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Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic
Scientia Horticulturae 261 (2020) 108931
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Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic
T
Vytória Piscitelli Cavalcantia, Neilton Antonio Fiusa Araújoa, Natália Bernardes Machadoa, Paulo Sérgio Pedroso Costa Júniorb, Moacir Pasquala, Eduardo Alvesc, Kátia Regina Freitas Schwan-Estradad, Joyce Dóriaa,* a
Agriculture Department, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil Biology Department, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil c Phytopathology departament, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil d Agronomy Department, State University of Maringá (UEM), Av. Colombo, 5790 – Jardim Universitário, 87020-220, Maringá, PR, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Allium sativum Bacillus amyloliquefaciens Bacillus pumilus Candida labiduridarum Biological control
This study aimed to detect potential bacteria and yeast for prevention of Sclerotinia sclerotiorum infection via biocontrol in garlic. Two yeasts (Pichia kudriavzeviiand Candida labiduridarum) and four bacteria (Bacillus acidiceler, B. macauenses, B. amyloliquefaciens and B. pumilus) were tested. The effect of volatile and diffusible antifungal metabolites on S. sclerotiorum mycelial growth in vitro was evaluated. Garlic cloves were immersed in a suspension of each microorganism (1 × 108 cells ml−1), then the phytopathogen was inoculated and the cloves were kept in a moist chamber for 15 days, at which point lesion diameter was evaluated and electron micrographs were obtained. The results showed a higher percentage of inhibition of S. sclerotiorum growth by volatile metabolites produced by C. labiduridarum, B. macauenses, B. amyloliquefaciensand B. pumilus than by those produced by the other agents, with variation of 74.61%–87.61%. A high reduction in phytopathogen growth due to B. amyloliquefaciens(84%) was observed, suggesting that B. amyloliquefaciens produces antifungal metabolites that inhibit phytopathogen development. The reduction in disease-affected area was most significant in garlic cloves treated with B. pumilus (86.74%) and C. labiduridarum (61.47%). Electron micrographs showed garlic clove surface colonization by all tested microorganisms and phytopathogen hyphae colonization. Research on B. amyloliquefaciens, B. pumilus and C. labiduridarum biocontrol of S. sclerotiorum in garlic is of interest.
1. Introduction Garlic (Allium sativum L.) is a species originating from Asia belonging to the Alliaceae family. It is a medicinal plant used in the treatment of various human diseases such as infections, cancer prevention and the ability to lower blood pressure and cholesterol, as well as being used as an antifungal, antihelmintic, antihypertensive, mild antihypertensive, antidiabetic, antioxidant, hepatoprotective, anti-inflammatory and wound healing agent (Borlinghaus et al., 2014; Corzomartínez et al., 2007; García Gómez and Sánchez-Muniz, 2000; Londhe et al., 2011; Yeh and Liu, 2001). In addition to its medicinal applications, garlic is a spice widely consumed around the world and is also being used for pest and disease control in plants (Slusarenko et al., 2008) due to its phytochemical
composition, which contains essential oils, sulfur compounds, carbohydrates, proteins, mineral salts and vitamins, and garlic cloves are the most commonly used portion because in them are concentrated the active constituents (Chagas et al., 2012; Kusano et al., 2016). The occurrence of phytopathogens is a factor that can depreciate product quality, also causing reductions in production. Therefore, it is fundamental to control the presence and manifestation of pests and diseases throughout the production process. To avoid white rot (Sclerotium cepivorum), many producers are advancing the date of garlic planting. During this period, the soils are warmer than in traditional planting time and are unfavorable to the development of the plant pathogen (Pinto et al., 2000). However, hot and humid conditions favor the development of other phytopathogens, such as the fungus Sclerotinia sclerotiorum, which causes white mold and is a general disease-causing
Corresponding author. E-mail addresses: [email protected] (V.P. Cavalcanti), [email protected] (N.A.F. Araújo), [email protected] (N.B. Machado), [email protected] (P.S.P. Costa Júnior), [email protected] (M. Pasqual), [email protected] (E. Alves), [email protected] (K.R.F. Schwan-Estrada), [email protected] (J. Dória). ⁎
https://doi.org/10.1016/j.scienta.2019.108931 Received 28 March 2019; Received in revised form 9 September 2019; Accepted 8 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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species in more than 500 species worldwide, being the most devastating and cosmopolitan plant pathogen (Boland and Hall, 1994; Bolton et al., 2006; Jaccoud Filho et al., 2017; Mcdonald and Boland, 2017). Several researchers have reported yield losses caused by S. sclerotiorum in many crops, and it can reach 100% of loss (Jaccoud Filho et al., 2017). Because garlic is a host species of S. sclerotiorum (Boland and Hall, 1994; Jaccoud Filho et al., 2017), planting under conditions that favor growth of this pathogen may lead to damage garlic crop, in addition to allowing the maintenance of this fungus in soil, leading to infection of other susceptible crops. Some farmers, specially from the south and southeast regions of Brazil, has reported the occurrence of this fungus on garlic field (data not published). Thus, we aimed to take a step ahead and focussed to prevent the damage of this pathogen in garlic crop. Chemical control is commonly used, although it presents great risks to the environment and human health, increases production costs and does not present satisfactory results since chemicals act mainly by controlling ascospores disseminated in air and do not act efficiently on the germination of sclerotia in soil (Cardoso et al., 2017). In addition, phytopathogen control is not effective through a single cultural or chemical practice, such as the use of fungicides or cultural rotation with resistant plants (de Sousa and Blum, 2013). Thus, the demand for diversified methods for phytopathogen control has risen. Biological control is a highly studied method because of its efficiency. It is more economical and causes fewer impacts than the use of agrochemicals, presenting itself as a great alternative in organic agriculture practice. Biological control agents are used to reduce the percentage of infected plants and the levels of disease. Trichoderma harzianum (Avila Miranda et al., 2006; de Sousa and Blum, 2013), Pseudomonas fluorescens, Bacillus subtilis and Bacillus pumilus (Elshahawy et al., 2018) have been proved to control white-rot in garlic, with controlling efficiency comparable or better than chemical fungicide. The efficacy of biocontrol agent can be tested by paired cultures method by measuring the reduction of the mycelial growth. The isolates of the genus Trichoderma (Isaias et al., 2014), Pseudomonas and Bacillus (Abou-Aly et al., 2015) produce secondary metabolites, which significantly reduce the growth of phytopathogens. These metabolites include chitinase, siderophores, ammonia, hydrogen cyanide and volatile antibiotics (Abou-Aly et al., 2015). Yeasts and Bacillus spp. are also used as biocontrol agents, exhibiting satisfactory results in the control of several different phytopathogens (Cavalcanti et al., 2018). Therefore, the aim of this essay was to detect yeasts and bacteria of the genus Bacillus with potential for biological control of Sclerotinia sclerotiorum in Allium sativum by observing the (1) production of antifungal volatile and diffusible compounds, through in vitro tests, and the (2) reduction in the area affected by the disease treating garlic cloves with the antagonistic microorganisms.
