A study of antifungal properties of violacein-forming strain Janthinobacterium sp. B-3515 as well as its secondary metabolite, violacein, against Alternaria brassicicola F-1864 is presented. Regardless of the presence of bacteria, mycelium growth in the first two days proceeded at the same rate. The effect of the bacterial strain was manifested after the third day of incubation. In general, during co-culture, the bacterial strain statistically significantly reduced the average growth of the mycelium of the mould fungus by 10

Gubina, E. D.

Lyakhovchenko, N. S.

Nikishin, I. A.

Pribylov, D. A.

Senchenkov, V. Yu.

Sirotin, A. A.

Solyanikova, I. P.

DSpace at Belgorod State University

IOP Conference Series: Earth and Environmental SciencePAPER • OPEN ACCESSAssessment of the antifungal activity of theviolacein-forming strain Janthinobacterium sp. B-3515 against the mould fungus Alternariabrassicicola F-1864To cite this article: N S Lyakhovchenko et al 2021 IOP Conf. Ser.: Earth Environ. Sci. 908 012006 View the article online for updates and enhancements.You may also likeSustainable Material: DevelopmentExperiment of Bamboo CompositeThrough Biologically Binding MechanismI F Ridzqo, D Susanto, T H Panjaitan et al.-Ganoderma boninense control in palm oilplantations using Trichoderma harzianumin various MediaAries Sukariawan, Eka Bobby Febrianto,Sakiah et al.-The application of biotic elicitor onArtemisia annua L. to increase artemisinincontentI Darwati, D Manohara, Rohimatun et al.-This content was downloaded from IP address 188.170.217.49 on 07/06/2022 at 09:26Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distributionof this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.Published under licence by IOP Publishing LtdDSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120061      Assessment of the antifungal activity of the violacein-forming strain Janthinobacterium sp. B-3515 against the mould fungus Alternaria brassicicola F-1864 N S Lyakhovchenko1, I A Nikishin1, E D Gubina1, D A Pribylov1,  V Y Senchenkov1, A A Sirotin1 and I P Solyanikova1,2 1 Belgorod State National Research University, Belgorod, 308015 Russia 2 G K Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS,    Pushchino, 142290 Russia  E-mail: lyakhovchenko@bsu.edu.ru Abstract. A study of antifungal properties of violacein-forming strain Janthinobacterium sp. B-3515 as well as its secondary metabolite, violacein, against Alternaria brassicicola F-1864  is presented. Regardless of the presence of bacteria, mycelium growth in the first two days proceeded at the same rate. The effect of the bacterial strain was manifested after the third  day of incubation. In general, during co-culture, the bacterial strain statistically significantly reduced the average growth of the mycelium of the mould fungus by 10%. The average growth of A. brassicicola F-1864 was decreased in the presence of an aqueous solution of violacein  in the nutrient medium (1%, 3%, and 5%). The pigment in 5% concentration had the greatest effect, as the difference between the average growth of the control group and the experimental group was 18%. The mycostatic activity of bacteria of genus Janthinobacterium and violacein against mould fungus Alternaria brassicicola F-1864 was shown for the first time. 1. Introduction The mould fungus Alternaria brassicicola has been shown to be the causative agent of seed pod disease in Brassica oleracea crops [1]. Many varieties of cabbage (Brassica sp.) are widely cultivated in Europe, Asia, New Zealand, and North America as a staple food for humans and cattle.  However, no known cultivar is completely resistant to A. brassicicola to date [2]. One of the ways how the fungus penetrates plant cells of the host is through the action of several serine esterases that are bound to the spore surface on the cuticle [3]. On this basis, some fungicides may be aimed at inhibiting the enzymatic activity of A. brassicicola spores, but if aggressive agents are used, the plant itself may be affected. Current trends in the development of plant protection products aim at biologization, namely  the replacement of active chemical pesticides with biological ones (e.g. antagonist microorganisms and their secondary metabolites). This direction requires the search for new effective producers  of antibiotic substances and the development of a set of optimal parameters for obtaining biologically active substances. One of the most promising compounds for the development of plant protection agents could be the blue-violet pigment violacein (figure 1). DSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120062       Figure 1. The structural formula of violacein [5].  