Biological Control of Lettuce Drop (Sclerotinia minor) Using Antagonistic Bacillus Species

2. Materials and Methods

2.1. Bacteria

B. subtilis JAN25-4 accession OR731944.1, B. megaterium JAN33-2 accession OR731941.1 (NCBI submission sub13929898/ 29.10.2023) and B. safensis CAM24K3 with accession PP797578.1 and B. mojavensis CAM23K1 accession PP797576.1 (NCBI submission sub14450584/ 08.06.2024, were isolated from soil intercropping with camelina (Camelina sativa L.) and vetch (Vicia sativa L.) on the experimental field of Agricultural University Plovdiv.

2.2. Amylolytic Activity of Tested Bacillus Strains

The isolated four different strains of the genus Bacillus were qualitatively and quantitatively evaluated for the presence of amylolytic activity by the agar diffusion method (Agati et al., 1998) [22]. Starch agar plate, g/L: wheat starch – 10; peptone – 5; yeast extract – 5; MgSO₄.7H₂O – 0.25; FeSO₄.7H₂O – 0.01; agar –15; pH 6.8 ± 0.2. In the agar-diffusion method, the nutrient medium is poured in 15 cm³ and after solidification in the petri dish, 4 wells with a diameter of 8 mm are made. For each strain, two samples were prepared: CM-culture medium from a 24-h suspension of the strain and CFS-cell-free supernatant obtained by centrifugation of culture fluid at 5000 x g (Universal 320 R, Marvel Ltd.). The bacterial suspension or CFS from each isolate was instilled in 100 µl. After incubation at 30°C for 48 h, the Petri dishes were treated with Lugol's solution (3% KI) to form a blue-colored starch-iodine complex. A halo area is observed around the amylolytic bacterial colonies. The result is reported by measuring the diameter of the formed halo zone in mm.

2.3. Proteolytic Activity of Bacillus Strains

The proteolytic activity of tested bacteria was tested by inoculation on milk agar according to Nabrdalik (2010) [23] containing: casein 0.5%, yeast extract 0.25%, dextrose 0.1%, skimmed milk powder 2.5% and agar 1.5%. Bacterial isolates were pre-activated in TSA broth. The proteolytic activity of each strain was investigated as culture fluid - CM obtained from 24 h culture and cell-free supernatant - CFS obtained by centrifugation of culture fluid. Wells with a diameter of 9 mm were formed on agar medium with added skimmed milk, in which 100 µl of the previously prepared cultures and cell-free extracts were placed. The samples were cultured at 30°C and the result as the diameter of the proteolytic zones measured in mm was reported after 24, 48, and 72 hours (Petkova et al. 2020) [24].

3. Quantification of the Synthesized Secondary Metabolites with Phytohormonal Activity

The synthesis of microbial phytohormones by soil microorganisms is associated with signal changes in the root and stimulation of plant growth. The presence of a particular phytohormone in the microbial culture supernatant is insufficient to prove the molecule's functional role in its interaction with the plant. A number of studies have observed a correlation between plant growth and hormone concentration measured in the culture medium or in colonized plant tissues in experiments and in situ (Spaepen, 2015) [25].

3.1. Quantification of Indole-3-Acetic Acid Using the Salkowski Reagent

For quantification of indole-3-acetic acid (IAA) produced, bacterial isolates were grown in a tube in medium with or without 0.1% (w/v) L-tryptophan (L-Trp) and incubated in the dark at 30°C and for 5 days as was published previously by Petkova et al. 2022 [26]. One milliliter of the cells was pelleted by centrifugation at 3000 g for 5 min, and 0.5 mL of the supernatant was mixed with 0.5 mL of Salkowski's reagent (2 mL of 0.5 M iron (III) chloride and 98 mL of 35% perchloric acid) (Gordon & Weber 1951) [27]. After 30 min, color development (red) was quantified using a spectrophotometer (Unico 1200-Spectrophotometer, USA) at 530 nm. A calibration curve using pure indole-3-acetic acid was established to calculate IAA concentration. IAA production of each bacterial isolate was determined by inoculating L-tryptophan medium containing 0.1% (w/v) L-tryptophan with and incubated in the dark at 20 C. After incubation, the IAA produced was quantified spectrophotometrically.

3.2. Screening for Dissolution of Inorganic Phosphates

Screening for dissolution of inorganic phosphates was performed using Pikovskaya's medium (PVK) as was published by Patil, 2014 [28] containing: glucose, 10 g; Ca(PO₄)₂, 5 g; (NH₄)₂SO₄ , 0.5 g; NaCl, 0.2 g; MgSO₄·7H₂O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO₄·H₂O, 0.002 g; and FeSO₄·7H₂O, 0.002 g.