Table 1 Microorganisms and their proper sources and places of origin. Code CCMA CCMA CCMA CCMA CCMA CCMA
0026 0027 0057 0058 0084 0098
Species
Location
Source
Pichia kudriavzevii Candida labiduridarum Bacillus acidiceler Bacillus macauenses Bacillus amyloliquefaciens Bacillus pumilus
Passos, MG, Brasil Arcos, MG, Brasil Passos, MG, Brasil Passos, MG, Brasil Arcos, MG, Brasil Arcos, MG, Brasil
Moist soil Moist soil Marigold pepper Marigold pepper Pineaple Souari nut
2.2. Volatile antifungal metabolite production The effect of volatile metabolites produced by yeast and bacterial isolates on S. sclerotiorum growth in vitro was evaluated using a completely randomized design with six treatments consisting of each yeast or bacteria and the phytopathogen as a control, with 4 replicates of 1 Petri dish each. This experiment was repeated under the same conditions within one year. A fungal mycelial growth disk was placed in the center of a 9 cm diameter Petri dish containing nutrient agar medium (NA) (meat extract (3 g), soy peptone (5 g), agar (13 g) and distilled water (1000 ml)), and the bacteria and yeast were cultured for two days in another Petri dish of the same size containing YEPD medium. Then, the plates with the respective microorganisms were attached with clear adhesive tape and placed in a biological oxygen demand incubator (BOD) at 25 °C with a photoperiod of 12 h, leaving the Petri dish of the phytopathogen superimposed on the antagonist Petri dish (Vieira et al., 2017). Measurement of mycelial growth was performed on the eighth day, the amount of time required for the control to reach the edge of the plate. The mycelial growth diameter of the phytopathogen was evaluated by measuring two orthogonal axes using two diametrically opposed measurements. The results were expressed as the percentage of mycelial growth inhibition of Sclerotinia sclerotiorum and were calculated through the measurement of how much smaller the mycelial growth diameter was compared with that of the control. The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011). 2.3. Diffusible antifungal metabolite production The experiment was performed on Petri dishes containing NA medium, where 5 mm diameter disks containing S. sclerotiorum growth were placed 2 cm from the edge of the Petri dish, and a growth loop of bacteria and yeast was spread as a 3 cm streak in the center of the dish (Rosa et al., 2010). Each microorganism constituted a treatment, using a control containing only the phytopathogen. The design was completely randomized, with four replicates performed for each treatment, each replicate consisting of one Petri dish. This experiment was repeated within one year under the same conditions. The treatments were incubated in BOD at a temperature of 25 ± 2 °C and a photoperiod of 12 h. Mycelial growth measurement was performed eight days after incubation, when growth in the control reached the edge of the plate, by measuring the mycelial growth in the direction of the central streak and in opposite direction. The results of phytopathogen mycelial growth in the direction of the antagonistic microorganisms and in opposite direction were expressed as mm. The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011).
2. Material and methods 2.1. Acquisition and culture of the antagonistic microorganisms and pathogen Yeasts and bacteria were obtained from the Agricultural Microbiology Cultures Collection (CCMA) of Federal University of Lavras (UFLA), and the collection codes of each species with the respective origin sites and substrates are described in Table 1. For the first tests, bacteria were chosen according to genera and species described in the literature as biocontrol agents in plants. The microorganism isolates were cultivated in yeast extract - peptone - dextrose medium (YEPD), consisting of glucose (20 g), peptone (20 g), yeast extract (10 g), agar (15 g) and distilled water (1000 ml). The Sclerotinia sclerotiorum isolate was obtained from Allium sativum bulbs that showed disease symptoms. For isolation, the pathogen present in the garlic bulb lesion was collected and grown in potato - dextrose - agar medium (PDA).