There is evidence that violacein produced by many bacteria (Chromobacterium violaceum, Janthinobacterium lividium, etc.) has antibacterial, antifungal, antiviral (against herpes simplex virus), and antitumor properties [5]. In addition, the pigment had a stimulating effect on the formation of immunoglobulin G by the ovary cells of the Chinese hamster [6]. Violacein showed antifungal activity against the phytopathogenic mould fungus Rosselinia necatrix [5]. The bacteria Janthinobacterium lividium were found to be active against the mould Batrachochytrium dendrobatidis, the causative agent of frog disease (chytridiomycosis), which is a serious problem for the ecological condition of water bodies [7]. The study aims to evaluate the antifungal activity of the violacein-forming bacterium Janthinobacterium sp. B-3515 and pigment against the mould fungus Alternaria brassicicola F-1864. 2. Materials and methods The antagonistic potential of indigenous violacein-forming strain Janthinobacterium sp. B-3515 against mould fungus Alternaria brassicicola F-1864 was evaluated by co-culture method [8]  on agarized Sabouraud’s medium composition (g/l): glucose, 40.0; peptone, 10.0; yeast extract, 5; agar, 18.0. A suspension of cells of B-3515 strain equal to 0.1 optical density (OD), prepared using a Microscan Turbidity Meter (manufactured by Siemens, USA), was introduced into agar wells (100 µl per well), made in a solid nutrient medium with a sterile 11 mm diameter drill bit. The volume of nutrient medium in each Petri dish was 20 ml. As a control, the mould fungus A. brassicicola F-1864 was cultured without Janthinobacterium sp. B-3515. Mold fungus F-1864 was replaced in the centre of a Petri dish using the imprinting method [9]. The cultures were incubated at 25ºC for 5 days. Every 24 hours, the colony diameter of the fungus was measured and averaged according to the formula (1) [10]:  𝑆∑  (1) where S is root mean square, V is a date, n – is the number of measurements. To obtain violacein, the culture of Janthinobacterium sp. B-3515 was grown in a liquid nutrient medium (3% peptone) for 5 days without stirring so that a biofilm was formed on the interface.  After incubation, the biomass of the pigmented culture was separated from the culture liquid by centrifugation at 4,400 g for 10 min (using a Neofuge 1600R centrifuge, Heal Force, China).  The supernatant was discarded, the precipitate was mixt with 50 ml of 95% ethanol and stirred for  1 hour at 35ºC (ThermoStable IS-30 shaker incubator, Daihan Scientific, Korea). The mixture was centrifuged again, the supernatant pigmented solution was collected and another 50 ml of ethanol  was added. The mixture was resuspended and centrifuged as descript above. The supernatant was collected so as the total volume of the crude ethanol extract was 100 mL. The obtained solution  was evaporated using a vacuum rotary evaporator (N-1000R). The evaporated violacein was dried in a desiccator (ThermoStable OF-50, Daihan Scientific, Korea) at 60ºC until the moisture evaporated completely. The resulting crystalline pigment powder was collected and the aqueous violacein solution was analysed using a UV-1900i spectrophotometer, Shimadzu, Japan at λ = 200-700 nm.  Antifungal activity of aqueous solutions of crude dry extract of violacein (concentrations were  1%, 3%, and 5%) against A. brassicicola F-1864 was determined by disc-diffusion method [8].  DSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120063      For this purpose, a suspension of mould spores equal to 0.1 optical density at λ = 600 nm (Microscan Turbidity Meter, Siemens, USA) was “lawn-passed” into Petri dishes each containing 20 ml of agarized Sabouraud nutrient medium. Sterile paper disks (“white tape”), 9 mm in diameter, were soaked in 40 ml of violacein solution and placed in cups in 6 replicates. The cultures were incubated for 72 hours at 25ºC. The average radii of the growth inhibition zones of the mould fungus were used to judge the antifungal activity of violacein. The effect of the pigment on the growth of the fungus was investigated by linear growth of mycelium, in which aqueous solutions of violacein were added to the agar (1,000 µl per cup).  