Seedborne mycoflora of faba bean (Vicia fabae L.) and evaluation of plant extract and Trichoderma species against mycelium growth of selected fungi

Fungal diseases are among the biotic factors limiting the production of faba bean in Ethiopia. The objective of this study was to isolate and identify seedborne mycoflora associated with faba bean seed samples, determine their effects on seed germination and disease transmission, and evaluate the antimicrobial activities of seven plant extracts and four Trichoderma spp. against the pathogen isolated from the seed. Fifty seed samples were collected from different farmers’ saved seeds of five major faba bean-producing varieties of the Ambo district and were tested by agar plate methods as recommended by the International Seed Testing Association (ISTA). A total of 7 fungal species belonging to 6 genera, viz. Fusarium oxysporum (Schlechlendahl), Fusarium solani (Mart.) Sacc, Aspergillus spp. Penicillium spp. Botrytis spp. Rhizoctonia solani (Kühn) and Alternaria spp. were isolated and identified. Among these, Fusarium spp., Aspergillus spp, and Penicillium spp. were the most predominant fungi in all seed samples. Seed-to-seedling transmission test results confirmed that F. oxysporum, F. solani and R. solani were major causal pathogens that caused root rot and damping-off disease in faba beans and were transmitted from seeds to seedlings. A higher germination rate was observed in Golja-GF2 (97%), and a lower germination rate was observed in Kure Gatira-KF8 (81%). A study on in vitro evaluation of plant extract and Trichoderma spp. against F. oxysporum, F. solani and R. solani revealed that plant extracts at 5%, 10% and 20% concentrations significantly inhibited the mycelial growth of all tested fungi. Inhibitory effects on the three tested fungi (R. solani, F. solani and F. oxysporum) were recorded on T. longibrachiatum (87.91%), T. atroviride (86.87%), Trichoderma virens (86.16%) and T. harzianum (85.45%). The inhibitory effect of the aqueous plant extracts on mycelial growth increased with an increase in concentration, and the hot water extracts showed higher effects compared to the cold water extract in all tested fungi. This study showed that the highest inhibitory effect of Allium sativum L. extracted at a 20% concentration against mycelial growth inhibition of the three test fungi (F. oxysporum, R. solani and F. solani) was 84.60%, 83.61% and 83.47%, respectively. However, Nicandra physalodes (L.) Gaertn.) extracts at the same concentration showed the lowest inhibitory effects on the three tested fungi (74.94%, 73.94% and 73.24%).


Introduction
Faba bean (Vicia fabae L.) is one of the most important food legumes in the world due to its high nutritive value both in terms of energy and protein contents containing 24-30% [1][2][3], and it is also an excellent nitrogen fixer [4]. It ranks fourth in the world after garden peas, chickpeas and lentils. Its production worldwide is concentrated in nine major agroecological regions, namely, northern Europe, the Mediterranean, the Nile valley, Ethiopia, Central Asia, East Asia, Oceana, Latin America, and North America [5].
It is an important high-land pulse crop of Ethiopia, which covers 520,519 ha of cultivated land with an annual production of 930,633 tons [6]. The crop accounts for the largest share of the area under pulse production in Ethiopia. The growing importance of this crop as an export in Ethiopia has led to a renewed interest by farmers to increase the area under production [7,8].
Despite its wide cultivation and huge importance, the average yield of Vicia fabae L. is quite low in Ethiopia. The average national yield is approximately 2.1 t ha − 1 [9], which is very low compared to the average yield of 3.7 t ha − 1 in major producer countries [10]. The productivity of this crop is far below its potential because of several biotic and abiotic constraints [11]. According to Sahile et al. [8], diseases are the most important biotic factors limiting the production of faba bean in Ethiopia. It is attacked by more than 100 pathogens [12]. More than 17 pathogens have been reported in Ethiopia [13]; among these, fungi are the largest and perhaps the most important groups affecting all parts of these plants at all growth stages [14].
Fungal diseases such as chocolate spot (Botrytis fabae), rust (Uromyces fabae), black root rot (Fusarium solani), and foot rot (Fusarium avenaceum) are among the fungal pathogens that contribute to the low productivity of the faba bean [15].
Seed dressings are used to eliminate most surface infestations of seeds but have relatively little effect on internally borne organisms [16]. An urgent need for alternatives to fungicides for the control of seedborne fungi is important in recent years; much attention has been given to nonchemical systems for seed treatment to protect them against seedborne pathogens. Plant extracts have played a significant role in the inhibition of seedborne pathogens and the improvement of seed quality and field emergence of seeds. Many authors have reported the effective and safe use of plant extracts for controlling seedborne fungi [17][18][19][20].
In the current study, the effects of seedborne fungi on seed germination, seed-to-seedling transmission and their management are important for the improvement of crop production and productivity. Therefore, this study was initiated with the following specific objectives: to assess the fungal pathogens associated with farmers' saved seeds in the study areas; to determine the level of seed infection by fungal pathogens associated with farmers' saved seeds in the study area; to determine the effect of seed infection on seed germination and disease transmission; and to evaluate the efficacy of some plant extracts and Trichoderma spp. against fungi associated with faba bean seeds.