3.3. ZnO Dissolution Screening

Isolates were tested for zinc solubilization ability using a modified ZnO medium (Khande et al. 2017) [29]. The medium included: 10.0 g glucose, 1.0 g ammonium sulfate, 0.2 g potassium chloride, 0.2 g dipotassium hydrogen phosphate, 0.1 g magnesium sulfate, 0.1% insoluble zinc from a ZnO source in 1000 ml distilled water at pH 7.0. From a 24 hour culture of isolates, 100 μl were dropped onto 10 mm petri wells containing the said medium and incubated at 30ºC for 72 hours. Colony diameter and halo areas counted were recorded. The solubility index was calculated using the clear zone diameter/colony diameter formula (Ramesh et al. 2014) [30].

4. Antimicrobial Activity against Sclerotinia minor

4.1. The Production of Siderophores

CAS (Chrome Asurol S) blue agar was prepared using 60.5 mg CAS dissolved in 50 ml distilled water and mixed with 10 ml iron (III) solution (1 mM FeCl and 10 mM HCI). Under stirring, this solution was slowly added to 72.9 mg HDTMA. (Hexadecyl-trimethyl-ammonium bromide) dissolved in 40 ml water. The resultant dark blue liquid was autoclaved for 20 min and also autoclaved a mixture of 750 ml water, 15 g agar, and the pH was adjusted with NaOH to 6.8. The CAS reaction rate was determined by measuring the intensity of color-change in the CAS-blue agar from blue to purple or orange color.

4.2. Dual Culture Assay for the Screening of Biocontrol of Syderophore-Produsing B. mojavensis

Bacterial isolate of B. mojavensis were tested for in vitro antagonistic activity against the S. minor by dual-culture assay as described by Sakthivel and Gnanamanickam (1986) [31]. B. mojavensis was plated in CAS (Chrome Azurol S) blue agar. A fungal disc (≈9 mm²) was placed in the centre of a potato dextrose agar (PDA) plate, which contained 0.7% agar. The plates were incubated at 28°C for 7 days. The growth of S. monor was observed under microscope and compared with the control plate with and without examined Bacillus mojavenisis strain.

4.3. Isolation of Sclerotinia minor Pathogen and Antimicrobial Activity against Sclerotinia minor

The tested bacterial strains were tested to determine their antifungal activities against S. minor. The pathogen was cultivated at 28 °C on yeast extract medium (10 g yeast extract, 20 g glucose and peptone, and 20 g agar) for 7 days. Then, the conidia were collected by washing with cold sterile distilled water and used to prepare an inoculum at a concentration of 1x 10⁴ spores/ml. S. minor infected plants were used in the plant pathogenicity tests according to Koch's postulates for the infection of the lettuce leaves is shown in Figure 1 A. The pathogen was isolated from the damage plants (Figure 1A) and cultured on yeast extract medium (5 g/L yeast extract, 10 g/L glucose and 20 g/L agar) at 28 °C for 5 days. Then, the 9 mm dicks were cut used to re-infects the healthy lettuce leaves after surface sterilization (Figure 1B). After infection, the fungal phytopathogen caused wilting and yellowing of lower leaves on 5–7 days of incubation (Figure 1 B). White, cottony fungal growth mycelia were observed on the basal plant surfaces and black structures called sclerotia detected within the cottony growth (Figure 1B).

Figure 1. A)S. minor infected plants B) Inoculation of lettuce leaves after surface sterilization with S. minor dicks and symptoms of the disease C) Displays the state of a control lettuce leaf.

Figure 1. A)S. minor infected plants B) Inoculation of lettuce leaves after surface sterilization with S. minor dicks and symptoms of the disease C) Displays the state of a control lettuce leaf.

3.4. Screening for Dissolution of Inorganic Phosphate and ZnO

Acid phosphatases and phytases synthesized by rhizosphere microorganisms are involved in the organic solubilization of soil phosphorus (Thaler and Pages, 1998) [35]. The strains were found to possess high (solubilization zone greater than 2 cm) phosphate-solubilizing activity on PVK medium. The established ability of three of the investigated strains to improve the solubility of inorganic phosphates is an important characteristic of PGP microorganisms. The results of the present study showed that cell free supernatant of B. subtilis, P. megaterium, and B. safensis did not exhibit such activity (Figure 4). The highest dissolution index was observed in B. mojavesis cell free culture was 2.670 ± 0.264 cm (Figure 3 A). The established ability of the investigated strain to improve the solubility of inorganic phosphates is an important characteristic of PGP-microorganisms.