The volume of nutrient medium in each Petri dish was 20 ml. A. brassicicola F-1864 was used as a control without adding the studied solutions. The antifungal properties of the pigment were judged by the mean square diameters of the colony of mould fungus F-1864. Confidence intervals (∆) were calculated according to the formula (2) [11]:  ∆ = tst · m , (2) where tst is the standard value of the Student test, m is the representativeness error of the mean square and is calculated according to the formula (3) [11]:  𝑚√ , (3) where σ is the standard deviation and is calculated as (4) [11]:  𝜎∑ Х , (4) where V is the date, X is the arithmetic mean (or S is the root mean square of the colony diameter), and n is the number of measurements. Average mycelial growth was calculated using the formula (5,6) [11]:  X = (lgGv+1 – 1) · 100% , (5) if  lg𝐺∑ (6) The significance of the difference between the averages was calculated statistically using the difference-in-differences method [12]. 3. Results and discussion The study of antagonistic activity of strain Janthinobacterium sp. B-3515 revealed that the colony growth of A. brassicicola F-1864 in control and experimental groups was statistically insignificantly different within 3 days (figure 2). However, a statistically significant difference in the mean square diameters of the mould fungus colonies in both the control and the experimental groups at days 1 and 3 was observed, as the calculated Student’s t-test was higher than the table value at the error level of p < 0.001. The colony diameters of the control and experimental groups increased statistically significantly from 3 to 4. The calculated Student’s t-test was higher than the table values with an error level of p < 0.001 (control group) and p > 0.01 (experimental group). On day 5, the mean squared colony diameter of the mould fungus in the control group significantly increased, as the calculated Student’s t-test was higher than the standard test at p < 0.05). In contrast, in the experimental group, there was no mycelial growth on day 5, relative to day 4 (table 1).  DSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120064       Figure 2. Growth of Alternaria brassicicola F-1864 mould fungus colonies without Janthinobacterium sp. B-3515 (1) and in the presence of the bacterial strain (2).  The mean quadratic diameter of the mould fungus colonies in the experimental group was statistically insignificant compared to the control group up to 3 days of incubation. On the 4th and 5th days, mycelial growth in the presence of bacterial strain B-3515 significantly slowed down in comparison with the control, as the calculated Student’s t-test was higher than the table test at p < 0.001 (table 1).  Table 1. Mean square colony diameter of the mould fungus Alternaria brassicicola F-1864  as an indicator of the antifungal properties of strain Janthinobacterium sp. B-3515. Variants Incubation Incubation time, days Mean square,    S ± ∆ Error of variance,    Sd Student’s t-test, t Mean colony growth of A. brassicicola F-1864, X, % Control 1(1)  6.6 ± 1.7 Sd(1-2) = 0.75 t(1-2) = 32.1c 60 Sd(1-1.1) = 0.5 t(1-1.1) = 2.7a 3(2)  30.7 ± 0.08 Sd(2-3) = 0.75 t(2-3) = 10.2c 4(3)  37.7 ± 2.4 Sd(3-4) = 1.4 t(3-4) = 2 5(4)  41.0 ± 3.5 Sd(2-1.2) = 0.3 t(2-1.2) = 2.4 Experiment 1(1.1)  5.3 ± 1.4 Sd(1.1-1.2) = 0.4 t(1.1-1.2) = 59.8c 50 Sd(3-1.3) = 0.9 t(3-1.3) = 6.6c 3(1.2)  29.7 ± 0.08 Sd(1.2-1.3) = 0.3 t(1.2-1.3) = 4.5b 4(1.3)  31.0 ± 1.6 Sd(1.3-1.4) = 0.4 t(1.3-1.4) = 0 5(1.4)  31.0 ± 1.6 Sd(4-1.4) = 1.1 t(4-1.4) = 8.7c  a – the difference is statistically significant at the level of error p < 0.05; b – the difference is statistically significant at the level of error p < 0.01; c – the difference is statistically significant at the level of error p < 0.001.   051015202530354045501 3 4 5S, mmDayDSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120065      Thus, based on the data obtained, when A. brassicicola F-1864 and Janthinobacterium sp.  B-3515 wear co-cultured, the bacterium has a mycostatic effect. This statement is confirmed by  the fact that the average mycelial growth (figure 3) in the control group is 10% higher than in the experimental group (table 1).   