Description of the study areas
The seed samples were collected from the major faba bean growing farmers' association of Ambo district, and the laboratory study was conducted at Ambo University College of Agriculture and Veterinary Science. Ambo is located 120 km west of Addis Ababa at 8 • 98 ′ south latitude and 37 • 83 ′ E 37 • 83 ′ north longitude (Fig. 1). It has a total geographical area of 83,598.69 sq. km., with elevations A. Neme et al. ranging from 1380 to 3300 m a.s.l. The annual rainfall ranges from 900 to 1100 mm, and the temperature ranges from 10 to 27 • C, with an average of 18 • C (National Meteorological Service, unpublished data of 2018).

Seed sample collection and isolation of fungi
A total of 50 seed samples were collected in polythene envelopes from different farmers' saved seeds of five major faba beanproducing farmer associations, namely, Golja, Jijigu Weransa, Ya'i Cabo, Kure Gatira, and Dase Akililo, in Ambo district with consults of the district agricultural office. The purposive sampling method was used for selecting the farmers' association, and seed samples were taken according to the [21] sampling procedure. Each sample was enclosed in paper bags with proper labelling, taken to the laboratory, and then kept in a refrigerator at 4 • C for 24 h until the beginning of the identification tests.
Four hundred seeds were randomly taken from each seed sample for mycoflora study. Four replicates of 100 seeds per sample were surface sterilized using 0.5% sodium hypochlorite (NaOCl) solution for 10 min with constant agitation. Seeds were rinsed three times in sterile distilled water for a minute, placed on sterile Whatman filter paper to dry and inoculated with 10 seeds per plate aseptically in (90 mm diameter) Petri dishes containing PDA using a sterile pair of forceps.
Ten Petri dishes were plated with 100 seeds to represent the replicates of treatment, and these were arranged in a completely randomized design. All Petri dishes were incubated at 25 ± 2 • C for 7 days under refrigeration cycles of 12 h near ultraviolet (UV) light and 12 h of darkness for sporulation of fungi. After incubation, the growth characteristics, as well as the percentage of infection, were recorded. The isolated colony culture of fungi was maintained on potato dextrose agar (PDA) medium. The fungi were identified based on their morphological features, as used by Booth [22] and Barnett and Hunter [23].

Determination of seeds infected with fungi
The percentage of seeds with fungal infection was determined by counting the infected seeds and dividing by the total number of seeds and expressing them as a percentage. Thus,

Percent of seeds infected =
No seed infected with fungi Total no of seeds per plate × 100 Colonies obtained from each infected seed were subjected to cultural and microscopic morphological characterization for identification. The occurrence of fungi was determined by counting the number of times each fungus occurred divided by the total number of fungi and expressed as a percentage. Thus,

Percentage occurrence of fungi =
No of times each fungus occurred Total no of fungi per plate × 100 The identification of the fungi was based on cultural characteristics, mainly on the growth patterns and pigmentation. Further microscopic examinations were carried out for mycelial and conidia structures based on the user identification manual of illustrated genera of fungi [23,24]. The morphological characteristics were determined by taking a small amount of mycelium from ten-day-old pure culture plates using a sterile needle and transferring onto a cleaned glass slide. The culture was taken from five different positions of the culture on the plate, four from the adjacent side and one from the middle. The mycelium was stained with 0.1% lactophenol cotton blue and observed under a compound microscope.

