Macrophomina phaseolina (Tassi) Goid Culture Filtrates is a Lucrative Source for Weed Control

The mycoherbicides have been recognized as an effective approach for weed control as being eco-friendly, low cost, and of long-lasting. This research was carried out to evaluate the herbicidal effect of Macrophomina phaseolina, the causal of sesame charcoal rot. The fungal culture was extracted with water and the extract toxicity was evaluated by seed germination, detached leaf, and whole plant bioassays on several weed species of Iraq (Cynanchum actum, Malva parviflora, Sorghum halepense, Lolium rgidum, and Silybum marianum). The results revealed that the extract of the fungal culture was significantly inhibited the germination of the examined weeds seeds by more than 86.7 %. The detached leaf bioassay also indicated that the culture extract was of potent toxic effect to all examined weed species, regardless their types (broad or narrow and mono or dicotyledonous). The phytotoxicity symptoms appeared on the detached leaves were as tissue and chlorophyll disintegration, yellowing, necrosis, and death. The same toxic symptoms were also recorded on the whole plant bioassay in vivo but the weed species of the narrow leaves however, showed significantly much less effect. The phytotoxicity on the examined weed species ranged from 66 to 83%. Moreover, the fungal culture extract was also found to be of potent toxic effect to milk thistle (Silybum marianum) despite the application method as foliar spraying or root immersing with 100% and 20% of the extract respectively. The results of this research showed for the first time the effect of M. phaseolina culture filtrates as a potent herbicide potential on different weed species.


Introduction
Weeds are an ever-present critical challenge to the agribusiness and the globe. The weeds cause potential economic yearly losses in agriculture that reach up to 10% of the world production. The weeds impose costs on agriculture production by reducing the crop quantities and qualities or increasing its production costs [1]; [2]. Hence, having effective weed control programs in agriculture is very important to reduce losses in production and assure food security. Several methods for weed control have been recommended and the chemical herbicides alone account for almost 50% of the agrochemical markets [3].
The charcoal rot pathogen (Macrophomina phaseolina) of sesame has been reported to attack a wide host range. It causes a plant disease also named summer wilt, or black rot [16]; [17]. Moreover, the Macrophomina phaseolina was reported to elaborate several phototoxic metabolites named aspertin, isoaspertin, phomalacton, phaseolinic acid, phonenon, phaseolinone. In addition to (-) -botryodiphodin, and phaseolinone [16]; [18]; [19]. No previous attempts have been mentioned to evaluate the herbicidal potentiality of M. phaseolina metabolites for weeds control. The present study was therefore conducted to explore and high light the effect of M. phaseolina culture extract as a bio-herbicide.

Isolation of Fungus
The fungus used in this research was isolated by aseptically culturing of little bits of surface-sterilized sesame plant tissue of charcoal rot symptoms on potato dextrose agar (PDA) culture. The plates were incubated at 25± 2. º C. The associated fungus was affirmed morphologically as M. phaseolina (5,8). The fungus was purified and sub cultured into PDA slants and kept in the refrigerator at 4ºC for further use.

Preparation of aqueous extract of M. phaseolina culture filtrates (AEMCF)
The AEMCF was prepared according to the technique described by [4]. Potato sucrose broth PSB was prepared (200 gram of sliced potato tuber and 20-gram sucrose/liter distilled water) and distributed equally into Erlenmeyer flasks (100 ml per 250ml flask) and autoclaved at 121 °C and 15 psi for 15 minutes, cooled and each flask aseptically inoculated with three discs of a 5mm taken from the M. phaseolina culture margin on PDA. The flasks were incubated at 25 ±2 º C for 21 days with daily handshaking. The cultures of each three flasks were blended by electric blender and aseptically filtrated through the Whatman No.1 under section pressure. The extract filtrates about 70% of the total used broth media were divided into three portions and treated as follows: 1. The first portion was filtrated aseptically through 0.45 μm Millipore membrane under suction pressure condition. 2. The second portion was autoclaved for 10 minutes (121 º C and 15psi). 3. The third portion was autoclaved for 20 minutes (121 º C and 15psi). To check for the viability of the fungal cells in the extracts of the above treatments a sample of each portion (0.25 ml) was aseptically cultured on the surface of the PDA plate and incubated for a week at 25 ±2 º C. Each portion was evaluated for its toxicity on the sesame detached leaf bioassay as previously described (23). Given the bioassay results, the first and second portions showed relatively the highest toxicity. Hence the M. phaseolina culture cell-free filtrates were prepared by a similar method of the second portion for every further investigation to assess the effect of the AEMCF as herbicide. The effect of the AEMCF was assessed according to the following bioassays: 2.2.1. Seed germination. The seed germination bioassay was carried out according to the procedure described before [20]. Seeds of Mallow, Bindweed, and Johnson grass were washed separately and surface sterilized by soaking in a 5 % bleach solution (17% NaOCl) for three minutes and washed thoroughly with sterile water. Enough surface-sterilized seeds of each weed were taken aseptically and divided into two portions. One portion was soaked overnight in sterilized water and the other was soaked in the AEMCF. Five seeds of each soaked weed were placed on a circle at the age of pre-wetted sterilized 3 filter paper on the Petri plat replicated three times. The other three replicates of the sterile water-soaked weed seeds (5 seeds per plat of each weed) were served as control. Plats were labelled and randomly distributed according to completely randomized design (CRD) and incubated in the growth room at 25±2 ºC and 10 hours daily light for 10 days. The germination % was calculated by the following formula: Seed germination (%) = No. of germinated Seed 15 (total No. of the examined seeds) × 100