2.4. Evaluation of yeasts and Bacillus spp. on S. sclerotiorum control in garlic The effect of yeasts and bacteria of the genus Bacillus on the control 2
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of Sclerotinia sclerotiorum in vivo was evaluated using garlic bulbs of the Giant Purple cultivar. The experiment was carried out based on the methodologies described by Rahman et al. (2016) and Mello et al. (2011) with some adaptations, as described below. Six treatments consisting of each individual organism and two controls were carried out; for the controls, one was treated with sterile distilled water inoculated with the phytopathogen, and one was treated with sterile distilled water without inoculation of the phytopathogen. The design was completely randomized with six replicates used for each treatment, each consisting of two garlic cloves. This experiment was repeated twice under the same conditions within one year. From the Petri dishes containing the yeast and bacteria with 4 days of growth, aqueous suspensions were prepared. Fifty milliliters of sterile distilled water was added to the Petri dishes, and the suspension was made using a Drigalski loop, adjusting to a concentration of 1 × 108 cells ml−1. For microbiolization, the garlic bulbs were immersed in the suspension for 1 h and then placed to dry in Petri dishes for 30 min. After this time, the inoculation of the phytopathogen was performed using 3 mm diameter discs with 8 days phytopathogen mycelial growth that were placed on the surface of the garlic cloves and kept in a humid chamber at room temperature (28 ± 3 °C) for 15 days. The humid chambers were made of Petri dish bases, where the garlic cloves were incubated with a cotton swab dipped in sterile distilled water in the center of the plate, packed in a plastic bag sprinkled with sterile distilled water (Dantas et al., 2003). At 15 days, the garlic cloves were removed from the humid chamber, and the lesion diameter was measured using an electronic caliper. Measurements were taken in two directions, and the means of the measurements were used to evaluate the development of the disease. The results of lesion diameter measurement were expressed in millimeters, and the reduction in affected area was calculated through the measurement of how much smaller the lesion diameter was than that of the control with the phytopathogen and expressed as percentage (%). The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011).
Karnovsky solution (2.5% glutaraldehyde, 2.5% formaldehyde in 0.05 M sodium cacodylate, pH 7.2, 0.001 M CaCl2) for at least 24 h. The fixed fragments were transferred to a cacodylate solution (0.05 M) and washed three times for 10 min. They were then washed in distilled water three times and dehydrated in solutions with increasing concentrations of acetone (25, 50, 75, 90 and 100%). Subsequently, they were dried to the critical point with liquid CO2 in a Balzers CPD 030 apparatus and then mounted on aluminum brackets (stubs) and covered with gold (Balzers SCD 050 evaporator) for observation in a LEO EVO 40 scanning electron microscope (Alves, 2005). 3. Results 3.1. Volatile antifungal metabolite production The inhibition of growth of S. sclerotiorum colonies by volatile compounds produced by the microorganisms showed a greater percentage of inhibition by the compounds produced by C. labiduridarum, B. macauenses, B. amyloliquefaciens, B. pumilus than by those produced by the other agents, with the inhibition of fungal growth varying from 74.61% (C. labiduridarum) to 87.61% (B. pumilus) (Fig. 1). The lowest inhibition rate was that of by B. acidiceler (3.78%). 3.2. Diffusible antifungal metabolite production In relation to the production of diffusible antifungal metabolites, Fig. 2 shows the fungus growth (cm) from the center of the graph to the end in the direction of each antagonistic microorganism (▲) and in the opposite direction to the antagonistic microorganism (●). Thus, we observed that there was inhibition of the growth of S. sclerotiorum colonies in the direction of B. amyloliquefaciens, suggesting the production of compounds by the bacteria that inhibit the development of the colony. This effect was more pronounced for B. amyloliquefaciens than that observed for other microorganisms. The yeast C. labiduridarum and the bacteria B. macauenses and B. pumilus also showed antifungal metabolite production, which caused less inhibition of phytopathogen growth when compared to that of B. amyloliquefaciens. The bacterium B. acidiceler and the yeast P. kudriavzevii showed no inhibition of colony growth, suggesting the lack of production of antifungal compounds or a low efficiency of the compounds produced (Fig. 2). No inhibition of fungal colony growth was observed in the opposite direction to the antagonist microorganism (Fig. 2, circles). The
2.5. Scanning electron microscopy At the end of the experiment described above, garlic clove fragments from each treatment were collected to evaluate the interaction of phytopathogens with microorganisms by scanning electron microscopy imaging. The fragments collected were immediately fixed in modified
Fig. 1. Growth inhibition (%) of Sclerotinia sclerotiorum in response to volatile and diffusible antifungal metabolite production. * Bars with capital (volatile metabolites) and lower case (diffusible metabolites) letters do not differ according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
3
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Fig. 2. Development of S. sclerotiorum colonies in Petri dishes together with the antagonistic microorganisms. (A) Growth of the phytopathogen (cm) to the microorganisms’ side (▲) and growth of the phytopathogen (cm) to the opposite side from the microorganisms (●). * Lower case letters (▲) and upper case letters (●) do not differ from each other according to Tukey’s test (p < 0.05). (B) Scheme of diffusible antifungal metabolite production experiment, showing the directions of the phytopathogen growth measures carried out to the microorganisms' side and to the opposite side. This experiment was repeated twice with similar results.
