CC BY
17
2023 Т. 53 № 3 / Техника и технология пищевых производств / Food Processing: Techniques and Technology
ISSN2074-9414 (Print) ISSN 2313-1748 (Online)
https://doi.org/1Q.216Q3/2Q74-9414-2Q23-3-2457 https://elibrary.ru/ZPULFC
Original article Available online at https://fptt.ru/en
Antagonistic Activity of Extremophilic Bacteria Against Phytopathogens in Agricultural Crops
Lyudmila K. Asyakina1'* , Yuliya R. Serazetdinova1 , Anna S. Frolova1 , Natalya V. Fotina1 , Olga A. Neverova1 , Andrey N. Petrov2
1 Kemerovo State University^**, Kemerovo, Russia 2 Russian Biotechnological University^*1, Moscow, Russia
*Lyudmila K. Asyakina: [email protected], https://orcid.org/0000-0003-4988-8197 Yuliya R. Serazetdinova: https://orcid.org/0000-0002-3044-3529 Anna S. Frolova: https://orcid.org/0000-0003-3988-8521 Natalya V. Fotina: https://orcid.org/0000-0002-7655-0258 Olga A. Neverova: https://orcid.org/0000-0002-0309-5709 Andrey N. Petrov: https://orcid.org/0000-0001-9879-482X
© L.K. Asyakina, Yu.R. Serazetdinova, A.S. Frolova, N.V. Fotina, O.A. Neverova, A.N. Petrov, 2023
Abstract.
Wheat is a vital agricultural crop whose phytopathogens include fungi of the genera Fusarium and Alternaría. 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.
Keywords. Triticum aestivum L., agricultural productivity, phytopathogens, biopesticides, extremophilic bacteria, consortium of microorganisms, environmental safety
Funding. This study was part of the state assignment entitled "Basic research on the development of biopesticides from extremophilic and endophytic microorganisms to help agricultural crops overcome abiotic and biotic stress in Kemerovo Oblast - Kuzbass" (code FZSR-2023-0003).
For citation: Asyakina LK, Serazetdinova YuR, Frolova AS, Fotina NV, Neverova OA, Petrov AN. Antagonistic Activity of Extremophilic Bacteria Against Phytopathogens in Agricultural Crops. Food Processing: Techniques and Technology. 2023;53(3):565-575. https://doi.org/10.21603/2074-9414-2023-3-2457
Received: 20.03.2023 Revised: 19.04.2023 Accepted: 02.05.2023
https://doi.org/10.21603/2074-9414-2023-3-2457 Оригинальная статья
https://elibrary.ru/ZPULFC https://fptt.ru
Антагонистическая активность экстремофильных микроорганизмов в отношении фитопатогенов сельскохозяйственных культур
Л. К. Асякина1-* , Ю. Р. Серазетдинова1 , А. С. Фролова1 , Н. В. Фотина1 , О. А. Неверова1 , А. Н. Петров2
1 Кемеровский государственный университет1*®*, Кемерово, Россия
2 Российский биотехнологический университет**®*, Москва, Россия
Поступила в редакцию: 20.03.2023 *Л. К. Асякина: [email protected],
Принята после рецензирования: 19.04.2023 https://orcid.org/0000-0003-4988-8197
Принята к публикации: 02.05.2023 Ю. Р. Серазетдинова: https://orcid.org/0000-0002-3044-3529
А. С. Фролова: https://orcid.org/0000-0003-3988-8521 Н. В. Фотина: https://orcid.org/0000-0002-7655-0258 О. А. Неверова: https://orcid.org/0000-0002-0309-5709 А. Н. Петров: https://orcid.org/0000-0001-9879-482X
© Л. К. Асякина, Ю. Р. Серазетдинова, А. С. Фролова, Н. В. Фотина, О. А. Неверова, А. Н. Петров, 2023
Аннотация.
Пшеница (Triticum aestivum L.) - важная сельскохозяйственная культура, фитопатогенами которой являются грибы рода Fusarium и Alternaria. Для борьбы с ними применяют синтетические пестициды, негативно влияющие на окружающую среду и здоровье человека. Разработка безопасных и эффективных аналогов - биопестицидов - является актуальным вопросом. Цель работы заключалась в разработке консорциума на основе экстремофильных микроорганизмов, выделенных из природных источников, для защиты пшеницы от заболеваний, вызванных грибами рода Alternaria и Fusarium. Объектами исследования являлись образцы 10 изолятов экстремофильных микроорганизмов. Биохимическую идентификацию изолятов проводили с использованием автоматического микробиологического анализатора Vitek 2 Compact. Изоляты оценивали по показателям антимикробной активности в отношении Escherichia coli и антагонистической активности в отношении фитопатогенов по методу встречных культур. На основании полученных данных конструировали микробные консорциумы и оценивали их эффективность и способность защищать пшеницу от фитопатогенов. Из 10 исследованных изолятов наибольшую активность проявляли 5 штаммов, 3 из которых являлись биосовместимыми: Leclercia sp., Sphingomonas paucimobilis и Lactobacillusplantarum. На основании данных микроорганизмов составили 4 консорциума. Установлено, что совместное применение микроорганизмов повышает их антогонистическую активность: площадь, не занятая фитопатогеном, увеличивалась на 4,2 % по отношению к среднему значению отдельных микроорганизмов, входящих в состав консорциума. Наиболее эффективным являлся консорциум с соотношением штаммов Leclercia sp., S. paucimobilis и L. plantarum 2:1:1 соответственно. Консорциум оказывал фитостимулирующее действие на проростки пшеницы (всхожесть варьировалась в пределах 73,2-99,6 %) и позволял избежать морфометрических изменений при обработке семян, зараженных фитопатогенами.
