Source of the causal pathogen of soybean root rot
The highly pathogenic isolates of Rhizoctonia solani used in this study were previously isolated and tested for pathogenicity by (Sallam Nashwa et al. 2008) who identified the isolates using the morphological features of mycelia, as described by Barnett and Hunter (1986). Pure fungal isolates were then grown on potato dextrose agar (PDA) slants at 25±2 °C, then kept in a refrigerator at 4 °C for further studies.
Isolation of endophyte fungi from soybean plants
Healthy roots of soybean plants were collected, washed with tap water, and cut into small pieces, which were then surface sterilized in 2% sodium hypochlorite for 3 min and 70% ethanol for 3 min, washed with sterile distilled water 3 times, and left to dry in a laminar flow chamber. The pieces were transferred to a Petri dish containing PDA medium and incubated at 27±2 °C for 10 days. Pure cultures were transferred onto PDA slants.
Antagonistic capability of various endophytic fungi isolates against Rhizoctonia solani
The five tested endophytic fungi isolates were grown on PDA medium and incubated for 5 days at 28 °C for use as inocula. PDA Petri plates (9 cm in diameter) were inoculated at 3 cm from the edge with disks of R. solani (5 mm in diameter), followed by inoculation with the tested fungi on opposite sides of R. solani. Four replicates were used for each treatment. The control treatment consisted in plates with R. solani alone, without the inoculation of endophytic fungi. The plates were incubated for 7 days at 28 °C. When the growth of the pathogen was covered, the plate in control treatment in the linear growth of the tested pathogen was recorded in the treatments fungi (Zein El-Abdean et al. 2013). The percentage of mycelial growth inhibition was calculated using the following formula: percentage of mycelial growth inhibition = [T–F/T] × 100, where T is the mycelia growth in the control and F is the mycelia growth in the tested isolate. The antagonistic fungi that afforded a high percentage of mycelial growth reduction were identified using a 28S rRNA molecular method.
Identification of Trichoderma spp. using polymerase chain reaction nucleotide sequencing (PCR-Seq)
Three isolates were used in this study. The nuclear rDNA region spanning the ITS1 ITS2 regions was used for the first amplification, which was performed in a total reaction volume of 50 μl including the following reagents: PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin), 200 μM of each deoxyribonucleotide triphosphate, 0.4 μM of each primer with 10 μl of the template DNA solution, and 1 U of Tth DNA polymerase (Toyobo). The PCR mixture was overlaid with 30 μl of mineral oil. The following thermal cycling was performed on a thermal cycler (PC-700; ASTEC): an initial denaturing step at 95 °C for 2 min; 30 cycles of 30 s at 72 °C for extension; and a final extension cycle of 7 min at 72 °C. One microliter of the first amplification mixture was used for the second amplification using the nested primer set ITS1 (White et al. 1990) and P3. The components of the reaction mixture and the thermal cycling conditions used for the second amplification were the same as those used for the first one. The PCR products from the second amplification were subjected to preparative electrophoresis in 1.5% agarose gels in Tris acetate EDTA (TAE) buffer. All amplifications yielded a single visible DNA product. The DNA product band was excised from the ethidium-bromide-stained gel and purified using a JETSORB kit according to the manufacturer’s protocol. Direct sequencing of PCR products was performed on an Applied Biosystems 373A sequencer using a PRISM Dye Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer’s protocol and using the ITS1 and ITS2 primers (White et al. 1990). The nucleotide sequence data of the ITS2 and ITS2 regions were subjected to pairwise alignment via the method of Lipman and Pearson (1985) using the program GENETYX-MAC (Software Development).
Evaluation of the antagonistic effect of fungi under greenhouse conditions
The inoculum was prepared in bottles containing barley grain medium using disks (5 mm) of R. solani isolates. The bottles containing barley medium were autoclaved at 121 °C and 1.5 kg/cm2 for 20 min before inoculation with the pathogen. After inoculation, the bottles were incubated at 25±2 °C for 15 days. For soil infestation, R. solani barley grains were mixed with sterilized soil at a concentration of 3%. Four replicates were used for each tested isolate (5 seedlings/pot). Pot experiments were carried out to study the effects of the 3 selected antagonistic fungi, for controlling the root rot incidence in soybean plants. Seeds of the Giza 111cv. soybean cultivar were sown in R. solani-infested soils as described above (5 seeds/pot), and the antagonistic fungi were applied as a soil treatment by adding a 3% solution to pots 7 days after infestation with R. Solani. Untreated pots served as the control. At 30 days after sowing, root rot disease severity was recorded as described previously (Abo-Elyousr et al. 2014b). The experiments were arranged in a randomized complete block design with 4 replicates. Disease severity percentage (DS%) was estimated as follows: DS% = [ΣA/A (4T)] × 100
Where A is the disease rating on each plant, d max is the maximum disease rating possible, and 4T is the total number of plants (T) multiplied by the maximum discoloration grade 4. According to Dorrance et al. (2003), with slight modifications, the different degrees of disease in plants were classified into 4 categories: 0 = no root rot; 1 = 1 to 33% of roots with visible lesions or root rot; 2 = approximately 34 to 50% of the roots exhibited rot or damage; 3 = 51 to 80% of the roots exhibited rot; and 4 = pre-emergence damping-off and few if any roots.