Fig. 3. Illustration of antifungal metabolite production experiments for in vitro control of Sclerotinia sclerotiorum. A - Bacillus amyloliquefaciens; B - Bacillus macauenses; C - Candida labiduridarum; D - Pichia kudriavzevii; E - Bacillus pumilus; F - Bacillus acidiceler.
differences in inhibition of fungus growth between treatments in the test of antifungal metabolite production can be observed in Fig. 3. The percentage of fungal growth inhibition caused by B. amyloliquefaciens was greater than 80% (Fig. 1), showing that its use in the biological control of Sclerotinia sclerotiorum may be promising.
the control garlic cloves (Table 2). The highest phytopathogen control was occasioned by treatment with B. pumilus, where the greatest reduction in area affected by the disease was recorded (86.8%) when compared to that of the other treatments, ranging from 30.18% for B. amyloliquefaciens to 61.47% for C. labiduridarum. The lesions caused by S. sclerotiorum on A. sativum cloves and the reduction in area affected by the disease in the different treatments can be observed in Fig. 5. Scanning electron microscopy images showed the different microorganisms colonizing superficial layers of the garlic cloves, as well as their interaction with the phytopathogen (Fig. 6). The electron micrographs also allow us to note the interaction between the antagonistic microorganisms and the phytopathogen through hyphae colonization, which may cause changes in the morphology of S.
3.3. Evaluation of yeasts and Bacillus spp. on S. sclerotiorum control in Allium sativum A reduction in the symptoms of S. sclerotiorum disease in garlic cloves treated with the microorganisms was also observed (Table 2 and Fig. 4). Smaller lesion diameters were observed in garlic cloves treated with B. pumilus (0.69 mm) and Candida labiduridarum (2.02 mm) than in 4
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volatile compounds since there was no contact between the phytopathogen and the antagonist and emphasizing the simplicity and efficiency of the evaluation of production of these compounds. Although the effect of volatile compounds produced by bacteria can be considered low (15 to 26%) to moderate (28 to 53%) in phytopathogenic fungi control, some species produce effective compounds that act to decrease mycelial growth and conidial germination of phytopathogens (Campos et al., 2010). The results obtained in this study showed variations between 45.4 and 74.61% inhibition of mycelial growth of S. sclerotiorum by the volatile compounds produced by the yeasts Pichia kudriavzevii and Candida labiduridarum, respectively, and between 3.78 and 87.61% by Bacillus acidiceler and B. pumilus, respectively (Fig. 1). Fernando et al. (2005) also verified the production of volatile organic compounds with antifungal activity in bacteria isolated from canola and soybean plants, which acted to inhibit the formation of sclerotia and the germination of ascospores, in addition to limiting the mycelial growth of Sclerotinia sclerotiorum in vitro and in soil tests, reducing levels of disease. Fialho et al. (2011) verified the control of S. sclerotiorum in vitro and the reduction in disease incidence in bean seeds through volatile organic compounds produced by the yeast Saccharomyces cerevisiae. Among the bacteria identified by Zou et al. (2007), which produce volatile compounds with antifungal activity, two strains of Bacillus pumilus showed inhibition in the mycelial growth of Paecilomyces lilacinus ranging from 65.2 to 100%. Campos et al. (2010), in their review, observed isolates of Bacillus amyloliquefaciens and Bacillus pumilus with
Table 2 Lesion diameter (mm) of Sclerotinia sclerotiorum in garlic cloves treated with different microorganisms. Microorganisms
Lesion diameter (mm)
Pichia kudriavzevii Candida labiduridarum Bacillus acidiceler Bacillus macauenses Bacillus amyloliquefaciens Bacillus pumilus Control with phytopathogen
3,13 2,02 2,53 3,53 3,66 0,69 5,24
bcz ab bc c c a d
z Means followed by the same letter did not differ from each other according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
sclerotiorum hyphae and help to contain infection by them, as observed for the yeast Pichia kudriavzevii and for the bacteria Bacillus acidiceler and B. amyloliquefaciens (Fig. 6). 4. Discussion In vitro tests have been efficient methods to identify volatile compound production by microorganisms that have the potential to be used in biological control of primary phytopathogens. Four microorganisms evaluated for volatile antifungal metabolite production were efficient in inhibiting phytopathogen colony growth, indicating the production of
Fig. 4. Percentage reduction in area affected by the disease caused by Sclerotinia sclerotiorum in Allium sativum cloves treated with different antagonistic microorganisms. * Bars with equal letters do not differ from each other according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
Fig. 5. Illustration of the effect of microorganisms on Sclerotinia sclerotiorum control in garlic. 5
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Fig. 6. Scanning electron micrographs of the interaction of yeasts and Bacillus with Sclerotinia sclerotiorum on garlic cloves. A - Phytopathogen colonizing garlic without inoculation of potential biocontrol agents; B - Pichia kudriavzevii colonizing the fungal hyphae (arrow); C - Candida labiduridarum and bacteria on the garlic surface (arrow indicating the yeast); D - Bacillus acidiceler with the arrow indicating its colonization in the phytopathogenic hyphae; E - Bacillus macauenses colonizing the garlic surface; F - Interaction between Bacillus amyloliquefaciens and the phytopathogen; G - Surface colonization of the garlic clove by Bacillus pumilus.
