to spraying at a rate of 50 ml/100 L water. Plots that had not been treated (only water sprayed) served as the control.
2.5.1. The following characteristics were evaluated:
a. Disease parameters:
The percentages of disease incidence (DI) and severity (DS) were calculated as mentioned above, in addition, the efficacy of the tested treatments was estimated using the following equations:
b. Yield components traits:
The plants were collected by hand approximately 150 days after seeding and left to dry for 5 days under natural conditions before the following parameters were examined; The number of pods per plant, Weight of pods / plant (g), the weight of 100 grains (g) in each plot and yield/plot (Kg) are all recorded. In addition, the percentage increase in all parameters was determined using El-Kholy's (2014) formula:
c. Chemical components determination:
All the following chemical assays for the faba bean plants were carried out in both Central Laboratory of Organic Agriculture and Central Lab. for Agricultural Climate, Agricultural Research Center (ARC), Giza, Egypt.
1. Protein content determination:
The micro-Kjeldahl method was used to determine the total nitrogen in the seed and multiplied by 6.25 to obtain the percentage to crude according to AOAC (2005).
2. Determination of Chlorophyll (SPAD value):
At the flowering stage, after 75 days from sowing, the Chlorophyll content was measured by SPAD - meter Model L13000L (Ling et al., 2011)
3. Determination of total phenols:
The amount of total phenols in extracts was determined by Folin – Ciocateu method as modified by Singleton et al. (I999).
4. Determination of total flavonoids content:
The flavonoid content is estimated in milligrams of rutin equivalents per gram of the digested sample (mg RE/g) as described by (Rice-Evans et al., 1996) method.
5. Enzymes determination:
5.1. Determination of peroxidase (PO):
Faba bean leaf samples were weighed from 0.1-0.5 g and then stored at 20 °C until processed as described by Ni et al. (2001). The activity of the peroxidase enzyme was estimated accordance to the instructions provided by Devi (2000).
5.2. Determination of polyphenol oxidase (PPO):
The activity of polyphenol oxidase was estimated according to Devi (2000).
5.3. Determination of chitinase:
Chitinase enzyme activity was assessed by procedure of (Boller and Mauch, 1988). Chitinase activity was measured as mM N-acetylglucose amine equivalent released/g fresh weight tissue/60 minutes.
5.4. Determination of β-1,3-glucanase:
β-1, 3-glucanase enzyme activity was determined as described by Sun et al. (2006) and estimated to be expressed as glucose equivalent mM/g fresh weight tissue/60 minutes.
d. Statistical analysis:
All the collected data were statistically analyzed and contrasted using the least significant difference (L.S.D.) as mentioned by Snedecor and Cochran (1989).