Antagonistic Activity of Extremophilic Bacteria Against Phytopathogens in Agricultural Crops

. Wheat is a vital agricultural crop whose phytopathogens include fungi of the genera Fusarium and Alternaria . Synthetic pesticides, which are used to combat them, have a negative impact on the environment. Therefore, there is a need for developing safe and effective biopesticides. We aimed to create a consortium of extremophilic microorganisms isolated from natural sources to protect wheat from the diseases caused by Alternaria and Fusarium fungi. Ten isolates of extremophilic microorganisms were tested for their antimicrobial activity against Escherichia coli and their antagonistic activity against phytopathogens. Based on the results, we developed microbial consortia and evaluated their effectiveness in protecting wheat from phytopathogens. Five of the strains under study showed the highest activity, three of which were biocompatible, namely Leclercia sp., Sphingomonas paucimobilis , and Lactobacillus plantarum . Four consortia were created from these microorganisms, of which consortium B (with a 2:1:1 ratio of the strains, respectively) proved the most effective. In particular, it increased the area free from the phytopathogen by 4.2% compared to the average values of its individual microorganisms. Also, the consortium had a phytostimulating effect on wheat seedlings (germination of 73.2–99.6%) and protected the seeds infected with phytopathogens from morphometric changes. The resulting consortium can be used as a biopesticide since it is highly effective in protecting wheat from Alternaria and Fusarium pathogens.


Introduction
Wheat (Triticum aestivum L.) is a vital agricultural crop that makes a significant contribution to the food security.However, its yield and nutritional value are greatly reduced by various diseases caused by phytopathogenic microorganisms [1,2].
Fusariosis is the most common disease in wheat.It is a pathological condition of cultivated and wild plants caused by microscopic fungi of the genus Fusarium.This phytopathogen deforms wheat ears and causes them to prematurely lose pigmentation [3,4].The grain shrinks, becomes brittle, and its germination capacity decreases [5].Moreover, mycotoxins accumulate in the grain, posing a threat to human and animal health [6,7].According to literature, mycotoxins reduce the resistance of wheat to other phytopathogens [8].
Alternaria blight is another common wheat disease caused by pathogens of the genus Alternaria.These microscopic fungi cause black spots of mycelium to form on the ears, disrupting the crop's normal development [9].In some cases, Alternaria pathogens directly affect the grains, causing their shell to darken.This does not affect their ability to germinate, but increases their allergenicity [10].
Synthetic pesticides are most often used to combat these and other phytopathogens that cause infectious diseases in wheat [11].However, their use is associated with a number of environmental problems.Pesticides are stable compounds that can persist in the environment for a long time, causing pollution of soils, ground and surface waters, as well as the atmosphere [12][13][14].When used for extended periods, they accumulate in agricultural soils, causing qualitative and quantitative changes in the microbiome of the rhizosphere and phylosphere.In particular, they decrease the diversity of bacteria and fungi, as well as affect the nitrogenfixing and colonizing abilities of rhizobacteria [15,16].This has a negative impact on cultivated crops such as wheat.Moreover, of considerable concern is the potential of synthetic pesticides for bioaccumulation.They accumulate in the edible parts of the crops, causing harm to human health [17].
Thus, there is a need for alternative methods that exclude the use of synthetic pesticides and ensure the environmentally safe protection of wheat from phytopathogens.According to literature, such methods involve biological means of protection, for example, biopesticides obtained by microbial synthesis [18].Microorganisms in such preparations are capable of synthesizing a wide range of secondary metabolites that can control the development of infectious diseases in plants [19].
Biopesticides can be developed from extremophiphilic microorganisms.Their survival strategies in adverse environmental conditions are due to their unique qualities [20].For example, some extremophiles are able to secrete antibiotic substances to reduce the number of competing species [21].However, their antagonistic activity is associated with not only antibiotics, but also certain enzymes.For example, Pseudomonas sp.isolated from marine sediments produced chitinase, an enzyme that significantly inhibited the development of phytopathogenic fungi [22].Thus, high antagonistic activity makes extremophiles effective biocontrol agents.