antifungal activity against several phytopathogens, including Sclerotinia sclerotiorum. Satisfactory results were observed for these two bacteria in the in vitro assays, with a reduction in the mycelial growth of S. sclerotiorum by 87.61 and 80.75% by volatile antifungal metabolites produced by B. pumilus and B. amyloliquefaciens, respectively, and 82.35%
by diffusible antifungal metabolites produced by B. amyloliquefaciens. The diffusible antifungal metabolite test identified the bacterium B. amyloliquefaciens as highly efficient (above 80%) in inhibiting the hyphae growth of S. sclerotiorum and identified two more bacteria (Bacillus pumilus and B. macauenses) and one yeast (Candida labiduridarum) with 6
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moderate potential (31.37–37.25 %) in phytopathogen control. The production of secondary metabolites by microorganisms has also been extensively studied for application in the biological control of major plant diseases. It has been proven that some Bacillus amyloliquefaciens strains produce lipopeptides such as C17 bacillomycin D, anteiso-C13 and iso-C13 [Ile7] surfactins with an potent antifungal action, being proven to control Botrytis cinerea in tomato and cucumber (Masmoudi et al., 2017; Tanaka et al., 2015). Metabolite production may explain the high efficiency of this bacterium in inhibiting the growth of S. sclerotiorum colonies in vitro. No inhibition of fungal colony growth was observed in the opposite direction of antagonistic microorganisms (Fig. 2, circles), suggesting that the inhibition of phytopathogen development is associated with its proximity to the evaluated microorganisms and reinforcing the possibility of production of inhibitory compounds by bacteria and yeasts. Suneeta et al. (2016) observed control of Sclerotium rolfsii by Bacillus isolates through antibiosis, with the action of these microorganisms in the field occurring through the release of extracellular compounds, antimicrobial substances and plant hormones in the rhizosphere, causing a reduction in disease development, in addition to helping in plant growth and helping in the absorption of nutrients present in the soil. The behavior of B. amyloliquefaciens was satisfactory in both experiments with production of volatile and diffusible antifungal metabolites on in vitro evaluations, demonstrating its potential for application in phytopathogen control. This microorganism has already been used as an alternative to chemicals in control of brown rot, caused by Monilinia laxa and Monilinia fructicola, reducing more than 60.0 and 75.5% of disease incidence and severity in peach fruits under laboratory conditions, using a concentration of 107 CFU ml−1 (Gotor-Vila et al., 2017). In studies by Vinodkumar et al. (2017), Bacillus amyloliquefaciens inhibited 45% of the mycelial growth of S. sclerotiorum and 100% of its sclerotia production on in vitro tests. This bacterium also reduced the incidence of white mold (4.6%), in addition to promoting the growth of clove plants (Dianthus caryophyllus) that were immersed in bacterial suspension for in vivo tests (Vinodkumar et al., 2017). The application of B. amyloliquefaciens at a concentration of 108 cells ml−1 for Sclerotinia sclerotiorum control in Allium sativum cloves presented a 31.29% reduction in the area affected by white mold (Fig. 4), and in vitro, it showed an inhibition of 82.35% of the development of the pathogen (Fig. 1), again demonstrating the biological control potential of this bacterium. Antifungal action against Aspergillus parasiticus has been observed through low molecular weight extracellular compounds produced by the bacterium Bacillus pumilus, which act to inhibit spore germination and hyphae development as well as to inhibit the production of aflatoxins (Bottone and Peluso, 2003; Munimbazi and Bullerman, 1997). The application of B. pumilus to garlic presented satisfactory results in S. sclerotiorum biological control, which may be related to the production of antifungal metabolites. In addition, the Bacillus pumilus result in the in vivo assay with Allium sativum was more expressive than that in the in vitro parity tests. This result may be related to the induction of resistance caused by this bacterium in garlic, increasing the efficiency of the control of phytopathogens, and further studies are necessary to confirm this activity. The phytopathogen Sclerotinia sclerotiorum is difficult to control, and the lack of genetic resistance against this pathogen led Bochalya et al. (2016) to study the induction of systemic resistance in Indian mustard (Brassica juncea L.) through treatment with rhizobacteria, observing high rates of disease control with the application of Pseudomonas and Bacillus related to the high total phenolic and polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), peroxidase (PO) and chitinase activity. The inhibitory action caused by microorganisms on conidial germination and phytopathogen colony development is one of the main
mechanisms of action sought by biological control studies involving bacteria and yeasts. This action may be related to antibiosis or parasitism, in which the microorganism colonizes and impairs phytopathogen structures, as observed in some strains of Trichoderma in studies on the control of S. rolfsii (Hirpara et al., 2017). Against the fungus Sclerotinia sclerotiorum, there is a report of a species of Aspergillus (ASP-4 strain) that parasitizes and destroys the sclerotia (Hu et al., 2013), in addition to the species Coniothyrium minitans, which through mycoparasitism and antibiosis is able to degrade oxalic acid, an important virulence factor of this phytopathogen (Zeng et al., 2014). Additionally, actinobacteria of the genus Streptomyces isolated from Brazilian tropical soils were able to irreversibly inhibit the growth of S. sclerotiorum through the production of chitinolytic enzymes, such as exochitinase, endochitinase, glucanase and peptidase, which degrade the cell wall of hyphae and sclerotia of the fungus (Fróes et al., 2012). The colonization of phytopathogen hyphae by biocontrol agents