Разработанный консорциум обладает высокой эффективностью защиты пшеницы от патогенов рода Alternaria и Fusarium и может использоваться в качестве пестицида биологической природы.
Ключевые слова. Triticum aestivum L., продуктивность сельского хозяйства, фитопатогены, биопестициды, экстремофильные бактерии, консорциум микроорганизмов, экологическая безопасность
Финансирование. Работа была выполнена в рамках государственного задания по теме «Фундаментальные исследования по разработке биопестицидов, состоящих из экстремофильных и эндофитных микроорганизмов, для преодоления абиотического и биотического стресса сельскохозяйственными культурами в условиях Кемеровской области - Кузбасса» (шифр FZSR-2023-0003).
Для цитирования: Антагонистическая активность экстремофильных микроорганизмов в отношении фитопатогенов сельскохозяйственных культур / Л. К. Асякина [и др.] // Техника и технология пищевых производств. 2023. Т. 53. № 3. С. 565-575. (На англ.). https://doi.org/10.21603/2074-9414-2023-3-2457
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 phytopa-thogenic 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 Fu-sarium. 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 phytopa-thogens [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 [12-14]. 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 nitrogen-fixing 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 extremophi-philic 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.
We aimed to develop a consortium based on extre-mophilic microorganisms isolated from natural sources to protect T. aestivum L. from diseases caused
by Alternaria alternata (F-525), Fusarium graminea-rum, PH-1 (F-877), Fusarium graminearum (F- 892), and Fusarium sporotrichioides T11 (F-902).
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 ^L 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 cross-culture method [26]. For this, we placed daily cultures of the isolates onto one side of Petri dishes with potato-glucose 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 = - ^^jj x 100 (1)
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 biocompa-tibility 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 phy-topathogen suspension (2.5*105) 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 phytopatho-gens 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. 1 as Pantoea sp. (95% probability), isolate No. 4 as Leclercia sp. (88% probability), isolate No. 5 as Sphingomonas paucimobilis (87% probability), isolate No. 7 as Stenotrophomonas maltophilia (86% probability), isolate No. 9 as Lac-tobacillus plantarum (99% probability), and isolate No. 10 as Staphylococcus aureus (85% probability).
In our previous studies, isolate No. 2 was identified as Klebsiella oxytoca (98% probability), isolate No. 3 as Enterobacter aerogenes (86% probability), isolate No. 6 as Pseudomonasputida (87% probability), and isolate No. 8 as Bacillus megaterium (88% probability).
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
Table 1. Biochemical characteristics of gram-negative microorganisms
Таблица 1. Результаты исследования биохимических особенностей грамотрицательных микроорганизмов
No. Substrate Inoculate No.
1 4 5 7
1 Ala-Phe-Pro-arylamidase - - - +
2 Adonitol - + - -
3 L-pyrrolydonyl arylamidase - + - -
4 L-Arabitol - - - -
5 D-Cellobiose + + + -
6 Beta-galactosidase + + + -
7 H2S - - - -
8 Beta-N-acetyl-glucosaminidase - + - +
9 Glutamyl arylamidase pNA - - - -
10 D-glucose + + + -
11 Gamma-glutamyl-transferase + - - +
12 Fermentation/glucose + + - -
13 Beta-glucosidase - + + +
14 D-maltose + + - -
15 D-mannitol + + + -
16 D-mannose + + + -
17 Beta-xylosidase + + + -
18 Beta-alanine arylamidase pNA - - - -
19 L-proline arylamidase - - - +
20 Lipase - - - +
21 Palatinose - - - -
22 Tyrosine arylamidase - - + +
23 Urease - - - -
24 D-sorbitol + - - -
25 Saccharose/sucrose + + + -
26 D-tagatose - - + -
27 D-trehalose + + + -
28 Citrate (sodium) + - - +
29 Malonate - - - +
30 5-keto-D-gluconate - - - -
31 L-Lactate alkalinisation + + + +
32 Alpha-glucosidase - - - +
33 Succinate alkalinisation - - - +
34 Beta-N-acetyl-galactosaminidase - + - -
35 Alpha-galactosidase - - - -
36 Phosphatase + - - +
37 Glycine arylamidase - - + -
38 Ornithine decarboxylase - - - -
39 Lysine decarboxylase - - - +
40 L-histidine assimilation - - - -
41 Coumarate - + + -
42 Beta-glucoronidase - - - -
43 O/129 resistance (comp. vibrio) + - - -
44 Glu-Gly-Arg-arylamidase - - - +
45 L-malate assimilation + - - -
46 ELLMAN + + - -
47 L-Lactate assimilation - - - -
Table 2. Biochemical characteristics of gram-positive microorganisms
Таблица 2. Результаты исследования биохимических особенностей грамположительных микроорганизмов
No. Substrate Inoculate No.