Effect of seed borne myco-flora on seed germination
To examine the effect of seedborne fungi on seed germination and seed-to-seedling transmission, seedling bioassays were carried out in a growth chamber. The sand used in the studies was sterilized in an autoclave at 121 • C and a pressure of 15 lb for 1 h, after which it was ready for use and allowed to dry. One hundred seeds were randomly taken from each sample and were surface sterilized by soaking in 0.5% sodium hypochlorite (NaOCl) solution for 10 min with constant agitation followed by rinsing in three changes of sterile distilled water for a minute and then dried between two layers of sterilized Whatman filter paper. One hundred treated seeds were placed in a tray containing sterilized sand aseptically by using a sterilized pair of forceps at equidistance to avoid crosscontamination and then placed in a growth chamber at 28 ± 2 • C in alternating cycles of 12 h of darkness and 12 h of light. The seeds were kept moistened by adding approximately 3 mL of sterilized distilled water every two days throughout the incubation period. The effect of the seedborne fungi on the germination of faba bean seed samples was determined after 7 days of incubation. Seed germination was assessed by counting the seeds with seed leaves and dividing them by the total number of seeds in the Petri plates expressed as a percentage.

Germination percentage =
Number of seeds germinated × 100 Total no of seeds per plate The incidence of the fungi in coleoptiles and root tissues of seedlings was determined on the 7th, 14th and 21st day after plating in sterilized sand by pulling out the seeds/seedlings showing disease symptoms. Finally, the seedling infection percentages per plate were calculated [25].

Seedling infection percentages =
No. of seedlings affected by a pathogen × 100 Total number of seeds sown (4) In addition, the progress of the disease and symptoms developed were monitored in the plated plants. The transmission efficiency (TE) of fungi from seeds to seedlings was estimated from the incidence data with the following formula: Transmission efficiency of fungi from seeds to seedling = C × 100 S (5) where C is the infection percentage of seedlings by fungi in the transmission study and S is the seed infection percentage of the fungi during the laboratory assay on agar. In vitro antimicrobial assays of the plant extracts and bioagents were performed in the plant pathology laboratory of Ambo University College of Agriculture and Veterinary Science.

Evaluation of plant extract
The Gaertn. and N. tabacum were collected from the Toke Kutaye district Guder areas. C. longa L. was collected from the west Wellaga zone (Gimbi), and P. dodecandra was collected from the Jibat state forest, west Shewa. The samples were separated into their selected parts (leaf, clove and seed), washed thoroughly under tap water followed by sterilized distilled water, cut into a smaller size of approximately 1-3 cm long, air dried under shade at room temperature for 1-2 weeks, pounded using a sterile mortar and pistil into a fine powder and kept in the refrigerator at 4 • C until use [26,27].
The crude plant extract was obtained by socking/infusing 50 g of each air-dried powdered plant material in 500 ml of hot and cold distilled water separately (1:10 w/v) in a 1000 ml conical flask. For garlic and A. vera (L.) Burm.f, 50 g of garlic paste and 50 g of A. vera (L.) Burm.f were placed into a 1000 ml conical flask and then filled with hot and cold distilled water separately up to 500 ml to give 1:10 w/v. The flasks were kept on a rotary shaker for 30 min, and then extraction took place under cold conditions for 24 h [28]. Plant extracts were filtered through double layers of muslin cloth followed by Whatman filter paper. Finally, the aqueous extract at a concentration of 10% was used as an original concentration in the antifungal activity test and stored in an airtight bottle in a refrigerator at 4 • C for further usage [29].
Five plant parts (leaf, clove, gel, rhizome, and seed) were evaluated against mycelial growth of seedborne test fungi F. solani, F. oxysporum and R. solani by using the poisoned food technique at 5, 10 and 20% concentrations for which 5, 10 and 20 ml of crude extract stock solution were mixed with 95, 90 and 80 ml of sterilized molten PDA media. The medium was thoroughly shaken for uniform mixing of leaf extract, and 20 ml of agar media was poured into sterile Petri plates and allowed to solidify. Five millimeter agar disks of test fungi were cut from 7-day-old culture plates by using a sterile cork borer and placed in the centre of the Petri plate containing different concentrations of plant extract. The experiment was conducted in a complete randomized design (CRD), with 7 treatments and 3 replications for each test pathogen. The Petri plates containing only PDA medium without plant extracts served as a control. All inoculated Petri plates were incubated at 25 ± 2 • C for seven days, and the data on the mycelial growth of the fungus were recorded from 24 h of inoculation to 7 days of inoculation. The percentage inhibition of mycelial growth was calculated as per the formula given by [30].