was also observed in this work by the yeast Pichia kudriavzevii and by the bacteria Bacillus acidiceler and B. amyloliquefaciens, indicating a possible occurrence of parasitism in biological control of the fungus. In addition to antibiosis, induction of systemic resistance and mycoparasitism (Chaurasia et al., 2005; Droby et al., 2002; Gond et al., 2015), yeasts and bacteria also act through nutrient competition and niche exclusion since they have the ability to colonize rapidly because they are single-celled organisms with a higher growth rate than filamentous fungi that are multicellular (Sun et al., 2017; Úbeda et al., 2014). These diverse abilities developed by antagonistic microorganisms, such as antibiosis, mycoparasitism, and competition for nutrients and space, among others, play an important role in the biological control of phytopathogens, such as S. sclerotiorum, especially the fact that they can act through more than one of these action mechanisms, increasing their efficiency. In the electron micrographs, it was possible to observe colonization of the Allium sativum surface by the antagonistic microorganisms tested, indicating potential for nutrient and space competition and niche exclusion, as well as for biofilm formation (Fig. 6B–G). Rahman et al. (2016) observed biofilm formation by all Bacillus isolates tested against S. sclerotiorum. The biofilm is an important mechanism for the colonization on the surface of the roots and leaves that helps in diverse phytopathogen prevention. Chi et al. (2015) also verified biofilm formation by the yeast Pichia kudriavzevii, noting that this state, in addition to being involved in biological control activity, is a morphological form that is more resistant to high temperatures and oxidative stress. Kaushal et al. (2017) observed the biological control of white mold caused by Sclerotinia sclerotiorum in cauliflower (Brassica oleracea var. Botrytis L.) by applying a Bacillus pumilus cell suspension (1 × 108 CFU ml−1), suppressing the severity of the disease by 93% and increasing culture productivity by 36%, showing that Bacillus pumilus is a potential bacterium for both biocontrol and plant growth promotion. In the present study, the bacterium B. pumilus showed the greatest reduction in area affected by white mold on garlic cloves (Allium sativum), reaching 86.74% (Fig. 4). More research is needed to verify its effects in the field, both for S. sclerotiorum control and for A. sativum productivity. 5. Conclusion The results present some antagonistic microorganisms with potential for control of Sclerotinia sclerotiorum, especially Bacillus amyloliquefaciens and B. pumilus among the bacteria tested and Candida labiduridarum among the yeasts. Bacillus amyloliquefaciens probably produce compounds that were inhibitory to Sclerotium sclerotiorum development in vitro. Bacillus pumilus and Candida labiduridarum presented the greatest reduction in area affected by the disease in the in vivo test, showing potential for use in biological control of S. sclerotiorum in Allium sativum. These results open a broad future line of work, like the (1) identification of the metabolites produced by these microorganisms, to determine which components are responsible for the antifungal 7
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activity; (2) test of the most efficient population of these microorganisms, as well as their combination, and measure the antifungal activity and the ideal concentration of their metabolites; (3) test of different kinds of products formulation; and (4) perform tests applying these microorganisms, or their metabolites, in greenhouse and in field conditions to make sure that they are efficient biocontrol agents.
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Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic
T
Vytória Piscitelli Cavalcantia, Neilton Antonio Fiusa Araújoa, Natália Bernardes Machadoa, Paulo Sérgio Pedroso Costa Júniorb, Moacir Pasquala, Eduardo Alvesc, Kátia Regina Freitas Schwan-Estradad, Joyce Dóriaa,* a
Agriculture Department, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil Biology Department, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil c Phytopathology departament, Federal University of Lavras (UFLA), P.O. Box 303, 37200-000, Lavras, MG, Brazil d Agronomy Department, State University of Maringá (UEM), Av. Colombo, 5790 – Jardim Universitário, 87020-220, Maringá, PR, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Allium sativum Bacillus amyloliquefaciens Bacillus pumilus Candida labiduridarum Biological control
This study aimed to detect potential bacteria and yeast for prevention of Sclerotinia sclerotiorum infection via biocontrol in garlic. Two yeasts (Pichia kudriavzeviiand Candida labiduridarum) and four bacteria (Bacillus acidiceler, B. macauenses, B. amyloliquefaciens and B. pumilus) were tested. The effect of volatile and diffusible antifungal metabolites on S. sclerotiorum mycelial growth in vitro was evaluated. Garlic cloves were immersed in a suspension of each microorganism (1 × 108 cells ml−1), then the phytopathogen was inoculated and the cloves were kept in a moist chamber for 15 days, at which point lesion diameter was evaluated and electron micrographs were obtained. The results showed a higher percentage of inhibition of S. sclerotiorum growth by volatile metabolites produced by C. labiduridarum, B. macauenses, B. amyloliquefaciensand B. pumilus than by those produced by the other agents, with variation of 74.61%–87.61%. A high reduction in phytopathogen growth due to B. amyloliquefaciens(84%) was observed, suggesting that B. amyloliquefaciens produces antifungal metabolites that inhibit phytopathogen development. The reduction in disease-affected area was most significant in garlic cloves treated with B. pumilus (86.74%) and C. labiduridarum (61.47%). Electron micrographs showed garlic clove surface colonization by all tested microorganisms and phytopathogen hyphae colonization. Research on B. amyloliquefaciens, B. pumilus and C. labiduridarum biocontrol of S. sclerotiorum in garlic is of interest.
