Sustainable Management of Soil-Borne Bacterium Ralstonia solanacearum In Vitro and In Vivo through Fungal Metabolites of Different Trichoderma spp.

: The efﬁcacy of traditional control measures for the management of plant pathogens is decreasing, and the resistance of these pathogens to pesticides is increasing, which poses a serious threat to global food security. The exploration of novel and efﬁcient management measures to combat plant disease is an urgent need at this time. In this study, fungal metabolites from three Trichoderma spp. ( T. harzianum , T. virens and T. koningii ) were prepared on three different growth media (STP, MOF and supermalt (SuM)). The fungal metabolites were tested in vitro and in vivo from March–April 2020 under greenhouse conditions in a pot experiment utilizing completely randomized design to test their management of the bacterial wilt disease caused by R. solanacearum in tomato plants. The effect of the fungal metabolites on bacterial cell morphology was also investigated through scanning electron microscopy (SEM) analysis. In vitro investigation showed that the fungal metabolites of T. harzianum obtained on the STP medium were the most effective in inhibiting in vitro bacterial growth and produced a 17.6 mm growth inhibition zone. SEM analysis conﬁrms the rupture of the cell walls and cell membranes of the bacterium, along with the leakage of its cell contents. Generally, fungal metabolites obtained on an STP medium showed higher activity than those obtained on the other two media, and these metabolites were then evaluated in vivo according to three application times (0 days before transplantation (DBT), 4 DBT and 8 DBT) in a greenhouse trial to examine their ability to manage R. solanacearum in tomato plants. Consistent with in vitro results, the results from the greenhouse studies showed a level of higher anti-bacterial activity of T. harzianum metabolites than they did for the metabolites of other fungi, while among the three application times, the longest time (8 DBT) was more effective in controlling bacterial wilt disease in tomato plants. Metabolites of T. harzianum applied at 8 DBT caused the maximum decrease in soil bacterial population (1.526 log cfu/g), resulting in the lowest level of disease severity (area under disease progressive curve (AUDPC) value: 400), and maximum plant freshness (with a resulting biomass of 36.7 g, a root length of 18.3 cm and a plant height of 33.0 cm). It can be concluded that T. harzianum metabolites obtained on an STP medium, when applied after 8 DBT, can suppress soil bacterial population and enhance plant growth, and thus can be used as a safe, environmentally-conscious and consumer-friendly approach to managing bacterial wilt disease in tomato plants and possibly other crops.


Introduction
The tomato is the most consumed vegetable crop, and is ranked as the second most important after the potato. Because of its potential health benefits and economic importance,

Fungal Metabolites Preparations
Three fungal species were grown on three liquid media to produce anti-bacterial metabolites. The detailed recipe of the media is given in Table S1. From the freshly prepared fungal culture, five plugs were added to a 100 mL fungal seed medium (with a composition of 10 g of yeast extract, 4 g of agar, 40 g of maltose and 10 g/L of bactoneopeptone) in one 500 mL flask and kept on shaking incubation for 4 days at 25 • C [33]. Then, 5 mL of the obtained culture broth was used to inoculate each of the flasks containing 100 mL of the three liquid media used in this study and incubated at 25 • C for 2 weeks. After 2 weeks, the fungal metabolites were extracted using the extraction solvent ethyl acetate [33]. After drying the extraction solvent, the fungal metabolites were collected, preserved at 4 • C and used for their anti-bacterial potential against R. solanacearum.

In Vitro Test
The anti-bacterial efficacy of the fungal metabolites was checked using the agar well diffusion method [34]. The fungal metabolites were dissolved in methanol to a concentration of 150 mg mL −1 . In a petri plate, the LB medium, with 0.5 mL of bacterial suspension (10 8 cfu), was poured and allowed to cool to solidification. A total of five wells of uniform size were made in the solidified LB medium. Three wells were filled with 10 µL of the fungal metabolites obtained from the three growth media, one well was filled with a positive control (streptomycin (an antibiotic)) and one well was filled with a negative control (methanol). The plates were incubated at 28 • C for 24 h, and anti-bacterial activity was evaluated by measuring the inhibition zone. The test was repeated once, with five replicates in each test, and the results were presented as mean values.

