Isolates of P. capsici, P. ultimum, and T. harzianum were obtained from the Department of Plant Pathology, The University of Agriculture, Peshawar, Pakistan.
In vitro assay
In vitro antagonistic activity of T. harzianum was tested against P. ultimum and P. capsici according to the standard procedure of Dennis and Webster (1971). Mycelial disc of 5 mm (diameter) of T. harzianum and pathogens were taken from 7-day-old culture and placed in a petri dish containing potato dextrose agar (PDA) at an equal distance in opposite direction. The control (pathogen alone) was also run in this assay. All the petri dishes were incubated in the dark at 28 ± 2 °C for 5 days. In vitro antagonistic activity was measured in terms of percentage inhibition using the following formula.
$$ I=\left(C-T\right)/C\times 100 $$
where I refers to percent inhibition, C is control, and T radial growth of pathogen (mm) in the presence of T. harzianum.
Green house experiment
Tomato germplasm, i.e. money maker, was obtained from Tarnab Agriculture Station, Peshawar, Pakistan. Nursery was raised in earthen pots from 3-week-old seedlings, transplanted to the 8.8inches diameter pots containing 2.5 kg of sterilized soil having sand, clay, and silt at a ratio of 2:1:1. The pure cultures of P. capsici, P. ultimum, and T. harzianum were refreshed on PDA media for 4 days at 25 °C. The inoculum was prepared in potato dextrose broth and placed in a shaking incubator for 1 week at 25 °C ± 2 °C. The flask containing the culture media was then seeded with disks (7 mm diameter) of 4-day-old culture (Margaret et al. 2011). Holes were made in rhizosphere, and 5 ml of conidial suspension of 107 ml− 1 was poured in each hole.
Both pathogens and biological agent were applied to the rhizosphere after 10 days of transplantation in a greenhouse of the Centre for Biotechnology and Microbiology (CB&M), University of Swat, Pakistan.
The experiment was designed in randomized complete block design (RCBD) with six treatments and three replications. The treatments were categorized in to the following:
Un-inoculated control (healthy)
T. harzianum (alone).
Inoculated with P. capsici (alone)
Inoculated with P. ultimum (alone)
Inoculated with P. capsici and T. harzianum
P. ultimum with T. harzianum
The experiments were terminated after 40 days of inoculation. Plants of each pot were carefully uprooted, separately labeled, and brought to the laboratory. Data on different agronomic traits were recorded and analyzed by Statistic 8.1 Program. Means of the results were compared by using least significant difference (LSD) test (Steel et al. 1997).
Histopathological study was conducted. A comparative study of the root tissues in each treatment was carried out at 30 and 40 days after inoculation. The uprooted samples were thoroughly washed with tap water and excised into small pieces for fixation in F.A.A. (formaldehyde/acetic acid/alcohol, 3:1:6) and processed for histopathological studies (Sass 1958). Following fixation, the samples were dehydrated in 10, 20, 30, 40, and 50% ethanol. Roots were then transferred to butanol/ethanol/water solution prepared in the following ratio.
Roots were kept in each of the above solutions for 2 h at room temperature. Dehydrated root tissues were infiltrated and imbedded in paraffin wax at 52 °C for 10 days. Air bubbles were removed from the roots during the wax infiltration process. Sections of 12-μm thicknesses were cut with a rotary microtome which were then affixed on slides with the help of Mayer’s Albumin adhesive and stained with safranin and fast green (Sass 1958). The stained sections were mounted in Canada balsam and examined. Photographs were taken using Olympus digital camera at 4 ×, 10 ×, and 100 × magnifications.