Percent inhibition of test fungi
where C is the average increase in mycelial growth in the control plate and T is the average increase in mycelial growth in the treatment plate.

Evaluation of bioagents under in vitro conditions
Purified cultures of antagonistic Trichoderma isolates T. harzianum, T. longibrachiatum, T. atroviride, and T. virens used in this study were obtained from the Plant Pathology Department of Ambo University College of Agriculture and Veterinary Sciences. Stock cultures of Trichoderma spp. contained pertinent information regarding how they were isolated from the natural environment and maintained on Petri dishes containing PDA medium in a refrigerator at 4 • C for subsequent use.
Stock cultures of four Trichoderma species were recultured in sucrose peptone broth (SPB) for multiplication, as recommended by Kumar et al. [31]. The conidia (colony-forming unit, cfu) suspension of each species was prepared in sterile distilled water from a 7-day-old culture on PDA [32]. The fungal inoculum was harvested by flooding the culture with sterile distilled water (SDW) and then rubbing the culture surface with a sterile glass rod. The fungal propagule concentration in each suspension was determined by counting using a hemocytometer slide, adjusted to 10 8 cfu/ml and used in a dual culture test following Kumar et al. [31].
The antagonistic effect of Trichoderma species against F. oxysporum, F. solani and R. solani isolates was evaluated by using the dual culture technique [33]. All the isolate test fungi and Trichoderma spp. were grown separately on sterilized standard PDA at 25 ± 2 • C in an incubator for 5 days to obtain juvenile colonies for the studies of antagonism. After an incubation period of 5 days, 5-mm diameter mycelial plugs of each isolated test fungus were cut by using a sterile cork borer and placed at the periphery of culture plates amended with tetracycline (0.1 g/L) by leaving 2 cm away from the edge side of the Petri dish, and on the same day, antagonist Trichoderma species were placed with equal distance on the opposite side of the same previous Petri dishes. For each treatment, three replications were used. The control plate was maintained by inoculating the medium with the pathogen only. The plates were incubated at 25 ± 2 • C in an incubator [34]. The growth of the pathogen in both the test and control experiments was recorded. The percent inhibition of the isolated test fungi was calculated by using the following formula of Vincent [30]: where R1: the radial colony growth of test fungi in the control plate (mm); R2: the radial colony growth of test fungi in the dual culture plate (mm) and the width of the zone of inhibition (ZI) was measured as the smallest distance between the colonies in the dual culture plate [35][36][37].

Data analysis
The obtained data were statistically analysed according to analysis of variance (ANOVA); Duncan's multiple range test was used for mean separation using SAS software for Windows version 9.2.

Isolated fungi and determination of infected seeds
Seedborne mycoflora of faba bean was tested by using the agar plate method as recommended by ISTA. Of the 50 farmers' saved seed samples collected from the five major faba bean-producing farmer associations in Ambo District, a total of 7 fungal species, F. oxysporum, F. solani, Aspergillus spp., Botrytis spp., Penicillium spp., R. solani and Alternaria spp. belonging to 6 fungal genera were isolated (Table 1).

Identification of fungi and frequency of occurrence
In this study, visual and microscopic observations were used to characterize the selected colony cultures. Details of the hyphal and spore characteristics of the fungi are noted in Table 2. Based on their colony morphology, mycelial growth, pigmentation and microscopic observations from a total of 12,545 cultures, 2584 were F. oxysporium, 2465F. solani, 1953 Aspergillus spp., 1703 Penicillium spp., 1294 Botrytis spp., 1278 R. solani and 1268 Alternaria spp. The results showed the morphology and mycelium characteristics of isolated strains, which will aid in the identification of isolated fungal strains in the future.

