Acetonitrile, methanol, and water of HPLC grade were purchased from Sigma-Aldrich Co. (Spruce Street, St. Louis, MO, USA). Glucose, magnesium sulphate, yeast extract, peptone, beef extract, dimethyl sulfoxide (DMSO), and agar were supplied from El-Gomhoria for pharmaceutical and chemicals Co., Alexandria, Egypt. Potato dextrose agar (PDA) was purchased from Oxoid (Basingstoke, Hampshire, RG24 8PW, UK). Other chemicals and solvents were purchased from El-Nasr Pharmaceutical Chemicals Co., Qalyub, Egypt, and used without further purification. All media were prepared immediately and autoclaved at 121 °C for 20 min.
Sampling area and collection of weed specimens
Plant weeds (Rumex dentatus and Sonchus oleraceus) with visible diseased symptoms of samples were collected from wheat field and placed in sterilized glass bottles. The symptoms of all diseased weeds were characterized by necrotic (an irregular brownish) spots with chlorotic halo around them. The seeds and leaves used in the present study were collected from weeds growing in fields of cultivated crops. Sampling locality was a cultivated area at Jarrar, Abu Hommos, Beheira Governorate, Egypt. The weed species were identified by the help of available literature (Täckholm 1974; Boulos 1999, 2002, 2005 and 2009).
Isolation and selection of phytopathogenic fungi
Phytopathogenic fungi were isolated from the infected leaves of R. dentatus and S. oleraceus weeds. The leaves were surface sterilized by 95% ethyl alcohol for 1 min, then dipped in 10% sodium hypochlorite for one minute, followed by washing in sterile water. The infected spots of leaves were directly transferred to a Petri plate containing PDA medium. Inoculated plates were incubated at 28 °C and observed after 7 days (Babu et al. 2003 and Aybeke 2017). Fungal colonies were selected for purification by repeated streaking on agar plates of the PDA. The pure colonies obtained were transferred to a fresh PDA slant, subcultured, and stored at 4 °C.
Identification of phytopathogenic fungi
Phytopathogenic fungi were identified morphologically on the basis of macroscopic (naked eye) and microscopic characteristics (Moubasher 1993) after culturing on the PDA medium at 28 °C for 7 days. For molecular characterizations of fungal isolates, total genomic DNA preparations of fungal cultures in potato dextrose broth (PDB) were made according to the protocol of Quick-DNA™ Fungal Microprep Kit (Zymo research #D6007). The extracted DNA preparations were examined using 1% agarose gel electrophoresis. The internal transcribed spacer (ITS) region (ITS1-5.8SrDNA-ITS2) of rRNA genes was amplified, using the primer pairs of ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) (White et al. 1990). The 5.8S rRNA gene and the two flanking internal transcribed spacer regions (ITS1-5.8S rDNA-ITS2) of rRNA genes were PCR amplified according to the protocol of Maxima Hot Start PCR Master Mix (Thermo K1051). The amplifications were performed in a ThermoHybaid PCR Sprint Thermal Cycler (Thermo Electron, USA). PCR products were purified according to the protocol of GeneJET™ PCR Purification Kit (Thermo K0701). Amplicons, along with the marker DNA (DNA Ladder), were visualized by 1 % agarose gel electrophoresis after staining with ethidium bromide to confirm the size and purity of the amplified PCR products. The amplified PCR products of ITS1-5.8SrDNA-ITS2 were sequenced on both strands using ITS1 and ITS4 primers at GATC Company by use ABI 3730xl DNA sequencer. The nucleotide sequences determined were deposited in GenBank. For the identification of the isolates, the nucleotide sequences obtained were compared with those sequences already deposited in the data bank of the National Centre for Biotechnology and Information (NCBI) using the nucleotide basic local alignment search tool (BLASTn) to find the most closely related sequences. The identification of the species was determined based on the best sequence alignment score. The obtained DNA fragments were also subjected to a phylogenetic study by means of comparative sequence analysis of the ITS regions including the 5.8S rDNA sequences. This was achieved by generating a neighbor-joining distance-based tree using the software MEGA 6.0.
Preparation of fungal inocula
For inoculum preparations, PDA Petri plates were inoculated by phytopathogenic fungi from slants and incubated at 28 °C for 7 days. Then, the culture surfaces on the agar plates were scraped with 10 ml of sterilized water, using isolation needle. The spores were obtained and the suspensions were counted using a hemocytometer to obtain the desired concentrations of 2.5 × 105 conidia/ml and used as inoculum sources.