Detached leaf (DL) bioassay.
The detached leaf (DL) procedure described by Sharma et al. (24) was adopted. Leaves of relatively the same size and age of each of the examined weeds (Mallow, Bindweed, and Johnson grass) were washed and surface sterilized by soaking in a 5 % bleach solution (17% NaOCl) for three minutes and washed thoroughly with sterile water. Three leaves of each weed were soaked for five minutes in the AEMCF and another three were soaked in sterile water served as control. Each leaf was placed on the sterilized Petri plate, the plates were labelled and randomly distributed according to CRD and incubated in the growth chamber at 25± 2 ºC and daily 10 hours light for 5 days. The results were recorded by measuring the phytotoxic damaged leaf tissue according to the formula reported before [6].
Damaged leaf area (%) = Width of damaged area Length of damaged area × Width of total leaf area Length of total leaf area × 100

Foliar Spray
The herbicide effect of the AEMCF was examined on the whole plants as follows: 2.3.1. In vitro. Young plants of 4-5 leaves of the relatively same size of milk thistle (Silybum marianum) were gently taken off with their whole root system as much as it could be from the field and washed thoroughly with tap water. The roots of each plant were immersed in 50 ml tap water in a 100ml glass vial. The vials with the plants were incubated in a growth room at 25 ± 2 º C and daily hours light for 72 hours before they were used. The incubated milk thistle plants were treated as follows: 2.3.2. Experiment 1. The foliar of three replicated plants were thoroughly sprayed with 100 % the AEMCF. Alternatively, the foliar of the other three replicated plants were thoroughly sprayed with sterile water served as control.

Experiment 2.
The root system of the other three replicated plants was immersed in 20 % AEMCF. Alternatively, the root system of the other three replicated plants was immersed in sterile water served as control.
The vials with plants of various treatments were randomly distributed in the growth room according to the CRD at 25º ± 2 º C and daily light for 10 hours and the results were collected after 10 days. The herbicide potential of the fungal extract was recorded as the percentage of tissue damaged according to the procedure described before [ according to CRD. The herbicide potential of the fungal extract was recorded as the percentage of tissue damaged following the procedure described before [21].

Statistical Analyses
This research was carried out at the laboratory and field of the Department of Plant Protection, College of Agriculture University of Anbar statistically. All data from laboratory and field bioassays were analyzed using analysis of variance and the LSD test to differentiate the treatment means at the 5% level of significance.

Results and Discussion
The fungus associated with the sesame infected plant was identified as M. phaseolina according to its morphology as previously described in several research papers [21]; [22]; [23]. The fungus showed growth of dens black mycelium mat after 21 days of incubation in the potato sucrose broth PSB. The sterilized extracted culture filtrates by filtration and autoclaving showed no growth on PDA after 10 days of incubation. This indicated that both sterilization methods completely got rid of any fungal propagules in the final filtrates. The preliminary examination for the phytotoxicity of the culture filtrates on detached sesame leaves revealed that the highest toxicity, as indicated by fast and clear chlorosis and necrosis of the sesame leaf tissue was that of the filtrating through the Millipore membrane and 10 minutes autoclaving. The culture autoclaved for 20 minutes showed relatively low and slow toxicity. Accordingly, it could be concluded that the phototoxic constituents of the culture filtrates of the M. phaseolina were water-soluble and relatively heat stable and it most likely was not of a protein entity (enzymatic). The results revealed that the AEMCF was highly inhibited the seed germination of examining weeds. The filtrates caused more than 86.7 % inhibition of all the examined weed seed species with highly significant differences in the germination of the treated and non-treated seeds (Figure 1) (Table 1).  These results agreed with the results of several other types of research on the effect of fungal metabolites on weeds seed germination in vitro [9]; [15]. The detached leaf bioassay also indicated a potential toxic effect of the AEMCF on both broad or narrow weed leaf types or that of mono and dicotyledonous (that included mallow, bindweeds, Johnsongrass, and bluegrass). The phytotoxicity of the AEMCF appeared as tissue and chlorophyll disintegration, yellowing, necrosis, and death on all examined weed species ( Figure  2). The phytotoxicity damage of the AEMCF was ranged between 75 % on Johnsongrass to 95.5% on Mallow (Table 2). These findings supported the previous research as many fungi filtrates of toxic effects on detached leaf bioassay [4]; [21].  Similar phytotoxic symptoms of the AEMCF were also shown on the whole plant bioassay in vivo at the plastic house (Table 2) and the field as well (Table 3)    Besides, the results of the bioassay of the AEMCF on the Milk Thistle plants (in vitro) clearly showed positive toxicity results despite the application methods as foliar sprayed or root immersed of 100 and 20% culture filtrates respectively. The toxic symptoms also appeared as the withering of the leaves, with chlorosis, necrosis, and drying (Table 4) ( Figure 5).  The results of all the bioassays in this research revealed for the first time a potent herbicidal effect of M. phaseolina culture filtrates against several winter weeds that widely spread in the middle of Iraq. Further researches are still needed to examine the effect of the AEMCF on the summer weeds as well. The toxic constituents of the culture filtrates against weeds were water-soluble, heat-stable, and relatively highly produced at an effective concentration. In this research, the herbicidal potential of M. phaseolina culture filtrates is highlighted for the first time and supports the previously reported findings that fungi of an attractive proposition for controlling weeds biologically [3]; [6]; [7]; [8]; [9]; [24]. The large application scale of the culture filtrates of M. phaseolina however, needs to be further studied to identify the active constituent (s) that may be used as analogs for the synthesis herbicide (s) as a natural product for weed management.

Conclusions
The current findings revealed for the first time a powerful herbicide potential of Macrophomina phaseolina culture filtrate on various weed species. The toxic constituents of the culture filtrates against weeds were water-soluble, heat-stable, and produced at evidently effected concentration.