Morphological Observation of the Bacterial Cells
The morphological destruction of the bacterial cells was evaluated using scanning electron microscopy (SEM). The bacterial samples were prepared by cutting small pieces of agar (with a thickness of 5 mm) from both the untreated control and from the inhibition zone of the best treatment and fixed in 2.5% (v/v) glutaraldehyde for one hour. After being washed with the phosphate buffer, the agar pieces were fixed in 1% (w/v) osmium tetroxide. The samples were then washed in a phosphate buffer and dehydrated in 30%, 50%, 70%, 90%, and 95% ethanol series, for 15 minutes each. After this, the samples were subjected to 100% ethanol and CO 2 . The fully dried samples were coated with gold sputtering and used for SEM analysis [35].

In Vivo Test
The fungal metabolites obtained on the STP medium (showing maximum in vitro anti-bacterial activity) were tested in vivo for their ability to manage R. solanacearum in tomato plants under greenhouse conditions. In brief, plastic pots with a diameter of 20 cm were filled with 1 kg of sterilized soil per pot. Thirty-five mL of a bacterial suspension (10 8 cfu mL −1 ) was poured in the center of each pot. For each of the three Trichoderma species tested, 15 mL of the fungal metabolites (9% w/v) obtained after a fungus cultivation on the STP medium were applied to the soil at three positions in the center of each pot 3 days after bacterial inoculation of the soil. One healthy tomato seedling of uniform size (25 days old) was transplanted into each pot. The fungal metabolites were applied separately at three different application times (i.e., 0, 4 and 8 days before transplantation (DBT)). The irrigation and fertilizer requirements were fulfilled according to the horticultural recommendations for tomato plants [36]. The experiment was repeated once, with five replications of the experiment conducted without any modification.

Data Parameters
The experiment was terminated after the 60th day since the transplantation was completed and the data on different plant growth parameters (i.e., fresh biomass, root length, plant height, bacterial population in soil and disease severity) were recorded.

Bacterial Population in Soil
To measure the number of bacteria in soil, four samples of soil cores were collected randomly from a 10-12 cm depth in each pot, and a composite sample was made by mixing the four samples together [37]. Three sub-samples were made from the composite samples and diluted serially up to 10 −7 . The 100 µL suspension from each sub-sample was poured on a petri plate containing 25 mL of the selective media tetrazolium chloride nutrient agar TZCNA [38]. The bacterial colonies with an off-white color and red center were counted and soil bacterial population was presented in terms of cfu g −1 of soil.

Area under Disease Progressive Curve (AUDPC)
The disease severity of the bacterial wilt affecting the tomato plants was recorded using a specified scale and converted to % disease severity [39]. The disease severity was measured 5 times every 12 days and converted to an AUDPC value using the AUDPC formula [40].

Statistical Analysis
Results were presented as mean values ± standard deviation. Analysis was done using ANOVA with IBMSPSS Statistix 20 software. Factorial arrangements were applied to both in vitro and in vivo data. Least significant difference (LSD) test was conducted to indicate significant differences among the treatments [41].