9 10
1 D-amygdalin + -
2 Phosphatidylinositol phospholipase C - -
3 D-xylose - +
4 Arginine dihydrolase 1 - +
5 Beta-galactosidase - -
6 Alpha-glucosidase + +
7 Ala-Phe-Pro Arylamidase - -
8 Cyclodextrin - -
9 L-Aspartate arylamidase - -
10 Beta galactopyranosidase + -
11 Alpha-mannosidase - -
12 Phosphatase - +
13 Leucine arylamidase + -
14 L-Proline arylamidase - +
15 Beta glucuronidase - -
16 Alpha-galactosidase - -
17 L-Pyrrolydonyl-arylamidase - -
18 Beta-glucuronidase - -
19 Alanine arylamidase + -
20 Tyrosine arylamidase - +
21 D-sorbitol + -
22 Urease - +
23 Polymixin b resistance + +
24 D-galactose + +
25 D-ribose + +
26 L-Lactate alkalinization - +
27 Lactose + +
28 N-Acetyl-D-Glucosamine + +
29 D-maltose + +
30 Bacitracin resistance + +
31 Novobiocin resistance + -
32 Growth in 6.5% NaCl + -
33 D-mannitol + +
34 D-mannose + +
35 Methyl-B-D-Glucopyranoside + -
36 Pullulan - -
37 D-raffinose + -
38 O/129 Resistance (comp.vibrio.) + +
39 Salicin + -
40 Saccharose/sucrose + +
41 D-trehalose + +
42 Arginine dihydrolase 2 - -
43 Optochin resistance + +
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
Klebsiella oxytoca Sphingomonas paucimobilis Stenotrophomonas maltophilia Bacillus megaterium Lactobacillus plantarum Enterobacter aerogenes Pseudomonas putida Staphylococcus aureus Leclercia sp, Pantoea sp.
6.0 H 6.0 H 6.0 H 6.0 H 6.0 H 7.0
H 7.0 H 8.0
H 8.0 H 9.0
0.0 5.0 10.0
Antimicrobial activity, mm
Figure 1. Antimicrobial activity of the isolated microorganisms
Рисунок 1. Результаты антимикробной активности выделенных микроорганизмов
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).
As can be seen, the most promising antagonist strains were E. aerogenes, Leclercia sp., S. paucimobilis, B. megaterium, and L. plantarum. The highest activity against the phytopathogenic fungi Alternaria alternata was shown by Leclercia sp. (31.3%), S. paucimobilis (33.7%), and L. plantarum (27.6%) on the 7th day of cultivation. The growth of Fusarium graminearum was inhibited by B. megaterium (26.8-28.0%) and E. aerogenes (31.2-32.3%). The highest antagonistic activity against the genus Fusarium (F. graminearum and Fusa-rium sporotrichioides) was observed in Leclercia sp. (51.0-54.8 and 63.0%, respectively), S. paucimobilis (68.4-70.8 and 58.5%, respectively), and L. lantarum (80.1-82.4 and 80.3%, respectively). Antagonistic activity against A. alternata was absent on the 7th day in K. oxytoca (3.5%), Pantoea sp. (7.3%), and S. aureus (9.4%). No inhibition zone was observed in S. maltophilia. The strain Pantoea sp. was not resistant to F. sporotrichioides. The microorganisms K. oxytoca and S. ltophilia showed low activity against F. gra-inearum (9.6-10.5%).