Figure 3. Average mycelial growth of Alternaria brassicicola F-1864 mould fungus without Janthinobacterium sp. B-3515 (control group) and in the presence of the strain (experimental group).  During the cultivation of Janthinobacterium sp. B-3515 as a producer, 0.4 g of dried violacein was obtained from 100 ml of the crude ethanol extract. Spectrophotometric analysis of the aqueous solution showed that the absorption maximum was at 576 nm, which approximated  the property of violacein (based on literature data, the absorption maximum of methanol extract of violacein is λ = 575 nm).   Figure 4. Average growth of mycelium of fungus Alternaria brassicicola F-1864 in the control sample (1), in the presence of 1% (2), 3% (3), and 5% (4) pigment in growth medium.  A study of the antagonistic activity of an aqueous solution of violacein revealed that the pigment mixed in Saburo nutrient medium (1,000 μl of the aqueous solution with pigment concentration of 1%, 3%, and 5%) had a weak mycostatic effect. Average growth of the mycelium of A. brassicicola F-1864 on the 5th day of incubation (figure 4) was statistically significantly reduced by 10% (for 1%), 11% (for 3%), and 18% (for 5%), compared to the control group. Thus, the investigation revealed a moderate mycostatic activity of the strain Janthinobacterium sp. B-3515 and its secondary metabolite, violacein, against  mould fungus Alternaria brassicicola F-1864. 6050010203040506070Control group Experimenal groupХ,%22121140510152025Х, %DSBAC 2021IOP Conf. Series: Earth and Environmental Science 908 (2021) 012006IOP Publishingdoi:10.1088/1755-1315/908/1/0120066      4. Conclusion In nature, microorganisms exist in permanent competition. This has led to the development of a multitude of ways to implement mechanisms of antagonistic interactions, including those which lead to the development of diseases in cultivated plants. However, compounds with proven antagonistic activity and the strains synthesizing them can be used as plant protection agents. In this work, the mycostatic efficacy of violacein-produced strain Janthinobacterium sp. B-3515 and its secondary metabolite against the mould fungus Alternaria brassicicola F-1864 were investigated. It was shown that during co-cultivation on agarized Sabouraud nutrient medium, the bacterium statistically significantly reduced the average mycelial growth by 10%, while the aqueous solution of violacein in the medium, in concentrations of 1%, 3%, and 5%, had a similar impact by 10%, 11%, and 18%, respectively. On this basis, it can be assumed that the mycostatic efficacy of an aqueous solution  of violacein increased with increasing of its concentration. The search for the optimal pigment concentration will continue. Acknowledgments This research was funded by the Ministry of Higher Education and Science grant No. FZWG-2020-0021. References  [1] Humpherson-Jones F M and Maude R B 1982 Annals of App. 100 61–71  [2] Macioszek V, Gapinska M, Zmienko A, Sobczak M, Skoczowski A, Oliwa J and Kononowicz A  2020 Cells 9 2329 [3] Berto P, Commenil P, Belingheri L and Dehorter B 1999 FEMS Microbiol. 180(2) 183–9  [4] Marinelli F, Genilloud O, Fedorenko V and Ron E Z 2015 BioMed. Res. 276964 [5] Duran N, Justo G Z, Ferreira CV, Melo P S, Cordi L and Martins D 2010 Biotechnology and  Applied Biochemistry 127–33 [6] Kido M, Idogaki H, Nishikawa K and Omasa T 2020 Cytotechnology 1–14 [7] Ackleh A S, Carter J, Chellamuthu V K and Ma B 2016 Ecological Modelling 320 158–69 [8] Egorov N S 1965 Microbial Antagonists and Biological Methods for Determining Antibiotic  Activity (Moscow: Higher School) p 211 [9] Dudka I A 1982 Methods in Experimental Mycology Reference book (Kyiv: Naukova   Dumka) p 535 [10] Lyakhovchenko N S et al. 2021 Microorganisms 9 p 102 [11] Snegin E A 2016 Workshop on Biometrics: Tutorial (Belgorod: Publishing house “Belgorod”,  NRU “BelSU”) p 56 [12] Moiseychenko V F, Trifonova M F, Zaveryukha A X and Eshchenko V E 1996 Fundamentals  of Scientific Research in Agronomy (Moscow: Kolos) p 336 

Assessment of the antifungal activity of the violacein-forming strain Janthinobacterium sp. B-3515 against the mould fungus Alternaria brassicicola F-1864

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Assessment of the antifungal activity of the violacein-forming strain Janthinobacterium sp. B-3515 against the mould fungus Alternaria brassicicola F-1864

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