Depiction of secondary metabolites and antifungal activity of endophytic fungi
Solubilization of minerals
Pikovskaya medium was used to test the phosphate-solubilizing ability of the fungi isolates by dissolution of precipitated tricalcium phosphate Ca3 (PO4)2. The fungi isolates were inoculated on the surface of PDA medium dried plates. The plates were incubated for 7 days at 28 °C. The solubilization index (SI) was calculated according to Edi-Premono et al. (1996).
As reported by Saravanan et al. (2003, b), the zinc-solubilizing ability of the tested fungi was detected based on the dissolution of precipitated zinc oxide on agar medium. On the surface of dried plates, a pinpoint inoculation of the fungal isolates was performed. Plates were incubated for 7 days at 28 °C. The SI was calculated as reported by Edi-Premono et al. (1996), as follows: ratio of the total diameter (colony diameter + halo zone diameter)/colony diameter.
Production of specific enzymes by the bioagent
Using the disk plate method, the enzymatic activities of potent fungal bioagent were visualized on agar plates containing specific enzyme substrates (Acuna-Arguelles et al. 1995). These pathogens were screened for multiple enzymes, such as cellulases, pectinases, proteases, and amylase, on suitable substrates. The size of the clearing zone that developed around the colonies corresponded to the enzymatic activity.
The tested fungal culture was inoculated on Czapek agar medium containing 2 NaNO3; 1.0 K2HPO4; 2 peptone; 0.5 MgSO47H2O; 0.5 KCl; and 20 agar (g/l) in 1000 ml of distilled water at pH 6.8 and enhanced with 1% citrus pectin as a substrate for pectinase. The Petri dishes containing screening agar medium were incubated for 24 h at 30 °C. One percent cetyl trimethyl ammonium bromide was added to observe the zone of clearance, which revealed the pectinase activity.
Medium containing carboxy methyl cellulose (CMC) (1% w/v) was used to detect the cellulase activity. The Petri plates were incubated for 24 h at 27 °C. After inoculation, the plates were screened for cellulase activity by flooding the plate with 1% Congo red solution for 15 min, followed by de-staining using a 1 M NaCl solution for 15 min. The clear zones around the growth of fungi indicated cellulase activity.
Medium containing starch (1% w/v) as a substrate was used to detect amylase activity. Plates were incubated at 27 °C for 24 h. The clear zones around the fungal growth were observed by staining the plate with 50 mM iodine.
Medium containing casein (1% w/v) as a substrate was used to test protease activity. Enzymatic activity was determined after the inoculation of the plate with the tested fungi. The formation of a clear zone around colonies after precipitation with 1 M HCl solution indicated protease activity (Rodarte et al. 2011).
Production and assay of indole acetic acid
The fungi strains were inoculated individually in 250 ml Erlenmeyer conical flasks containing 50 ml of potato dextrose broth medium supplemented with 0.4% tryptophan, as a precursor. The inoculated flasks were then incubated in agitated conditions of 150 rpm for 7 days. After incubation, the fungal growth was decanted by centrifugation at 10,000 rpm for 10 min and the cell-free supernatants were used as sources of indole acetic acid (IAA). The reaction mixture consisted of 1 ml of the cell-free supernatant and 2 ml of the Salkowski reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35% perchloric acid). The mixture was then incubated at 28 °C for 30 min. Quantification was performed calorimetrically at 530 nm by comparison to an IAA standard curve (Gordon and Paleg 1957).
Qualitative estimation of siderophores
The production of siderophores by fungi isolates was assayed via the plate assay method, as described by Schwyn and Neilands (1987). After growth, fungal isolates (a 5-mm disk of each isolate) were potted on Chrome Azurol S blue agar plates and incubated for 48 h at 28±2 °C. The development of a yellow-orange zone around the colony was taken as a positive indication of siderophore production. The extent of siderophore biosynthesis was measured as the diameter of the colored zone (Alexander and Zeeberi 1991).
Data were subjected to statistical analysis using the MSTATC program of variance, and means were compared using the least significant difference (LSD) test at P ≤ 0.05, as described by Gomes and Gomes (1984). The LSD at 5% probability was used for testing the significance of the differences among the mean values of the tested treatments.