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
Table 3. Antagonistic activity of the isolated microorganisms against phytopathogenic fungi Таблица 3. Результаты антагонистической активности выделенных микроорганизмов по отношению к фитопатогенным грибам
Antagonist strain Incubation time, Strain of phytopathogen, %
days Alternaria Fusarium Fusarium Fusarium
alternata graminearum graminearum sporotrichioides
(F-525) PH-1 (F-877) (F-892) T11 (F-902)
Pantoea sp. 3 3.8 ± 0.1 10.0 ± 0.5 10.4 ± 0.5 0
5 7.3 ± 0.2 12.1 ± 0.6 12.6 ± 0.6 0
7 7.3 ± 0.2 14.6 ± 0.7 15.0 ± 0.8 0
Klebsiella oxytoca 3 0 8.4 ± 0.4 7.2 ± 0.4 11.4 ± 0.4
5 2.8 ± 0.1 9.9 ± 0.5 9.1 ± 0.5 14.8 ± 0.5
7 3.5 ± 0.1 9.9 ± 0.5 10.5 ± 0.5 15.3 ± 0.5
Enterobacter 3 4.5 ± 0.1 6.4 ± 0.3 10.0 ± 0.5 9.7 ± 0.3
aerogenes 5 12.6 ± 0.4 18.4 ± 0.9 16.1 ± 0.8 16.2 ± 0.5
7 16.2 ± 0.5 32.3 ± 1.6 31.2 ± 1.6 21.0 ± 0.6
Leclercia sp. 3 14.2 ± 0.5 30.4 ± 1.5 33.0 ± 1.7 29.7 ± 0.9
5 18.6 ± 0.6 42.1 ± 2.1 39.6 ± 2.0 43.8 ± 1.3
7 31.3 ± 1.0 54.8 ± 2.7 51.7 ± 2.6 63.0 ± 1.9
Sphingomonas 3 12.9 ± 0.4 36.0 ± 1.5 35.1 ± 1.1 27.6 ± 1.1
paucimobilis 5 21.5 ± 0.7 44.2 ± 1.9 53.9 ± 1.6 46.3 ± 1.8
7 33.7 ± 1.1 68.4 ± 2.9 70.8 ± 2.2 58.5 ± 2.3
Pseudomonas 3 3.0 ± 0.1 15.7 ± 0.7 14.3 ± 0.4 0
putida 5 8.2 ± 0.3 17.0 ± 0.7 15.8 ± 0.5 3.8 ± 0.1
7 8.2 ± 0.3 17.8 ± 0.8 16.1 ± 0.5 6.7 ± 0.2
Stenotrophomonas 3 0 0 5.8 ± 0.2 15.0 ± 0.4
maltophilia 5 0 2.7 ± 0.1 7.4 ± 0.2 17.2 ± 0.4
7 0 10.2 ± 0.4 9.6 ± 0.3 21.4 ± 0.5
Bacillus 3 5.2 ± 0.2 7.6 ± 0.4 8.9 ± 0.3 2.5 ± 0.1
megaterium 5 8.6 ± 0.3 13.2 ± 0.6 17.3 ± 0.5 11.4 ± 0.4
7 17.8 ± 0.6 26.8 ± 1.2 28.0 ± 0.9 15.9 ± 0.6
Lactobacillus 3 12.7 ± 0.4 48.3 ± 2.2 52.1 ± 3.1 50.0 ± 2.8
plantarum 5 20.4 ± 0.7 75.1 ± 3.5 74.9 ± 4.5 71.8 ± 4.0
7 27.6 ± 0.9 82.4 ± 3.8 80.1 ± 4.9 80.3 ± 4.4
Staphylococcus 3 2.8 ± 0.1 11.3 ± 0.5 11.9 ± 0.2 0
aureus 5 5.0 ± 0.2 15.8 ± 0.7 14.7 ± 0.3 6.4 ± 0.2
7 9.4 ± 0.3 18.1 ± 0.8 19.0 ± 0.4 13.1 ± 0.5
strains of the genus Bacillus have been reported to have antagonistic activity against the genus Alternaria. Panebianco et al. found that the epiphytes Bacillus ce-reus 6C, Bacillus licheniformis 4L, Bacillus thuringien-sis 18D, and Bacillus velezensis 23A isolated from PGI Pachino tomatoes inhibited the development of A. alternata under the conditions of artificial infection [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-Diaz 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. plan-tarum. 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 biocompati-bility of the isolates (Table 4).
We found that the strain E. aerogenes was not compatible with Leclercia sp., S. paucimobilis, and L. plan-tarum, as it suppressed their growth. The strain Lecler-cia 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. megate-rium, while Leclercia sp. and L. plantarum contributed to their active growth. The microorganism B. me-gaterium 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. gram-inearum (F-892), with 9.4 and 5.2% below the average,
respectively. Consortium C's activity against A. alter-nata 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 phy-tostimulating effect, with an average of 24.8 germinated seeds, while consortium A had the lowest phytosti-mulating 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 cole-optile 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.