In Vitro Test
Fungal metabolites of the three Trichoderma spp. (T. harzianum, T. virens and T. koningii) obtained on three different growth media (STP, MOF and supermalt (SuM)) were tested for in vitro anti-bacterial activity against R. solanacearum. Significant differences in antibacterial activity were observed among the treatments (Figures 1 and 2). Among different fungal species, the fungal extracts of T. harzianum showed the highest anti-bacterial activity, and across different media, the fungal metabolites obtained on the STP media showed the highest anti-bacterial activity. The fungal metabolites obtained on the MOF and SuM media exhibited similar anti-bacterial activity from all three fungal species. After that of the positive control (streptomycin), the maximum growth inhibition zone was produced by the fungal metabolites of T. harzianum obtained on the STP media, followed by the T. virens fungal extracts obtained on the STP media. Growth inhibition zones of 17.6 mm, 13.1 mm and 12.6 mm were produced by T. harzianum metabolites obtained on the STP, MOF and SuM media, respectively. The inhibition zones for T. virens metabolites obtained on the STP, MOF and SuM media were recorded as 13.2 mm, 9.6 mm and 8.1 mm, respectively, while T. koningii metabolites obtained on STP, MOF and SuM media produced 10.3 mm, 7.4 mm and 7.2 mm growth inhibition zones, respectively. The negative control methanol did not show any activity and produced no inhibition zone. obtained on the STP, MOF and SuM media were recorded as 13.2 mm, 9.6 mm and 8.1 mm, respectively, while T. koningii metabolites obtained on STP, MOF and SuM media produced 10.3 mm, 7.4 mm and 7.2 mm growth inhibition zones, respectively. The negative control methanol did not show any activity and produced no inhibition zone.

Bacterial Cell Morphology
The morphological alterations in bacterial cells were observed through SEM analysis. The treated and untreated bacterial cells exhibited a clear difference in their morphology ( Figure 3). The morphology of bacterial cells treated with fungal metabolites (the most effective treatment of which was T. harzianum metabolites obtained on STP media) underwent tremendous destruction. The cell content leakage, cell wall degradation and membrane breakdown can be easily observed. The bacterial cells belonging to control group (without any treatment) showed normal rod-shaped uniform bacterial morphology without any interference. obtained on the STP, MOF and SuM media were recorded as 13.2 mm, 9.6 mm and 8.1 mm, respectively, while T. koningii metabolites obtained on STP, MOF and SuM media produced 10.3 mm, 7.4 mm and 7.2 mm growth inhibition zones, respectively. The negative control methanol did not show any activity and produced no inhibition zone.

Bacterial Cell Morphology
The morphological alterations in bacterial cells were observed through SEM analysis. The treated and untreated bacterial cells exhibited a clear difference in their morphology ( Figure 3). The morphology of bacterial cells treated with fungal metabolites (the most effective treatment of which was T. harzianum metabolites obtained on STP media) underwent tremendous destruction. The cell content leakage, cell wall degradation and membrane breakdown can be easily observed. The bacterial cells belonging to control group (without any treatment) showed normal rod-shaped uniform bacterial morphology without any interference.

Bacterial Cell Morphology
The morphological alterations in bacterial cells were observed through SEM analysis. The treated and untreated bacterial cells exhibited a clear difference in their morphology ( Figure 3). The morphology of bacterial cells treated with fungal metabolites (the most effective treatment of which was T. harzianum metabolites obtained on STP media) underwent tremendous destruction. The cell content leakage, cell wall degradation and membrane breakdown can be easily observed. The bacterial cells belonging to control group (without any treatment) showed normal rod-shaped uniform bacterial morphology without any interference.

Plant Growth
In a green house experiment, the fungal metabolites of three Trichoderma spp. (T. harzianum, T. virens and T. koningii) obtained on an STP medium and applied at three different times (0, 4 and 8 DBT) were evaluated for their ability to manage bacterial wilt disease in tomato plants. The results showed significant differences both for Trichoderma spp. and application times in terms of affecting plant growth parameters such as fresh biomass, root length and plant height ( Figure 4). Among the three application times, 8 DBT gave the best results, and among the three fungal metabolites, the T. harzianum metabolites gave the best results. The maximum fresh biomass (51.3 ± 3.6 g), root length (27.4 ± 2.4 mm) and plant height (46.3 ± 3.1) were achieved when T. harzianum metabolites were applied at 8 DBT. The minimum effect on plant growth was noticed for the treatment combination of T. koningii and 0 DBT that gave 25.2 ± 1.7 g, 10.5 ± 0.9 cm and 16.5 ± 2.1 cm in fresh biomass, root length and plant height, respectively. A similar trend in the results was noticed in the repeated experiment, where the application time 8 DBT and the fungal metabolites of T. harzianum gave the best plant growth as compared to other application times and fungal metabolites (Tables 1-3, Experiment II).