Effects of seedborne fungi on the germination of seeds
The effect of the seedborne fungi on the germination of seed samples was determined after 7 days of incubation. The results from the germination percentage of seed samples revealed that the germination percentage ranged from 81% to 97%. A significant difference in germination percentage among the seed samples was observed. The seed samples obtained from Golja kebele showed the highest percentage of germination (97%). The germination percentage of seeds differed significantly from location to location and from farmer to farmer. Seed germination of a major faba bean-producing farmer's association, Golja, was significantly higher (97%), followed by Jijigu Weransa (89%), but Kure Gatira had the lowest germination percentage (81%) ( Table 4).
Seed collected from Golja kebele in the farmer of seed sample code GF 2 showed the highest germination (97%), followed by the seed sample code GF 5 , GF 1 and JF 7 with germination (96%, 94% and 93%), respectively, but the seed collected from Kure Gatira kebele in the farmer of seed sample code KF 8 showed the lowest germination (81%), followed by the seed sample code YF 6 , DF 8 and KF 7 with germination (82%, 82% and 83%), respectively. The results also revealed that the sample with the highest fungal prevalence resulted in the lowest germination (Table 4). Among the seedborne fungi transmitted from seed to seedlings, distinct symptoms of damping off, seed rot and seedling blight were observed for Fusarium spp. and R. solani. All transmitted seedborne fungi may serve as the primary source of infection to the faba bean crop. These fungi serve as the primary inoculum for the spread of diseases and have epidemiological significance. The widespread distribution of Fusarium, Aspergillus and Penicillium species may be attributed to the wide occurrence of these fungi on a wide range of substrates and their efficient spore dispersal mechanism. In the present study, F. oxysporium and F. solani were isolated from almost all seed samples with high frequency, but R. solani rarely occurred ( Table 5). As observed from the results, three of the pathogens isolated from seed samples were F. oxysporium, F. solani and R. solani, which commonly cause root rot and damping-off disease of faba bean and are transmitted from seeds to seedlings in this experiment.

Antifungal activities of plant extracts
The in vitro antifungal effects of seven plants extracted with hot and cold water against the mycelial growth of F. oxysporium, F. solani and R. solani of test fungi by the poisoned food technique are shown as follows. The results presented in Table 6 revealed that all plant extracts significantly inhibited the mycelial growth of the three tested fungi (p < 0.05) at all tested concentrations and both types of aqueous extracts. However, the effects varied with the concentration of the extracts. The mycelial growth of the test fungi decreased as the concentration percentage of plants increased in both hot and cold water extracts, but when comparing the hot water extract with the cold water extract, the greatest inhibitory effect was shown by the hot water extract compared with the cold water extract, as shown in Table 6. Based on this, the impacts of various treatments on the mycelium growth of tested fungi in comparison with control/untreated plate the mycelium growth of the tested fungi has been influenced differently by the plant extracts in comparison to each other and test fungal pathogens (Supplementary material 1, Table S1-7).
The antagonistic effects of different plant extracts at different concentrations (5%, 10% and 20%) on mycelial growth and the inhibition zone of the tested fungi were significantly (P ≤ 0.01) different between the treatments. Among the plant extracts at 20% concentration, A. sativum L. caused the lowest mycelial growth of F. solani, F. oxysporum and R. solani (6.57 mm, 6.58 mm and 6.92 mm, respectively), followed by C. longa L. (7.00 mm, 7.08 mm and 7.42 mm, respectively) and P. dodecandra (7.42 mm, 7.58 mm and 7.92 mm, respectively), whereas the highest mycelial growth was recorded at the same concentrations extract of N. physalodes (L.) Gaertn. (9.42 mm, 9.58 mm and 9.83 mm, respectively). At a 5% concentration, the mycelial growth of the three test pathogens was recorded above 14.42 mm in all plant extract tests. Based on their mycelial growth, the inhibition zone percentages of F. oxysporum, F. solani and R. solani corresponding to seven plant extracts at 5%, 10% and 20% concentrations.
For both extraction methods (5%, 10% and 20%), the concentration revealed significant mycelial growth and inhibition zone percentage compared to each other (Table 6). Among them, at 20% concentration, the plants extracted in hot water caused the lowest mycelial growth of F. solani, F. oxysporum and R. solani (7.33 mm, 7.52 mm and 7.83 mm, respectively), followed by 10% concentration (11.17 mm, 11.67 mm and 12.00 mm, respectively), whereas the highest mycelial growth was recorded at the same extraction method of 5% concentration (15.33 mm, 15.83 mm and 16.17 mm, respectively). Based on their mycelial growth, the inhibition zone percentages of F. oxysporum, F. solani and R. solani affected at 5%, 10% and 20% concentrations of both extraction methods are shown in Table 6.