Table 4. Biocompatibility of the isolated microorganisms Таблица 4. Результаты исследования биосовместимости выделенных микроорганизмов
Strain Enterobacter Leclercia sp. Sphingomonas Bacillus Lactobacillus
aerogenes paucimobilis megaterium plantarum
Enterobacter aerogenes - - + -
Leclercia sp. - + - +
Sphingomonas paucimobilis - + - +
Bacillus megaterium - - + -
Lactobacillus plantarum - + + -
Table 5. Composition of consortia Таблица 5. Состав консорциумов микроорганизмов
Consortium Composition of consortium
Consortium А Leclercia sp., Sphingomonas paucimobilis, Lactobacillus plantarum in a ratio of 1:1:1
Consortium В Leclercia sp., Sphingomonas paucimobilis, Lactobacillus plantarum in a ratio of 2:1:1
Consortium С Leclercia sp., Sphingomonas paucimobilis, Lactobacillus plantarum in a ratio of 1:2:1
Consortium D Leclercia sp., Sphingomonas paucimobilis, Lactobacillus plantarum in a ratio of 1:1:2
Table 6. Antagonistic activity of the isolated microorganisms against phytopathogenic fungi Таблица 6. Результаты антагонистической активности выделенных микроорганизмов по отношению к фитопатогенным грибам
Consortium Day Strain of phytopathogen, %
Alternaria alternata (F-525) Fusarium graminearum PH-1 (F-877) Fusarium graminearum (F-892) Fusarium sporotrichioides T11 (F-902)
Consortium A 3 12.9 ± 0.4 42.2 ± 1.4 42.8 ± 1.4 40.1 ± 1.3
5 17.0 ± 0.5 60.8 ± 2.0 59.3 ± 2.0 49.1 ± 1.6
7 21.5 ± 0.7 69.0 ± 2.3 62.3 ± 2.1 70.2 ± 2.3
Consortium B 3 15.2 ± 0.5 50.9 ± 1.5 53.1 ± 1.8 55.3 ± 1.9
5 26.4 ± 0.8 76.9 ± 2.3 75.6 ± 2.6 73.2 ± 2.5
7 35.1 ± 1.1 90.2 ± 2.7 87.6 ± 3.0 86.2 ± 3.0
Consortium C 3 13.4 ± 0.4 37.2 ± 1.2 36.5 ± 1.1 34.6 ± 1.0
5 19.9 ± 0.6 55.4 ± 1.7 50.0 ± 1.5 41.5 ± 1.3
7 28.7 ± 0.8 60.9 ± 1.9 67.8 ± 2.1 48.0 ± 1.5
Consortium D 3 10.8 ± 0.4 35.0 ± 1.2 32.4 ± 1.0 38.1 ± 1.2
5 18.3 ± 0.6 48.2 ± 1.7 48.0 ± 1.6 55.7 ± 1.8
7 31.2 ± 1.0 52.3 ± 1.9 52.9 ± 1.7 60.3 ± 1.9
Table 7. Growth of wheat treated with consortia and phytopathogenic fungi Таблица 7. Показатели роста пшеницы, обработанной консорциумами и фитопатогенными грибами
Treatment Average number of germinated seeds, pcs Average coleoptile length, mm Total length of seedling roots, mm Average number of roots per plant, pcs
Alternaria alternata (F-525)
Consortium А 19.2 ± 1.4 39.1 ± 1.8 187.4 ± 9.5 3.8 ± 0.3
Consortium B 24.8 ± 1.9 48.3 ± 2.6 190.1 ± 10.2 3.9 ± 0.4
Consortium C 22.0 ± 1.6 40.8 ± 2.1 197.1 ± 10.1 3.5 ± 0.2
Consortium D 24.2 ± 1.5 45.6 ± 2.2 201.3 ± 10.9 3.3 ± 0.2
Fusarium graminearum (F-877) PH-1
Consortium А 24.2 ± 1.3 46.7 ± 2.3 206.2 ± 10.2 4.1 ± 0.3
Consortium B 24.9 ± 2.0 45.1 ± 2.5 215.6 ± 10.5 4.0 ± 0.2
Consortium C 23.9 ± 1.2 41.2 ± 2.1 186.2 ± 10.3 3.0 ± 0.1
Consortium D 21.4 ± 2.1 43.3 ± 2.2 179.7 ± 9.6 3.4 ± 0.1
Fusarium graminearum (F-892)
Consortium А 20.1 ± 1.8 36.4 ± 1.9 179.2 ± 9.6 3.9 ± 0.4
Consortium B 24.6 ± 2.3 37.2 ± 2.4 210.6 ± 10.2 4.3 ± 0.5
Consortium C 23.1 ± 1.2 41.3 ± 2.3 201.4 ± 9.5 3.4 ± 0.4
Consortium D 20.2 ± 1.6 38.3 ± 2.1 195.1 ± 10.2 3.2 ± 0.2
Fusarium sporotrichioides (F-902) T11
Consortium А 20.5 ± 1.6 34.2 ± 1.9 195.7 ± 9.7 4.1 ± 0.3
Consortium B 24.9 ± 2.1 44.9 ± 2.1 200.2 ± 9.4 5.1 ± 0.2
Consortium C 21.8 ± 1.8 40.1 ± 1.9 210.4 ± 10.3 3.3 ± 0.1
Consortium D 18.3 ± 1.7 35.1 ± 1.8 167.3 ± 9.4 3.2 ± 0.1
Control
Without treatment 24.7 ± 2.1 39.7 ± 2.5 189.4 ± 10.17 3.8 ± 0.1
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).