Plant Growth
In a green house experiment, the fungal metabolites of three Trichoderma spp. (T. harzianum, T. virens and T. koningii) obtained on an STP medium and applied at three different times (0, 4 and 8 DBT) were evaluated for their ability to manage bacterial wilt disease in tomato plants. The results showed significant differences both for Trichoderma spp. and application times in terms of affecting plant growth parameters such as fresh biomass, root length and plant height (Figure 4). Among the three application times, 8 DBT gave the best results, and among the three fungal metabolites, the T. harzianum metabolites gave the best results. The maximum fresh biomass (51.3 ± 3.6 g), root length (27.4 ± 2.4 mm) and plant height (46.3 ± 3.1) were achieved when T. harzianum metabolites were applied at 8 DBT. The minimum effect on plant growth was noticed for the treatment combination of T. koningii and 0 DBT that gave 25.2 ± 1.7 g, 10.5 ± 0.9 cm and 16.5 ± 2.1 cm in fresh biomass, root length and plant height, respectively. A similar trend in the results was noticed in the repeated experiment, where the application time 8 DBT and the fungal metabolites of T. harzianum gave the best plant growth as compared to other application times and fungal metabolites (Tables 1-3, Experiment II).

Plant Growth
In a green house experiment, the fungal metabolites of three Trichoderma spp. (T. harzianum, T. virens and T. koningii) obtained on an STP medium and applied at three different times (0, 4 and 8 DBT) were evaluated for their ability to manage bacterial wilt disease in tomato plants. The results showed significant differences both for Trichoderma spp. and application times in terms of affecting plant growth parameters such as fresh biomass, root length and plant height (Figure 4). Among the three application times, 8 DBT gave the best results, and among the three fungal metabolites, the T. harzianum metabolites gave the best results. The maximum fresh biomass (51.3 ± 3.

Soil Bacterial Population
At the beginning and the end of the experiment, the soil bacterial population was counted. The difference in the number of bacteria from the beginning to the end is classified by the decrease in bacterial population. The results showed that soil treatment with fungal extracts significantly affected the soil bacterial population. The three application times of the fungal extract also showed significant differences regarding the decrease in soil bacterial population. Among application times, the 8 DBT was the most effective at suppressing soil bacterial population, especially when combined with T. harzianum metabolites, which were the most highly active of the fungal metabolites in suppressing soil bacterial population. T. harzianum metabolites applied at 8 DBT showed a maximum decrease of 1.526 log cfu/g in soil bacterial population, followed by the decrease exhibited by T. virens. Meanwhile, among all the treatments, T. konigii metabolites applied at 0 DBT exhibited the lowest decrease (0.686 log cfu/g) in soil bacterial population ( Figure 5A). Similar results were shown by the treatments when the experiment was repeated (Figure 5B).
At the beginning and the end of the experiment, the soil bacterial population was counted. The difference in the number of bacteria from the beginning to the end is classified by the decrease in bacterial population. The results showed that soil treatment with fungal extracts significantly affected the soil bacterial population. The three application times of the fungal extract also showed significant differences regarding the decrease in soil bacterial population. Among application times, the 8 DBT was the most effective at suppressing soil bacterial population, especially when combined with T. harzianum metabolites, which were the most highly active of the fungal metabolites in suppressing soil bacterial population. T. harzianum metabolites applied at 8 DBT showed a maximum decrease of 1.526 log cfu/g in soil bacterial population, followed by the decrease exhibited by T. virens. Meanwhile, among all the treatments, T. konigii metabolites applied at 0 DBT exhibited the lowest decrease (0.686 log cfu/g) in soil bacterial population ( Figure 5A). Similar results were shown by the treatments when the experiment was repeated ( Figure  5B).