Evaluation of bioagents on mycelial growth of selected fungi under in vitro conditions
The inhibitory effects of T. harzianum, T. longibrachiatum, T. atroviride, and Trichoderma virens against the mycelial growth of the three test fungi F. oxysporum, F. solani and R. solani in the dual culture method are presented in Fig. 2 and Supplementary material 1, Table S8-13. The antagonistic effects of Trichoderma spp. against the mycelia growth of F. oxysporum were in the range of 5.50 mm-6.53 mm. T. longibrachiatum showed the highest inhibitory effect on mycelial growth (5.50 mm), followed by T. atroviride (6.00 mm), Trichoderma virens (6.50 mm) and T. harzianum (6.53 mm).
These results showed that the growth inhibition of F. solani by Trichoderma spp. was in the range of 5.00 mm-5.83 mm. T. longibrachiatum also inhibited the mycelial growth of F. solani, where the growth inhibition was 5.00 mm, followed by T. atroviride

Discussion
According to experimental work on the seedborne fungi associated with faba bean seeds, seven species of fungi were isolated and identified. Faba bean is susceptible to several fungal diseases that decrease production and lower the quality of seeds. The results of the study revealed that F. oxysporum, F. solani, Aspergillus spp. Penicillium spp. Botrytis spp. R. solani and Alternaria spp. were observed and identified. These results are in agreement with the findings of other researchers [15,[38][39][40][41]. Among the isolated fungi, Fusarium and Aspergillus species were the most predominant fungi in all seed samples. Seed-to-seedling transmission tests indicated that F. oxysporum, F. solani and R. solani isolates were the most common fungal isolates that significantly induced damping-off and root rot disease on faba bean plants. This result is similar to the findings of Elliot and Crowford [42], who found these fungal pathogenic organisms to be the most devastating rot-causing organisms in bean crops in different locations in the world [43]. These results showed that several root rot and wilt pathogens, such as F. oxysporum, R. solani and F. solani, are reported to attack faba bean roots and stem bases, causing serious losses in seed germination and seedlings. These results agree with those recorded by Abdel-Kader et al. [44]. Seedborne pathogen invasion reduces germination and nutrition and is also responsible for producing mycotoxins and loss of quality [45]. Seedborne diseases serve as primary inocula for the infection of the next developing crops, thereby reducing yields and qualities of the produce and playing a role in spreading the diseases to new areas [44]. The seedborne pathogens associated with seeds externally or internally may cause various infections, such as seed necrosis, reduction or elimination of germination capacity, and seedling damage, resulting in the development of disease at later stages of plant growth by systemic infection [46]. Infected seeds play a key role in the dissemination of plant pathogens and disease establishment [47]. The results of these studies are similar to those obtained by earlier workers and show that Fusarium is the most dominant species isolated from maize [48][49][50], and in Egypt, the main pathogens responsible for damping-off and wilt incidence of beans are R. solani (Kühn) and F. oxysporum f. sp. phaseoli [51]. Biological control of the three isolated test fungi by seven plant extracts and four Trichoderma spp. have been evaluated. The results revealed that all the tested plant extracts at 5%, 10% and 20% concentrations significantly inhibited the mycelial growth of all test fungi, and the four Trichoderma species also exhibited the strongest antagonistic activity. The inhibitory action of the aqueous plant extracts on mycelial growth increased with an increase in concentrations, and the hot water extracts provided higher effects/toxicity than the cold water extract in all test fungi. The results highlight the highest inhibitory effect of A. sativum L. extracted at 20% concentration against mycelial growth inhibition of the three test fungi (F. oxysporum, R. solani and F. solani), but N. physalodes (L.) Gaertn. extract at the same concentration showed the lowest inhibitory effects on the three test fungi. These results revealed that the antifungal activities of the extracts were enhanced by increasing the concentration from 5 to 20% (w/v); hence, the inhibition activities of the extracts were concentration dependent. This is in agreement with the reports of Ilondu [52], Chiejina and Ukeh [53], and Jasso et al. [54], who indicated that an increase in antifungal activities corresponded to an increase in the concentration of plant extracts.
The traditional medicinal use of plants extracted in this study, i.e., Kombolcha leaf (Maytenus senegalensis Loes.), Argissa (Aloe vera (L.) Burm.f), Abshoo leaf (N. physalodes (L.) Gaertn.), tabaco leaf (Nicotiana tabacum L.), Turmeric rhzome (Curcuma longa L.) and endod seeds (Phytolacca dodecandra L'Hér.) has been reported in Ethiopia [58][59][60][61]. In vivo and in vitro studies have shown that A. vera species has several biological activities, such as antifungal, antioxidant and antiviral properties [62]. Due to the restrictive application of fungicides, natural alternatives to synthetic fungicides, such as the use of essential oils, have shown efficacy in reducing decay [63]. The jel of A. vera could be applied as a treatment before harvesting to inhibit fungal spoilage and reduce the incidence of decay after harvesting storage of table grapes [64]. Aqueous extract of A. sativum has been used to treat plantlets of Harpagophytum procumbens by presoaking plantlets in the extract prior to planting out into the pot or by applying the extract as a soil drench after planting plantlets into the pot [65]. In a field trial, fungal disease severity reduction was achieved by the extraction of M. senegalensis Loes at a 5% concentration against Phytophthora infestans [66].
Alkaloid mixture, petroleum ether, and withanicandrin of N. physaloides Gaertn showed antifungal activities against S. cerevisiae and C. albicans [67]. Different compounds isolated from N. tabacum, such as alkaloids, nicotine, and capsidiol, have higher inhibitory activity against the fungus Phytophthora nicotianae [68]. C. longa has been reported to have toxic activity on fungi involved in the deterioration of crops by interfering with the development of mycelia [69]. It has been reported that the ethanol and hexane extracts of C. longa have significant antifungal activity against pathogenic fungi, such as Botrytis cinerea, Chaetomium olivaceum, Fusarium graminearum, and Mycogone perniciosa [70,71]. The methanol extract of its root had an antifungal effect against Candida albicans, Cryptococcus neoformans, Microsporum gypseum, and Trichophyton mentagrophytes [72]. Organic compound emitted from Trichoderma spp. inhibit mycelial growth of P. infestans grown on laboratory media and on potato tubers [73].