Conclusion
We identified six extremophilic microorganisms and studied the antagonistic activity of ten isolates against the model bacterium Escherichia coli. The samples showed low bactericidal properties. Next, we studied their activity against phytopathogenic fungi such as Alternaria alternate (F-525), Fusarium graminearum PH-1 (F-877), Fusarium graminearum (F-892), and Fusarium
antagonistic activity against the phytopathogenic fungi. We found that consortium B, which consisted of Leclercia sp., S. paucimobilis, and L. plantarum in a ratio of 2:1:1, increased the antagonistic properties of its individual microorganisms. Also, this consortium had a phytostimulating effect on wheat seeds, increasing the average coleoptile length by 10.5% compared to the control and contributing to an average germination rate of 24.8 seeds. In addition, the joint treatment of seeds with the consortium and the phytopathogens did not cause any visual morphometric defects in wheat. Thus, this consortium proved highly effective in protecting wheat from Alternaria and Fusarium pathogens.
Contribution
The authors were equally involved in writing the manuscript and are equally responsible for plagiarism.
Conflict of interest
The authors declare no conflict of interest regarding this publication.
Критерии авторства
Авторы в равной степени участвовали в написании рукописи и несут равную ответственность за плагиат.
Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов в данной публикации.
References/Список литературы
1. Sabouri H, Kazerani B, Fallahi HA, Dehghan MA, Alegh SM, Dadras AR, et al. Association analysis of yellow rust, fusarium head blight, tan spot, powdery mildew, and brown rust horizontal resistance genes in wheat. Physiological and Molecular Plant Pathology. 2022;118. https://doi.org/10.1016/j.pmpp.2022.101808
2. Drakopoulos D, Kagi A, Six J, Zorn A, Wettstein FE, Bucheli TD, et al. The agronomic and economic viability of innovative cropping systems to reduce Fusarium head blight and related mycotoxins in wheat. Agricultural Systems. 2021;192. https://doi.org/10.1016/j.agsy.2021.103198
3. Shude SPN, Mbili NC, Yobo KS. Epiphytic yeasts as potential antagonists against Fusarium head blight of wheat (Triticum aestivum L.) caused by Fusarium graminearum sensu stricto. Journal of the Saudi Society of Agricultural Sciences. 2022;21(6):404-411. https://doi.org/10.1016/j.jssas.2021.11.001
4. Zhang D, Chen G, Zhang H, Jin N, Gu C, Weng S, et al. Integration of spectroscopy and image for identifying fusarium damage in wheat kernels. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2020;236. https:// doi.org/10.1016/j.saa.2020.118344
5. Martínez M, Biganzoli F, Arata A, Dinolfo MI, Rojas D, Cristos D, et al. Warm nights increase Fusarium Head Blight negative impact on barley and wheat grains. Agricultural and Forest Meteorology. 2022;318. https://doi.org/10.1016/ j.agrformet.2022.108909
6. Gagkaeva TYu, Gavrilova OP, Orina AS. First detection of Fusarium globosum in small grain cereals on Ural and Siberian territory. Plant Protection News. 2019;99(1):10-18. (In Russ.). https://doi.org/10.31993/2308-6459-2019-1(99)-10-18
7. Asan-Ozusaglam M, Celik I. White pitahaya as a natural additive: potential usage in cosmetic industry. Foods and Raw Materials. 2023;11(1):57-63. https://doi.org/10.21603/2308-4057-2023-1-552
8. Wegulo SN, Baenziger PS, Nopsa JH, Bockus WW, Hallen-Adams H. Management of Fusarium head blight of wheat and barley. Crop Protection. 2015;73:100-107. https://doi.org/10.1016/j.cropro.2015.02.025
Control (seed treatment with water without pathogen)
Seed treatment with consortium B and Fusarium graminearum (F-892)
Figure 2. Wheat seedlings treated with consortium B and Fusarium graminearum (F-892)
Рисунок 2. Внешний вид проростков пшеницы, обработанных консорциумом B и Fusarium graminearum (F-892)
sporotrichioides T11 (F-902). According to the results, the most promising antagonist strains were Enterobacter aerogenes, Leclercia sp., Sphingomonaspaucimobilis, Bacillus megaterium, and Lactobacillus plantarum. We also found that most of the antagonist strains showed