Study objects and methods
We studied the extremophilic bacteria previously isolated from natural sources [23].
Four of the isolates were identified before, while the remaining six were identified using a Vitek 2 Compact automatic microbiological analyzer (Biomerieux, France).For this, microorganisms were cultivated on Columbian blood agar (Himedia, India) for 48 h at 28°C.The resulting cultures were used to prepare suspensions with a McFarland density of 2.70-3.30[24].
The antagonistic activity of the strains against bacterial cultures was tested on the model microorganism Escherichia coli.For this, isolates were grown in the MPB medium at 28°C for 48 h.Then, 1 mL of the culture liquid was centrifuged at 5000 rpm for 5 min, and the supernatant was removed.E. coli were inoculated into Petri dishes with a sterile MPA medium.Then, we cut out wells 6 mm in diameter and filled them with 50 µL of the supernatant.The dishes with the wells were placed in a thermostat and kept for 24 h at 28°C.The results were interpreted by measuring the diameter of inhibition zones [25].
The antagonistic activity of the isolates against the phytopathogenic fungi was assessed by the crossculture method [26].For this, we placed daily cultures of the isolates onto one side of Petri dishes with potatoglucose agar (Himedia, India) and agar blocks with the phytopathogenic fungi on the other side.The Petri dishes were kept in a thermostat at 28°C, and the inhibition zones were monitored after 3, 5, and 7 days.The control was the Petri dishes with the phytopathogen without the antagonist culture.Radial growth inhibition was calculated according to the formula as follows: Radial growth inhibition 1 -× 100 dr ds where dr is the diameter of the fungus mycelium in a Petri dish with the antagonist culture, mm; ds is the diameter of the fungus mycelium in the control, mm.To create a consortium, we evaluated the biocompatibility of the most promising strains of microorganisms by their co-cultivation.For this, pure cultures of the isolates were cultivated in MPB medium at 28°C for 48 h.Then, the culture liquid was centrifuged for 5 min at 5000 rpm.Isolate No. 1 was evenly applied onto a Petri dish with the MPA medium, and the supernatant of isolate No. 2 was added into wells 6 mm in diameter.The cultures were cultivated at 28°C for 24 h, followed by the monitoring of inhibition zones.This method was used for all the isolates [27].
The antagonistic activity of the consortia was assessed as described above.
To measure the consortia's ability to reduce the toxic effects of the phytopathogens on wheat (Triticum aestivum L.), the seeds were treated with a mixture of the consortium and the phytopathogen in a ratio of 1:1.Prior to this, the seeds were sterilized with a 5% sodium hypochlorite solution for 10 min, washed 5 times with sterile distilled water, and dried for 2 h in a sterile laminar box (Laminar Systems, Russia).
To infect the seeds, they were treated with a phytopathogen suspension (2.5×10 5 ) prepared by washing off the mycelium and spores of the fungus grown on slant agar at 28°C for 48 h.The seeds were soaked in the suspension for 2 h and then dried under sterile conditions.A consortium of microorganisms for treating the seeds was prepared in a similar way, with the isolates cultivated at 28°C.After the treatment, the seeds were dried and placed on Petri dishes with moistened filter paper discs (25 seeds per dish).The seeds were incubated in a climate chamber (Binder, Germany) at 25°C and 40% humidity.The control was the seeds that were not treated with the phytopathogens or the consortium [28].
Each experiment was performed in triplicate.Mathematical processing was carried out using the Microsoft Office software package.