Conclusions and recommendations
The results obtained from this study showed that the extracts of different screened plants exhibit antifungal effects against F. oxysporum, F. solani and R. solani. In particular, hot water extracts of A. sativum L., M. senegalensis Loes., A. vera (L.) Burm.f, N. physalodes (L.) Gaertn., N. tabacum L., C. longa L. and P. dodecandra L'Hér. offer effective bioactive compounds for mycelial growth of the test fungi. The tested Trichoderma spp. also showed a good antagonistic effect against the three test fungi. These results indicate that plant extracts and Trachoderma spp. can be a potential alternative management option against plant disease and can be used as a seed treatment or seed coating. Hot water extracts of tested plants were revealed to be relatively more effective against the pathogen than cold water extracts. Active compounds of all test plants are extractable with water, and most are extracted with hot water. The results of this study revealed that cold and hot aqueous extracts of all the plants possess diverse antifungal activity against the test fungi. The differentiating activities against the selected isolate of these plant extracts encourage the development of broad-spectrum antifungals in the future. All the tested plant extracts and Trichoderma spp. contain antifungal agents and can be used against seedborne fungi in faba bean but need further purification for better efficacy. The results also confirmed that continuous effort to search for excellent plant extracts and antagonistic microorganisms is primarily needed. The results of the present study can be further exploited and tested under greenhouse and field experiments to formulate an integrated disease management schedule for root rot and damping-off disease of faba bean.

Author contribution statement
Amsalu Neme, Ararsa Leta, Amin Mohammed Yones: Conceived and designed the experiments; performed the experiments; analysed and interpreted the data and wrote the paper.
Muhidin Tahir: Analysed and interpreted the data and wrote the paper.

Data availability statement
Data will be made available on request.

Additional information
Supplementary content related to this article has been published online at [URL].

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.