AUDPC
The data recorded on disease severity of the tomato plants grown in soil treated with fungal extracts at different application times were changed to AUDPC. Results showed significant differences among the treatments. The plants grown in untreated control soil showed the maximum AUDPC value and died completely. Compared to the control, the plants grown in treated soil exhibited lower AUDPC values. Among fungal spp., the metabolites of T. harzianum were the most effective in lowering the AUDPC value, and among application times, 8 DBT was the most effective in lowering the AUDPC value. The fungal metabolites of T. harzianum applied at 8 DBT had the lowest AUDPC value (400). The maximum AUDPC value (1750) was shown by plants grown in soil treated with T. koningi applied at 0 DBT ( Figure 6A). The experiment was repeated without any modification and similar results were obtained ( Figure 6B). The data recorded on disease severity of the tomato plants grown in soil treated with fungal extracts at different application times were changed to AUDPC. Results showed significant differences among the treatments. The plants grown in untreated control soil showed the maximum AUDPC value and died completely. Compared to the control, the plants grown in treated soil exhibited lower AUDPC values. Among fungal spp., the metabolites of T. harzianum were the most effective in lowering the AUDPC value, and among application times, 8 DBT was the most effective in lowering the AUDPC value. The fungal metabolites of T. harzianum applied at 8 DBT had the lowest AUDPC value (400). The maximum AUDPC value (1750) was shown by plants grown in soil treated with T. koningi applied at 0 DBT ( Figure 6A). The experiment was repeated without any modification and similar results were obtained ( Figure 6B).