Results and discussion
The biochemical identification was carried out for 6 microorganisms (Tables 1 and 2).
We identified isolate No. Escherichia coli was used as a model microorganism to study the antimicrobial activity of the isolates (Fig. 1).Antimicrobial activity is an ability of microorganisms to produce substances that inhibit the development of other microorganisms.It can be used to prevent the growth of pathogenic microflora in an area, especially in agriculture to increase the survival rate of plants [29,30].
According to the results, 5 strains did not show any antimicrobial activity against E. coli, namely K. oxytoca, S. paucimobilis, S. maltophilia, B. megaterium, and L. plantarum.The inhibition zones of the other strains varied from 1.0 to 3.0 mm.Since most of the microorganisms under study did not have bactericidal properties, further tests aimed to measure their antagonistic activity against fungal phytopathogens (Table 3).
Most antagonist strains showed maximum activity against the phytopathogens on the 7th day of cultivation.However, the activity of some strains peaked on the 5th day of cultivation and remained at the same level, e.g., the activity of Pantoea sp. and P. putida against A. alternata (7.3 and 8.2%, respectively), or the antagonicity of K. oxytoca against F. graminearum PH-1 (F-877) (9.9%).
Our data are consistent with the results reported in modern scientific literature.For example, various   [31].The L2 strain of B. megaterium inhibited the sporulation (by 96.02%) and growth of the mycelium of this phytopathogen [32].
The genus Pseudomonas has been reported to suppress Alternaria.According to Gupta et al., Pseudomonas fluorscens exhibited antimicrobial properties against Alternaria brassicae [33].In addition, the isolate stimulated the growth of agricultural crops.
High antimicrobial activity of S. maltophilia against Alternaria was observed by Jankiewicz et al. [34].According to the authors, it was due to the release of an active chitinolytic enzyme belonging to the family of 18 glycosyl hydrolases into the substrate.S. maltophilia also showed antagonicity against the fungal phytopathogens Rhizoctonia and Fusarium.
Bacteria of the genus Pseudomonas have been reported to exhibit antagonistic activity against phytopathogens of the genus Fusarium.For example, Chavéz-Díaz et al. described the ability of three Pseudomonas isolates from the rhizosphere of Mexican maize to inhibit the growth of the phytopathogen mycelium and increase the rate of seed germination [35].The seedlings treated with the isolates had a more deveped root system and aerial part.Literature also reports the effective inhibition of Fusarium by a strain of L. plantarum.This microorganism is able to colonize wheat ears and suppress fungal diseases, increasing the nutritional properties of the grain [36].Pantoea sp. and Enterobacter sp. were also found to reduce the impact of the Fusarium phytopathogens on the root system of cultivated plants, both in greenhouse and field conditions [37].
Thus, the microorganisms that we isolated in this study have great potential in the fight against phytopathogens.
To create consortia, we evaluated the biocompatibility of the isolates (Table 4).
We found that the strain E. aerogenes was not compatible with Leclercia sp., S. paucimobilis, and L. plantarum, as it suppressed their growth.The strain Leclercia sp. had a positive effect on the growth of S. paucimobilis and L. plantarum.S. paucimobilis metabolites inhibited the growth of E. aerogenes and B. megaterium, while Leclercia sp. and L. plantarum contributed to their active growth.The microorganism B. megaterium was only compatible with S. paucimobilis.L. plantarum metabolites adversely affected the growth of E. aerogenes and B. megaterium.Based on the results, we selected those strains that did not exhibit antagonistic activity against each other, namely Leclercia sp., S. paucimobilis, and L. plantarum.We created four variants of consortia based on these strains (Table 5).
The antagonistic activity of the consortia against the phytopathogenic fungi of the genera Alternaria and Fusarium are shown in Table 6.
As can be seen, consortium B showed high anta gonistic activity against the phytopathogenic fungi.In particular, the area free of A. alternata increased by 4.2% in relation to the average value achieved by individual microorganisms in the consortium.Consortium B's activity against the genus Fusarium increased by an average of 20.2% on the 7th day of cultivation.Consortium A, however, showed low antagonistic activity on the 7th day against A. alternata and F. graminearum (F-892), with 9.4 and 5.2% below the average, respectively.Consortium C's activity against A. alternata and F. graminearum PH-1 (F-877) decreased by 2.2 and 7.6%, respectively.Moreover, its activity against F. sporotrichioides was the lowest among the consortia under study, amounting to 48.0% (19.3% lower than the average value).Consortium D showed low antagonistic activity against the fungi of the genus Fusarium.In particular, the area free of F. graminearum PH-1 (F-877) and F. graminearum (F-892) decreased by 16.2 and 14.6%, respectively.All the consortia showed maximum activity against phytopathogens on the 7th day of cultivation.
Table 7 shows the consortia's ability to inhibit the phytopathogenic effect on wheat.When the seeds were treated by both the consortium and the phytopathogens, their germination varied within 73.2-99.6%.The consortia showed the strongest effect against F. graminearum PH-1 (F-877).
As can be seen, consortium B had the highest phytostimulating effect, with an average of 24.8 germinated seeds, while consortium A had the lowest phytostimulating effect, with an average of 21 germinated seeds.Consortium B had the greatest effect on wheat seedlings, contributing to a 10.5% higher average coleoptile length than in the control samples.However, when the seeds were inoculated with consortium A, the average coleoptile length was 39.1 mm, i.e., 1.5% shorter than in the control.Treating the seeds with consortium B increased the total length of the seedling roots by 7.2% compared to the control (treated with water).Consortium D, however, decreased this indicator by 1.9% compared to the control, leading to an average length of 185.9 mm.The smallest number of roots per plant was provided by consortia C and D (1.15 and 1.16% below the control, respectively).Consortium B, however, increased the average number of roots 1.13 times compared to the control.Figure 2 shows the seedlings treated with consortium B and F. graminearum (F-892), as well as the control sample without this treatment.
Noteworthily, we found no visual morphometric defects in any of the wheat samples treated with the consortia.The sprouts had a uniform color that did not differ from that of the control samples (untreated with the consortia and phytopathogens).

Table 1 .
Biochemical characteristics of gram-negative microorganisms

Table 2 .
Biochemical characteristics of gram-positive microorganisms

Table 4 .
Biocompatibility of the isolated microorganisms