maximum activity on the 7th day of cultivation. The
isolates were then tested for biocompatibility to form consortia. As a result, we selected those strains that did not exhibit antagonistic properties against each other, namely Leclercia sp., S. paucimobilis, and L. plantarum in the ratios of 1:1:1, 2:1:1, 1:2:1, and 1:1:2 (consortia A, B, C, and D, respectively). The consortia were tested for
9. da Cruz Cabral L, Delgado J, Patriarca A, Rodriguez A. Differential response to synthetic and natural antifungals by Alternaria tenuissima in wheat simulating media: Growth, mycotoxin production and expression of a gene related to cell wall integrity. International Journal of Food Microbiology. 2019;292:48-55. https://doi.org/10.1016/j.ijfoodmicro.2018. 12.005
10. Somma S, Amatulli MT, Masiello M, Moretti A, Logrieco AF. Alternaria species associated to wheat black point identified through a multilocus sequence approach. International Journal of Food Microbiology. 2019;293:34-43. https://doi. org/10.1016/j.ijfoodmicro.2019.01.001
11. Guler GO, Cakmak YS, Dagli Z, Aktumsek A, Ozparlak H. Organochlorine pesticide residues in wheat from Konya region, Turkey. Food and Chemical Toxicology. 2010;48(5):1218-1221. https://doi.org/10.1016/j.fct.2010.02.013
12. Syed-Ab-Rahman SF, Singh E, Pieterse CMJ, Schenk PM. Emerging microbial biocontrol strategies for plant pathogens. Plant Science. 2018;262:102-111. https://doi.org/10.1016/j.plantsci.2017.11.012
13. Choe SG, Maeng HR, Pak SJ, U SN. Production of Bacillus thuringiensis biopesticide using penicillin fermentation waste matter and application in agriculture. Journal of Natural Pesticide Research. 2022;2. https://doi.org/10.1016/ j.napere.2022.100012
14. Ajayi FF, Ogori AF, Orede VO, Peter E. Synergistic effect of Balanites aegyptiaca essential oil and storage materials on cowpea seeds. Foods and Raw Materials. 2022;10(2):353-364. https://doi.org/10.21603/2308-4057-2022-2-545
15. Lu C, Yang Z, Liu J, Liao Q, Ling W, Waigi MG, et al. Chlorpyrifos inhibits nitrogen fixation in rice-vegetated soil containing Pseudomonas stutzeri A1501. Chemosphere. 2020;256. https://doi.org/10.1016/j.chemosphere.2020.127098
16. Walder F, Schmid MW, Riedo J, Valzano-Held AY, Banerjee S, Büchi L, et al. Soil microbiome signatures are associated with pesticide residues in arable landscapes. Soil Biology and Biochemistry. 2022;174. https://doi.org/10.1016/ j.soilbio.2022.108830
17. Tao Y, Jia C, Jing J, Zhang J, Yu P, He M, et al. Occurrence and dietary risk assessment of 37 pesticides in wheat fields in the suburbs of Beijing, China. Food Chemistry. 2021;350. https://doi.org/10.1016/j.foodchem.2021.129245
18. Mrid RB, Benmrid B, Hafsa J, Boukcim H, Sobeh M, Yasri A. Secondary metabolites as biostimulant and bio-protectant agents: A review. Science of the Total Environment. 2021;777. https://doi.org/10.1016/j.scitotenv.2021.146204
19. Nysanth NS, Divya S, Nair CB, Anju AB, Praveena R, Anith KN. Biological control of foot rot (Phytophthora capsici Leonian) disease in black pepper (Piper nigrum L.) with rhizospheric microorganisms. Rhizosphere. 2022;23. https:// doi.org/10.1016/j.rhisph.2022.100578
20. Santos AP, Muratore LN, Solé-Gil A, Farias ME, Ferrando A, Blázquez MA, et al. Extremophilic bacteria restrict the growth of Macrophomina phaseolina by combined secretion of polyamines and lytic enzymes. Biotechnology Reports. 2021;32. https://doi.org/10.1016/j.btre.2021.e00674
21. Giudice AL, Fani R. Antimicrobial potential of cold-adapted bacteria and fungi from Polar Regions. In: Rampelotto PH, editor. Biotechnology of extremophiles: Advances and challenges. Cham: Springer; 2016. pp. 83-115. https://doi.org/10.1007/ 978-3-319-13521-2_3
22. Liu K, Ding H, Yu Y, Chen B. A cold-adapted chitinase-producing bacterium from Antarctica and its potential in biocontrol of plant pathogenic fungi. Marine Drugs. 2019;17(12). https://doi.org/10.3390/md17120695