Preparation of fungal bioactive metabolites with herbicidal action
For the production of fungal filtrates with herbicidal activity, the fungal culture broths were prepared using 250-ml Erlenmeyer conical flasks containing 100 ml of PDB medium inoculated by 2 ml of spore suspension (2.5 × 105 conidia/ml) at pH 6 and in static conditions at 28 °C (Babu et al. 2003 and Guo et al. 2020). After 7 days of incubation, fungal mycelia were separated by filtration on a pre-weighed Whatman filter paper (No. 4), washing twice with distilled water and dried to a constant weight at 60 °C and reweighed. Fungal dry biomass was measured gravimetrically as the difference in weight. The culture filtrate for each fungus (≈ 500 ml) was centrifuged at 10,000 rpm for 10 min and the supernatant was lyophilized.
Bioherbicidal activity of crude fungal filtrates
The lyophilized product (0.1 g) that was obtained from the fungal filtrate was dissolved in 50 ml distilled water and sterilized, using 0.45 μm Minisart membrane filter.
In vitro bioherbicidal activity
The in vitro bioherbicidal activities of the filtrates of phytopathogenic fungi were assayed by seed germination bioassay and seedling morphology. All seeds were treated by 0.5% sodium hypochlorite for 10 min and washed numerous times with sterile distilled water before germination assay immediately.
Evaluation of bioherbicidal potential of crude filtrate, using an in vitro seed germination bioassay was carried out on filter paper (Whatman No. 4) in Petri dishes (10 cm in diameter) against R. dentatus L., S. oleraceus L., Avena fatua L., Polypogon monspeliensis L., Setaria viridis L., Echinochloa crus-galli L. Beauv, E. colona L., and Plantago major L. seeds. Crude filtrate of each phytopathogenic fungus (2 ml daily) was poured on the filter paper in Petri plate containing 100 seeds and incubated for 10 days under alternation between light (12 h using 2000 Lux) and dark (12 h) at 25 °C. Germination (%), plumule heights, and radical lengths of seedlings were examined. Control treatments were carried out, using sterile PDB medium instead of crude filtrates. Each trial was examined with three replications.
In vivo bioherbicidal activity
The in vivo bioherbicidal activities of crude filtrates were tested by leaf disk puncture assay against leaves of the abovementioned weeds. Crude filtrate of each phytopathogenic fungus (20 μL) was applied on leaf disks of the selected plants. The disks (13-mm diameter) were cut from weed leaves, then located on wetted filter paper (Whatman No. 4) within Petri plates, and punctured by sterile needle in the center before addition of fungal filtrates. Leaf disks were kept under constant light conditions and 25 °C. After 5 days of incubation, symptoms were examined visually. The degree of phytotoxicity (%) was scored according to severity of the symptoms on leaf disks. Control treatments were carried out, using sterile PDB medium instead of crude filtrates (Guo et al. 2020). Each trial was examined with three replications.
Isolation of fungal extracts and GC/MS analysis
For preparation of fungal extracts, culture filtrates of phytopathogenic fungi (500 ml) were lyophilized. A weight of 0.1 g was dissolved in 50 ml distilled water and stored at 4 °C in the dark. The compositions of the extracts were analyzed (Ekman & Holmbom 1989) by gas chromatography/mass spectrometry (GC/MS) with the following specifications: A Trace GC Ultra/Mass Spectrophotometer ISQ (Thermo Scientific) instrument was equipped with flame ionization detector (FID) and a DB-5 narrow bore column. Helium (average velocity 39 cm s−1) was used as the carrier gas (flow rate of 1 ml/min), and the temperature program was 120 °C/min, raised at 6 °C/min to 320 °C, injector temperature was 260 °C, and detector temperature was 320 °C with post run (off) at 320 °C. The GC/MS was equipped with a ZB-5MS Zebron capillary column (length 30 m × 0.25 mm ID, 0.25-μm film thickness; Agilent). A sample (1 μl) was injected at 250 °C, with split/split-less injector (50:1 split ratio) in the split-less mode flow with 10 ml/min. All mass spectra were recorded in the electron impact ionization (EI) at 70 electron volts. The mass spectrometer was scanned from 50 to 500 m/z at five scans per second. Scan time is 1.5 s and mass range is 40 to 300 amu. Peak area percent was used for obtaining quantitative data with the Xcalibur software (Thermo Scientific) without the use of response factor correction. Constituents were identified by comparing their mass spectra with MS library (NIST, Mainlib, Reblib and Wiley) data (Adams 1995). Quantification of constituents was obtained by integrating the peak area of the chromatogram.
Experimental data are presented as mean ± standard error and the analysis of variance (ANOVA) of data was conducted and mean property values were compared (p ≤ 0.05) to Fisher’s least significant difference (LSD) method by Minitab 16.1.0 program (Minitab Inc., PA, USA).