Discussion
Managing phytopathogens through the metabolites of biocontrol agents that are biologically active is a potentially environmentally-conscious and consumer friendly approach [22]. In this study, the metabolites of three Trichoderma spp. obtained on three different growth media, were tested for their anti-bacterial potential in managing R. solanacearum both in vitro and in vivo in tomato plants.
The results from in vitro evaluation showed the strong bioactivity of the fungal extracts. All of the fungal metabolites were not equally active against R. solanacearum. Generally, the fungal metabolites of T. harzianum exhibited higher anti-bacterial activity, followed by the fungal metabolites of T. virens and T. koningi. This is due to the fact that different species (as well as different strains) of the fungus produce different secondary metabolites (SMs). It is reported that different Trichoderma isolates exhibited different extents of anti-microbial activity against pathogens [32]. The results obtained in this study are in line with previously reported results, in which metabolites of different Trichoderma spp. were evaluated in vitro against different plant pathogens, and it was found that T. harzianum metabolites were more active than other species [33]. The anti-bacterial activity of the fungal metabolites could be attributed to the several anti-bacterial compounds present in the metabolites. Different bioactive compounds have previously been reported from Trichoderma spp. and used against plant pathogenic bacteria [42][43][44][45]. Several studies have also reported the ability of SMs to inhibit nucleic acid and cell wall synthesis and destroy membranes [46][47][48]. The lysosime was reported to cause membrane breakdown and cell content leakage, which lead to cell death [49]. The analysis of bacterial cell morphology in this study also confirms that these mechanisms (such as the disintegration of bacterial cell wall and membranes) can be easily seen in SEM micrographs.
The results from the in vivo tests indicated that fungal metabolites, when applied to soil infested with R. solanacearum, can suppress the bacterial population in soil, reducing the disease severity and improving plant growth. Consistent with the results of the in vitro tests, the fungal metabolites of T. harzianum were more active than the metabolites of other fungus. This is because the fungal metabolites of T. harzianum may have stronger anti-microbial metabolites against R. solanacearum. The ability of the anti-biotic production is usually isolate or species dependent. A specific anti-biotic can be produced by different fungal species, and, in a similar way, a specific fungal species can produce several bioactive compounds [29]. The secretion of anti-microbial compounds is one of the major anti-microbial mechanisms of a biocontrol agent. It is reported that the use of fungal metabolites against the pathogens exhibited similar results that the same processes achieved through corresponding micro-organisms [28]. The improved plant growth resulting from the application of fungal metabolites can also be explained on the basis of the plant growth promoting properties of Trichoderma metabolites. Along with direct antagonistic or parasitic activity against pathogenic microbes, Trichoderma spp. also secrete various compounds that alter the host metabolism positively and affect its development and growth. It was reported that plant seeds exhibited increased productivity when exposed to conidia of Trichoderma spp., which suggests that metabolites from Trichoderma spp. can act as signaling compounds in addition to promoting plant growth effects [50]. Several Trichoderma strains are capable of producing secondary metabolites involved in auxin-dependent mechanisms to enhance root growth and increase plant biomass [51,52]. The role of fungal metabolites (i.e., koninginins A, B, C, E and G isolated from T. koningii and 6-pentyl-alpha-pyrone isolated from T. harzianum) as growth regulators in wheat plants was reported by several studies [53][54][55][56]. Trichocaranes A, B, C and D produced by T. virens significantly affected the growth of etiolated wheat coleoptiles [57]. A sesquiterpene metabolite cyclonerodiol obtained from culture filtrate of T. harzianum and T. koningii demonstrated a regulatory effect on plant growth [58,59]. The direct toxic effect to the R. solanacearum, as well as the plant growth promoting effect of compounds present in the fungal metabolites, collectively contributed to enhanced plant growth. As discussed above, the anti-bacterial compounds affect the bacterial population in a variety of ways, including targeting membranes and DNA and causing protein destructions [45,47,48]. This direct toxic effect of fungal metabolites contributes largely to a higher decrease in the soil bacterial population, resulting in a lower disease severity (or AUDPC) and the resulting enhanced plant growth.
It was noted that a longer application time (8 DBT) was superior to shorter application times (4 DBT and 0 DBT). This is because the longer application time helps in thorough mixing, as well as increasing the stability and compatibility of fungal metabolites in the soil, resulting in increased activity of the extract. Our results are in line with the findings of Khan et al. [60], where an organic soil amendment with different application times was used to test its management of R. solanacearum in tomato plants. The results suggested that a longer application time exhibited the best results because of the increased stability and compatibility of the treatments, which, in turn, release more anti-microbial compounds in the soil. The fungal extract was prepared on low cost media without the use of heavy mechanical or chemical involvement. The extract was active at a low concentration and effectively reduced soil bacterial population and improved plant growth. The fungi belonging to the Trichoderma genus are well known and widely used as biocontrol agents. Recently, the management of bacterial wilt disease was enacted through direct application of the Trichoderma spp. to the soil [31,32]. However, the metabolites were not evaluated beforehand for their ability to manage bacterial wilt disease in tomato plants and other crops. The results of this study indicated the potential of Trichoderma metabolites to manage R. solanacearum on tomato plants. However, such potential can be evaluated only in comparison with other protection techniques applied currently on tomato plants against this pathogen. This study will also provide a basis for the commercial preparation of Trichoderma metabolites in the market for the purpose of managing R. solanacearum in tomato plants and possibly other crops and, therefore, could be useful for global research and agricultural communities.

Conclusions
In this study, fungal metabolites of three Trichoderma spp. (i.e., T. harzianum, T. virens and T. koningii) obtained on three different growth media (STP, MOF and SuM) were tested in vitro for their anti-bacterial activity and in vivo for their anti-bacterial potential to manage bacterial wilt disease caused by R. solanacearum in tomato plants. Fungal metabolites of T. harzianum obtained on the STP medium were most effective in the management of R solanacearum. T. harzianum metabolites caused the maximum decrease in soil bacterial populations, resulted in lowered disease severity and enhanced plant growth. The results from this study suggest that T. harzianum metabolites can be used as eco-friendly, cost-effective and efficient tools to manage R. solanacearum in tomato plants and possibly other plants.