1. Introduction Garlic (Allium sativum L.) is a species originating from Asia belonging to the Alliaceae family. It is a medicinal plant used in the treatment of various human diseases such as infections, cancer prevention and the ability to lower blood pressure and cholesterol, as well as being used as an antifungal, antihelmintic, antihypertensive, mild antihypertensive, antidiabetic, antioxidant, hepatoprotective, anti-inflammatory and wound healing agent (Borlinghaus et al., 2014; Corzomartínez et al., 2007; García Gómez and Sánchez-Muniz, 2000; Londhe et al., 2011; Yeh and Liu, 2001). In addition to its medicinal applications, garlic is a spice widely consumed around the world and is also being used for pest and disease control in plants (Slusarenko et al., 2008) due to its phytochemical
composition, which contains essential oils, sulfur compounds, carbohydrates, proteins, mineral salts and vitamins, and garlic cloves are the most commonly used portion because in them are concentrated the active constituents (Chagas et al., 2012; Kusano et al., 2016). The occurrence of phytopathogens is a factor that can depreciate product quality, also causing reductions in production. Therefore, it is fundamental to control the presence and manifestation of pests and diseases throughout the production process. To avoid white rot (Sclerotium cepivorum), many producers are advancing the date of garlic planting. During this period, the soils are warmer than in traditional planting time and are unfavorable to the development of the plant pathogen (Pinto et al., 2000). However, hot and humid conditions favor the development of other phytopathogens, such as the fungus Sclerotinia sclerotiorum, which causes white mold and is a general disease-causing
Corresponding author. E-mail addresses: [email protected] (V.P. Cavalcanti), [email protected] (N.A.F. Araújo), [email protected] (N.B. Machado), [email protected] (P.S.P. Costa Júnior), [email protected] (M. Pasqual), [email protected] (E. Alves), [email protected] (K.R.F. Schwan-Estrada), [email protected] (J. Dória). ⁎
https://doi.org/10.1016/j.scienta.2019.108931 Received 28 March 2019; Received in revised form 9 September 2019; Accepted 8 October 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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species in more than 500 species worldwide, being the most devastating and cosmopolitan plant pathogen (Boland and Hall, 1994; Bolton et al., 2006; Jaccoud Filho et al., 2017; Mcdonald and Boland, 2017). Several researchers have reported yield losses caused by S. sclerotiorum in many crops, and it can reach 100% of loss (Jaccoud Filho et al., 2017). Because garlic is a host species of S. sclerotiorum (Boland and Hall, 1994; Jaccoud Filho et al., 2017), planting under conditions that favor growth of this pathogen may lead to damage garlic crop, in addition to allowing the maintenance of this fungus in soil, leading to infection of other susceptible crops. Some farmers, specially from the south and southeast regions of Brazil, has reported the occurrence of this fungus on garlic field (data not published). Thus, we aimed to take a step ahead and focussed to prevent the damage of this pathogen in garlic crop. Chemical control is commonly used, although it presents great risks to the environment and human health, increases production costs and does not present satisfactory results since chemicals act mainly by controlling ascospores disseminated in air and do not act efficiently on the germination of sclerotia in soil (Cardoso et al., 2017). In addition, phytopathogen control is not effective through a single cultural or chemical practice, such as the use of fungicides or cultural rotation with resistant plants (de Sousa and Blum, 2013). Thus, the demand for diversified methods for phytopathogen control has risen. Biological control is a highly studied method because of its efficiency. It is more economical and causes fewer impacts than the use of agrochemicals, presenting itself as a great alternative in organic agriculture practice. Biological control agents are used to reduce the percentage of infected plants and the levels of disease. Trichoderma harzianum (Avila Miranda et al., 2006; de Sousa and Blum, 2013), Pseudomonas fluorescens, Bacillus subtilis and Bacillus pumilus (Elshahawy et al., 2018) have been proved to control white-rot in garlic, with controlling efficiency comparable or better than chemical fungicide. The efficacy of biocontrol agent can be tested by paired cultures method by measuring the reduction of the mycelial growth. The isolates of the genus Trichoderma (Isaias et al., 2014), Pseudomonas and Bacillus (Abou-Aly et al., 2015) produce secondary metabolites, which significantly reduce the growth of phytopathogens. These metabolites include chitinase, siderophores, ammonia, hydrogen cyanide and volatile antibiotics (Abou-Aly et al., 2015). Yeasts and Bacillus spp. are also used as biocontrol agents, exhibiting satisfactory results in the control of several different phytopathogens (Cavalcanti et al., 2018). Therefore, the aim of this essay was to detect yeasts and bacteria of the genus Bacillus with potential for biological control of Sclerotinia sclerotiorum in Allium sativum by observing the (1) production of antifungal volatile and diffusible compounds, through in vitro tests, and the (2) reduction in the area affected by the disease treating garlic cloves with the antagonistic microorganisms.
Table 1 Microorganisms and their proper sources and places of origin. Code CCMA CCMA CCMA CCMA CCMA CCMA
0026 0027 0057 0058 0084 0098
Species
Location
Source
Pichia kudriavzevii Candida labiduridarum Bacillus acidiceler Bacillus macauenses Bacillus amyloliquefaciens Bacillus pumilus
Passos, MG, Brasil Arcos, MG, Brasil Passos, MG, Brasil Passos, MG, Brasil Arcos, MG, Brasil Arcos, MG, Brasil
Moist soil Moist soil Marigold pepper Marigold pepper Pineaple Souari nut
2.2. Volatile antifungal metabolite production The effect of volatile metabolites produced by yeast and bacterial isolates on S. sclerotiorum growth in vitro was evaluated using a completely randomized design with six treatments consisting of each yeast or bacteria and the phytopathogen as a control, with 4 replicates of 1 Petri dish each. This experiment was repeated under the same conditions within one year. A fungal mycelial growth disk was placed in the center of a 9 cm diameter Petri dish containing nutrient agar medium (NA) (meat extract (3 g), soy peptone (5 g), agar (13 g) and distilled water (1000 ml)), and the bacteria and yeast were cultured for two days in another Petri dish of the same size containing YEPD medium. Then, the plates with the respective microorganisms were attached with clear adhesive tape and placed in a biological oxygen demand incubator (BOD) at 25 °C with a photoperiod of 12 h, leaving the Petri dish of the phytopathogen superimposed on the antagonist Petri dish (Vieira et al., 2017). Measurement of mycelial growth was performed on the eighth day, the amount of time required for the control to reach the edge of the plate. The mycelial growth diameter of the phytopathogen was evaluated by measuring two orthogonal axes using two diametrically opposed measurements. The results were expressed as the percentage of mycelial growth inhibition of Sclerotinia sclerotiorum and were calculated through the measurement of how much smaller the mycelial growth diameter was compared with that of the control. The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011). 2.3. Diffusible antifungal metabolite production The experiment was performed on Petri dishes containing NA medium, where 5 mm diameter disks containing S. sclerotiorum growth were placed 2 cm from the edge of the Petri dish, and a growth loop of bacteria and yeast was spread as a 3 cm streak in the center of the dish (Rosa et al., 2010). Each microorganism constituted a treatment, using a control containing only the phytopathogen. The design was completely randomized, with four replicates performed for each treatment, each replicate consisting of one Petri dish. This experiment was repeated within one year under the same conditions. The treatments were incubated in BOD at a temperature of 25 ± 2 °C and a photoperiod of 12 h. Mycelial growth measurement was performed eight days after incubation, when growth in the control reached the edge of the plate, by measuring the mycelial growth in the direction of the central streak and in opposite direction. The results of phytopathogen mycelial growth in the direction of the antagonistic microorganisms and in opposite direction were expressed as mm. The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011).
