In vitro experiments
Isolation, purification, and identification of Fusarium oxysporum
Diseased tomato plants were collected from different regions of the Governorate of Elmenofya (Shebin El Kom, Kafer Dawood, and Bader), Egypt, showing various degrees of wilt symptoms. Infected roots were washed thoroughly with flowing tap water and cut into small pieces before plunging into sodium hypochlorite (0.5% chlorine) for 1 min. The surfaces were washed by distilled water, dried between 2 sterilized filter sheets, placed directly in Petri dishes on potato dextrose agar (PDA) medium, and incubated at 28°C for 3 days. Analysis was carried out when the fungal growth originated from the incubated materials. All the isolated fungi were extracted using single spore, or hyphal tip techniques were suggested by Dhingra and Sinclair (1985). The fungi were identified according to their morphological characteristics according to Booth (1985) and confirmed in Mycology center (MC), Assuit University, Egypt. The stock of isolates was stored on PDA slants and kept at 5–10°C in a refrigerator for further study.
Pathogenicity test of F. oxysporum
Pot and soil sterilization
Earthenware pots (20 cm in diameter) were sterilized by plunging them into a 5% formalin solution for 15 min and covered overnight with plastic sheets, then left to dry. Soil sterilization was carried out by autoclaving for 1 h at 121°C.
Nursery preparation
Susceptible marmand tomato cultivar obtained from the Research Institute of Horticulture, Agricultural Research Center (ARC), Giza, Egypt. Tomato seeds were sterilized by 1% sodium hypochlorite on the surface for 1 min, then washed in several changes of sterilized water, and left to dry. Sterilized seeds were sown on the surface in sterilized soil and irrigated with water for 30 days.
Inoculum preparation and soil infestation
Bottles containing the medium sand-barley were autoclaved and then the isolated fungus was inoculated after 2 weeks of incubation at 28°C. The fungus was thoroughly mixed with sterilized soil at a rate of 1gm inoculum/1 kg soil. Inoculated pots were watered regularly, 7 days before planting. The pots used as control were packed by a soil free-sand barley medium fungus (Shehata 2001).
Transferring of transplants and cultivation
Transplants were transferred to infested soil and planted under sterilized conditions. Five transplants were planted in sterilized un-infested soil. The wilting % after 30 days of transplant cultivation was recorded as follows:
$$ \mathrm{Wilting}\%=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{wilting}\ \mathrm{plants}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}}\times 100 $$
(1)
Isolation of Trichoderma spp. from rhizospheric region
Rhizospheric soils were collected from different regions of Egypt. Trichoderma spp. were isolated from the rhizosphere soil samples by dilution plate technique, using PDA and Trichoderma selective medium (TSM). Trichoderma spp. were purified, using the hyphal tip technique (Tuite 1996). The isolated species (T1, T2, T3…T23) were identified dependent upon growth, mycelium structure, conidiophores, phialides, and conidia on colony characters.
Dual culture experiment
Antagonistic efficacy of Trichoderma spp. isolates was tested against the isolated pathogenic fungus by dual culture experiment (Dennis and Webster 1971). Disc of growth agar medium of Trichoderma was placed against component F. oxysporum and the 6-day incubation at 28±2°C monoculture plates served as controls of both, 7 days after incubated radial growth, measured for F. oxysporum and Trichoderma spp. The colony diameter of test fungus was observed and compared with control on a dual culture plate. The percentage of the inhibition of radial growth (% RGI) was determined using formula:
$$ \mathrm{RGI}\%=\frac{\mathrm{C}-\mathrm{T}}{\mathrm{C}}\times 100 $$
(2)
where C is the growth of test pathogen with the absence of antagonist and T is the growth of test pathogen with of antagonist (Pandey and Vishwakarma 1998).
Identification of active Trichoderma isolates
The potent inhibitor of Trichoderma isolates against the tested phytopathogenic fungus was selected for identification up to colony character, growth, mycelium structure, conidiophores, phialides, and conidia (Kubicek and Harman 2002). Confirmed identification was done at MC.
Preparation of scanning electron microscopy (SEM)
Interaction among hyphae of pathogen F. oxysporum and bio-control agent T. atroviride was examined using the scanner electron microscope (SEM). To obtain hyphae touch sites, PDA plate was inoculated with a mycelial disk (5 mm) cut from the front edge of the 2 at a constant distance from the edge of the Petri plate. From colonies F. oxysporum and T. atroviride, the 2 fungi grow up to each other, their hyphae mixed. After 4 days of incubation, the plate cultures were examined under a light microscope to check the early stage of touch. The contact sites were labeled, and blocks of 1 cm agar were removed for SEM preparation, were fixed with osmium oxide, and then were dehydrated using a serial dilution of the ethyl alcohol finally acetone. A critical point drier (EMS 850) was then used to dry the processed samples, coated with gold using a sputter coater (EMS 550), and then SEM (JEOL100CX-ASID-4D) was used to examine the samples at the Regional Center for Mycology and Biotechnology Center, Egypt.
In vivo experiments
Effect of T. atroviride on tomato plants infected with F. oxysporum
Planting, growth conditions, and treatments
Susceptible tomato seeds (marmand) were sterilized on the surface for 1–3 min in 0.01% mercury chloride and washed with sterilized distilled water, then planted for 30 days in sterilized plastic pots containing sterilized soil before transplants were developed. The inoculum was prepared by introducing five discs of a 7-day-old culture of F. oxysporum grown on PDA medium into 500-ml size bottles containing sand-cornmeal medium (SCM), then incubated at 28oC for 14 days (Shehata 2001). The inoculum of F. oxysporum was added to sterilized pots (12 cm diameter × 20 cm height), containing 2.5kg sterilized soil at the rate of 10g inoculum/one kg soil, then watered whenever needed for 7 days. Healthy root systems of tomato transplants were soaked for 1–2 h in the 10% suspension of T. atroviride. Control roots were immersed in water only. Five pots, each containing 5 transplants, were used for each treatment. The fungicide Rizolex-T (Tolclofosm methyl+ Thiram) from Sumitomo Chemical Company Japan was used through this experiment for comparison at the recommended dosage (3 gm/l water) according to the Ministry of Agriculture, Egypt. The experimental design can be summarized as follows: C= (control), P= (pathogen), (F. oxysporum), P+F (pathogen + fungicide), and P+T (pathogen + T.atroviride). Data of this experiment were recorded 30 days after transferring. For each treatment, 3 plants were harvested and carefully washed by flowing water to dissolve debris from the soil. The following parameters were measured for the tomato plants: shoot length (cm), root length (cm), number of leaves, fresh and dry weight of shoot (gm), fresh and dry weight of root (gm), number of wilting plants, total number of plants, root surface area, and estimation of total phenol (ug. g-1 dry weight) and was performed in accordance with the method described by Folin Ciocalteau reagent (Malik and Singh 1980).
The disease incidence (DI) was measured as follows:
$$ \mathrm{DI}\%=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{wilting}\ \mathrm{plants}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{plants}}\times 100 $$
(3)
Statistical analysis
All values were the sum of the triple determinations. Data was statistically analyzed using the SPSS (1999) one-way variance analysis (ANOVA). The small difference was shortened as LSD and measured at P≤0.05.