ZnO is insoluble in water and soluble in most of acids (Klingshirn, 2007) [36]. Carboxylic groups of organic acids can complex metal cations and displace anions to convert insoluble forms of Zn, such as ZnO, into more soluble forms (Chen et al., 2009) [37]. Several laboratory studies have reported that microbs, which produce siderophores or CO₂, may contribute to environmental Zn solubilization, depending on species of organism and their growth conditions (Kushwaha et al. 2020; Saleem et al., 2023) [38,39]. B. mojavesis showed the Zn solubilising activity, while the remaining strains showed no zinc solubilizing ability (Figure 3 B).

3.5. The Production of Siderophores

A change to yellow was found in P .megaterum (2), B. safensis (3) and B. mojavensis (4), with a zone of 0.323 ± 0.078 cm, 0.167 ± 0.032, 0.598 ± 0.233 cm, respectively (Figure 4 A). Exogenous application on two layers CAS/PDA agar of the bacterial siderophore to fungal cultures resulted in decreased colony size, increased filament length, and changes in hyphal branching pattern (Figure 4 B and C).

Figure 4. A) Siderophores production by B. subtilis, P. megaterum, B. safensis, and B. mojavensis strains, expressed as a halo zone on CAS agar plate. B) S. minor filament growth, and С) changes in hyphal branching pattern after treatment with B. mojavensis siderophores on two layers CAS/PDA agar.

Figure 4. A) Siderophores production by B. subtilis, P. megaterum, B. safensis, and B. mojavensis strains, expressed as a halo zone on CAS agar plate. B) S. minor filament growth, and С) changes in hyphal branching pattern after treatment with B. mojavensis siderophores on two layers CAS/PDA agar.

In the current investigation, the siderophore production was detected the CAS reagent which revealed a change in blue to orange color by P .megaterum (2), B. safensis (3) and B. mojavensis (4). The result is in accordance with the findings of De Los Santos-Villalobos, 2012; Mehri et al. 2012 [40,41] that reported different doses of zinc and magnesium also play a major role in the induction of siderophore production by the formation of yellow-green fluorescent pigment around them on nutrient agar medium. Several studies revealed that siderophores are involved in the stimulation of plant growth and also protecting the plants against various biotic stress phytopathegens Ramos-Solano et al., 2010 [42] including Sclerotinia sclerotiorum (Lib.) de Bary Ribeiro et al. (2021) [43].

3.7. In Vitro Antifungal Activity of the Tested Bacillus Strains

Many active bacterial strains have been described as biocontrol agents due to their common ability to produce various antifungal metabolites. The antifungal activity of the four tested strains inhibited S. minor and the calculated inhibition effects are presented in Figure 5. From the obtained results, all four strains of tested bacteria completely inhibit the growth of S. minor (Figure 6).

S. minor produces mycotoxins which are unsuitable for human consumption. S. minor has become one of the most economically important and studied fungal plant pathogens. Environmental pollution caused by excessive use of chemical pesticides has led to considerable changes in people’s attitudes toward their substation with biological control with antagonistic microorganisms (Figure 5).

In vivo, the antibacterial efficacy of B. mojavensis against S. minor in lettuce showed inhibition on the seventh day after infection (DAI) (Figure 6). On the seventh DAI symptoms was observed with from the plant tissues without treatment with beneficial bacteria. In the absence of B. mojavensis, the fungal pathogen caused 100% infection with lesions of the lettuce plants (Figure 6). In the presence of the B. mojavensis, a decrease in infection was observed on the seventh DAI (Figure 6). The results demonstrate a promising potential for the suppression of the S. minor infection of Lactuca sativa and expand the knowledge on antimicrobial applications of Bacillus strains.

In conclusion, Bacillus sp. can promote plant growth and suppress phytopathogens by producing amylolytic, proteolytic enzymes nad siderophores. In the pot trials, when lettuce plants were grown in soils contaminated with S. minor, a 100% pathogen attack was determined when plants were without treatment B. mojavensis CAM23K1. Lettuce treated with bacteria reduces the effect of the pathogen. The antagonistic B. mojavensis CAM23K1 suppressed S. minor disease in the pot experiment and promoted lettuce growth and development.