23. Milentyeva IS, Fotina NV, Zharko MYu, Proskuryakova LA. Microbial treatment and oxidative stress in agricultural plants. Food Processing: Techniques and Technology. 2022;52(4):750-761. (In Russ.). https://doi.org/10.21603/2074-9414-2022-4-2403
24. Voitenkova EV, Matveeva ZN, Makarova MA, Egorova SA, Zabrovskaia AV, Suzhaeva LV, et al. Difficulties in identification of Comamonas kerstersii strains isolated from intestinal microbiota of residents of Republic of Guinea and Russian Federation. Russian Journal of Infection and Immunity. 2018;8(2):164-168. https://doi.org/10.15789/2220-7619-2018-2-163-168
25. Khalil T, Oklab MK, Al-Qahtanic WH, Alia F, Zahrad M, Shakeela Q, et al. Tracing probiotic producing bacterial species from gut of buffalo (Bubalus bubalis), South-East-Asia. Brazilian Journal of Biology. 2022;84. https://doi.org/10.1590/1519-6984.259094
26. Sornakili A, Thankappan S, Sridharan AP, Nithya P, Uthandi S. Antagonistic fungal endophytes and their metabolite-mediated interactions against phytopathogens in rice. Physiological and Molecular Plant Pathology. 2020;112. https://doi.org/ 10.1016/j.pmpp.2020.101525
27. Mahar A, Wang P, Ali A, Awasthi MK, Lahori AH, Wang Q, et al. Challenges and opportunities in the phytoremedia-tion of heavy metals contaminated soils: A review. Ecotoxicology and Environmental Safety. 2016;126:111-121. https://doi. org/10.1016/j.ecoenv.2015.12.023
28. Goswami M, Deka S. Isolation of a novel rhizobacteria having multiple plant growth promoting traits and antifungal activity against certain phytopathogens. Microbiological Research. 2020;240. https://doi.org/10.1016/j.micres.2020.126516
29. Gorbunov MYu, Mrachkovskaya AN. The use of growth stimulants to increase the commercial yield of petunia seedlings. Trends in the Development of Science and Education. 2018;(35-4):60-62. (In Russ.). https://doi.org/10.18411/ lj-28-02-2018-70
3Q. Asyakina LK, Dyshlyuk LS, Prosekov AYu. Reclamation of post-technological landscapes: International experience. Food Processing: Techniques and Technology. 2021;51(4):S05-S1S. (In Russ.). https://doi.org/1Q.216Q3/2Q74-9414-2Q21-4-S05-S1S
31. Panebianco S, Lombardo MF, Anzalone A, Musumarra A, Pellegriti MG, Catara V, et al. Epiphytic and endophytic microorganisms associated to different cultivar of tomato fruits in greenhouse environment and characterization of beneficial bacterial strains for the control of post-harvest tomato pathogens. International Journal of Food Microbiology. 2022;379. https://doi.org/l0.l0l6/j.ijfoodmicro.2022.l09S6l
32. Xie Z, Li M, Wang D, Wang F, Shen H, Sun G, et al. Biocontrol efficacy of Bacillus siamensis LZSS against brown spot disease of tobacco caused by Alternaria alternate. Biological Control. 2Q21;154. https://doi.org/l0.l0l6/j.biocontrol.2020. 10450S
33. Gupta S, Didwania N, Singh D. Biological control of mustard blight caused by Alternaria brassicae using plant growth promoting bacteria. Current Plant Biology. 2Q2Q;23. https://doi.org/l0.l0l6/jxpb.2020.l00l66
34. Jankiewicz U, Brzezinska MS, Saks E. Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens. Journal of Bioscience and Bioengineering. 2Q12;113(1):3Q-35. https://doi.org/l0.l0l6/jjbiosc.20ll.0S.023
35. Chavéz-Díaz IF, Cruz-Cárdenas CI, Sandoval-Cancino G, Calvillo-Aguilar FF, Ruíz-Ramírez S, Blanco-Camarillo M, et al. Seedling growth promotion and potential biocontrol against phytopathogenic Fusarium by native rhizospheric Pseudomonas spp. strains from Amarillo Zamorano maize landrace. Rhizosphere. 2Q22;24. https://doi.org/l0.l0l6/j.rhisph.2022. 1QQ6Q1
36. Baffoni L, Gaggia F, Dalanaj N, Prodi A, Nipoti P, Pisi A, et al. Microbial inoculants for the biocontrol of Fusarium spp. in durum wheat. BMC Microbiology. 2015;15. https://doi.org/l0.llS6/sl2S66-0l5-0573-7
37. Wang L-Y, Xie Y-S, Cui Y-Y, Xu J, He W, Chen H-G, et al. Conjunctively screening of biocontrol agents (BCAs) against fusarium root rot and fusarium head blight caused by Fusarium graminearum. Microbiological Research. 2015;177:34-42. https://doi.org/l0.l0l6/j.micres.20l5.05.005
