Collection and preparation of plant material
Healthy roots of P. hysterophorus were collected from Malakandir farms of the University of Agriculture Peshawar, Pakistan. Roots were kept in polythene bags and transferred to the laboratory for further processing. Healthy roots were again sorted and thoroughly rinsed with sterile distilled water to remove dirt and debris. Following rinsing, surface sterilization was carried out with 70% ethanol (v/v) for 1 min and 0.1% mercuric chloride (v/v) for 5 min, respectively. The roots were again thoroughly rinsed for five times, using sterilized distilled water to remove the excess of chemicals from the surface of roots. The roots were spread on sterilized tissue paper to dry for 30 min under sterilized conditions (laminar flow unit). The dried and sterilized roots were then used for the isolation of endophytic fungi (Priyadharsini and Muthukumar 2017).
Isolation and identification of endophytic fungi
The sterilized roots were then cut into pieces of 1 cm, then plated on to Sabouraud dextrose agar (SDA). The pH of SDA was adjusted in the range of 7.5–7.8. The inoculated plates were then incubated at 28 °C for 1–2 weeks to allow the growth of endophytic fungi. Pure cultures of endophytic fungi were obtained by transferring hyphal tips from the growing colonies to fresh SDA plates (Hamzah et al. 2018).
Identification of endophytic fungi was done on the bases of the morphological and cultural characteristics. Hyphae of the endophytic fungi were placed on slides, stained with lactophenol cotton blue, and observed under compound microscope. Cultural and microscopic characteristics were noted, and identification was done based on available keys (Gilman 2001).
Preparation of fungal filtrates
Flasks having potato dextrose broth (PDB) media were individually inoculated with 5-mm agar discs of each of the 3 endophytic fungi grown in a Petri dish. Inoculated flasks were incubated at 27 °C in an incubator for 15 days. After 15 days of incubation, fungal cultures from each flask were first filtered through Whatman filter paper no. 1 and then passed the filtrate through 0.22-μm filter papers with the help of vacuum filtration to remove any hyphae or spores present in the filtrate. Fungal cultural filtrates were preserved at 4 °C for further use.
In vitro studies were carried out to evaluate the bioherbicidal effects of cultural filtrates of the isolated endophytic fungi on the germination and early seedling growth of 3 weed species, namely Chenopodium album, Avena fatua, and Convolvulus arvensis. Seeds of these weed species were obtained from the Department of Weed Science, University of Agriculture Peshawar, Pakistan. Seeds were surface sterilized with 70% ethanol for 3 min, followed by 1% sodium hypochlorite (v/v) for 5 min. Seeds were then rinsed with sterilized distilled water for five times. Each weed species had 4 treatments and 3 replicates. A total of 36 Petri dishes (90 mm diameter) were used, each having 15 seeds on 2 layers of sterilized filter paper. Five milliliters of each cultural filtrate was added to each Petri dish. PDB was used as a negative control. The experimental design was completely randomized design (CRD), and each treatment was repeated 4 times. Petri dishes were placed in a growth chamber with alternating temperatures of 10/20 °C in the 8/16-h dark/light regime (Alshallash 2018). Data regarding percentage of germination, shoot length (mm), root length (mm), and plant biomass (mg) were recorded after 15 days.
Foliar spray pot experiment
Another experiment was conducted to evaluate the cultural filtrates of endophytic fungi on 2-week-old weed plants under controlled conditions. Pot experiments were carried out in a horticultural nursery of the University of Agriculture Peshawar during the month of October to November 2019. Eight seeds of each weed species were sown per 60-cm-diameter perforated plastic pot each having 2.5 kg soil, i.e., sand, clay, silt = 30.31%, 26.22%, 43.93%; pH 6.8 and EC of 0.288 dSm−1. Two weeks after emergence, pots were thinned to 4 healthy and equal-sized plants. Three-week-old plants were sprayed with fungal cultural filtrates (100%) at the rate of 100 ml per meter square area. Treated weed plants were examined for percentage of cellular respiratory activity, percentage of electrolyte leakage, chlorophyll content (mg/cm2), shoot length (cm), and biomass (g) on 1 and 7 days of the spray.
Estimation of chlorophyll content
Chlorophyll content of leaves was measured with the help of atLEAF chlorophyll meter. Chlorophyll meter was properly calibrated by following the manufacturer’s guidelines. Each leaf SPAD value obtained was the average of 6 readings. The values were then converted to milligrams per square centimeter of chlorophyll content (Novichonok et al. 2016).
Determination of cellular respiration
Cellular respiration is an indirect estimation of tissue viability, which is determined by using 2,3,5-triphenyl tetrazolium chloride (TTC). Viable cells reduced TTC to red-colored triphenyl formazan by accepting electrons from mitochondrial electron transport chain. The reduction in cellular respiration is the indication of tissue damage from fungal cultural filtrate. To estimate root oxidizability, 50 mg of tissue was treated with 5 ml of 0.4% TTC w/c and 5 ml of 0.06 M phosphate buffer. The mixture was then incubated at 40 °C for 2.5 h, followed by 2 ml of 2.0 N H2SO4. After incubation, the root was ground in 10 ml of absolute ethanol and absorbance was measured at 485 nm. The triphenyl formazan content was expressed as a percentage of the control, calculated according to the following equation.
The formazan content calculation was termed as cellular respiration.
% Cellular respiration inhibition = (Absorption of thawed tissue)/(Absorption of control) × 100
Relative electrolyte leakage
Relative electrolyte leakage (REL) of selected weed plants was measured, following the protocol of Kaur et al. (2010). A 200-mg leaf tissue was incubated in pure sterilized distilled water for 2 h at 25 °C in a test tube, and electrical conductivity (EC) was measured. The test tube containing leaf material was boiled for 30 min, and the conductivity was measured (E2) again. The relative electrolyte leakage (%) was calculated, using the formula: REL (%) = E1/E2 × 100.
The experimental design used in the present study was CRD. Each treatment was repeated 3 times. The data obtained was subjected to one-way ANOVA. Least significant difference (LSD) at p < 0.05 was carried out to compare the mean of treatments. For statistical analysis, software package Statistix 10.1 was used.