2. Material and methods 2.1. Acquisition and culture of the antagonistic microorganisms and pathogen Yeasts and bacteria were obtained from the Agricultural Microbiology Cultures Collection (CCMA) of Federal University of Lavras (UFLA), and the collection codes of each species with the respective origin sites and substrates are described in Table 1. For the first tests, bacteria were chosen according to genera and species described in the literature as biocontrol agents in plants. The microorganism isolates were cultivated in yeast extract - peptone - dextrose medium (YEPD), consisting of glucose (20 g), peptone (20 g), yeast extract (10 g), agar (15 g) and distilled water (1000 ml). The Sclerotinia sclerotiorum isolate was obtained from Allium sativum bulbs that showed disease symptoms. For isolation, the pathogen present in the garlic bulb lesion was collected and grown in potato - dextrose - agar medium (PDA).
2.4. Evaluation of yeasts and Bacillus spp. on S. sclerotiorum control in garlic The effect of yeasts and bacteria of the genus Bacillus on the control 2
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of Sclerotinia sclerotiorum in vivo was evaluated using garlic bulbs of the Giant Purple cultivar. The experiment was carried out based on the methodologies described by Rahman et al. (2016) and Mello et al. (2011) with some adaptations, as described below. Six treatments consisting of each individual organism and two controls were carried out; for the controls, one was treated with sterile distilled water inoculated with the phytopathogen, and one was treated with sterile distilled water without inoculation of the phytopathogen. The design was completely randomized with six replicates used for each treatment, each consisting of two garlic cloves. This experiment was repeated twice under the same conditions within one year. From the Petri dishes containing the yeast and bacteria with 4 days of growth, aqueous suspensions were prepared. Fifty milliliters of sterile distilled water was added to the Petri dishes, and the suspension was made using a Drigalski loop, adjusting to a concentration of 1 × 108 cells ml−1. For microbiolization, the garlic bulbs were immersed in the suspension for 1 h and then placed to dry in Petri dishes for 30 min. After this time, the inoculation of the phytopathogen was performed using 3 mm diameter discs with 8 days phytopathogen mycelial growth that were placed on the surface of the garlic cloves and kept in a humid chamber at room temperature (28 ± 3 °C) for 15 days. The humid chambers were made of Petri dish bases, where the garlic cloves were incubated with a cotton swab dipped in sterile distilled water in the center of the plate, packed in a plastic bag sprinkled with sterile distilled water (Dantas et al., 2003). At 15 days, the garlic cloves were removed from the humid chamber, and the lesion diameter was measured using an electronic caliper. Measurements were taken in two directions, and the means of the measurements were used to evaluate the development of the disease. The results of lesion diameter measurement were expressed in millimeters, and the reduction in affected area was calculated through the measurement of how much smaller the lesion diameter was than that of the control with the phytopathogen and expressed as percentage (%). The data were submitted to analysis of variance, and the means were compared by Tukey’s test using STATISTICA free version 10 software (Statsoft, INC., 2011).
Karnovsky solution (2.5% glutaraldehyde, 2.5% formaldehyde in 0.05 M sodium cacodylate, pH 7.2, 0.001 M CaCl2) for at least 24 h. The fixed fragments were transferred to a cacodylate solution (0.05 M) and washed three times for 10 min. They were then washed in distilled water three times and dehydrated in solutions with increasing concentrations of acetone (25, 50, 75, 90 and 100%). Subsequently, they were dried to the critical point with liquid CO2 in a Balzers CPD 030 apparatus and then mounted on aluminum brackets (stubs) and covered with gold (Balzers SCD 050 evaporator) for observation in a LEO EVO 40 scanning electron microscope (Alves, 2005). 3. Results 3.1. Volatile antifungal metabolite production The inhibition of growth of S. sclerotiorum colonies by volatile compounds produced by the microorganisms showed a greater percentage of inhibition by the compounds produced by C. labiduridarum, B. macauenses, B. amyloliquefaciens, B. pumilus than by those produced by the other agents, with the inhibition of fungal growth varying from 74.61% (C. labiduridarum) to 87.61% (B. pumilus) (Fig. 1). The lowest inhibition rate was that of by B. acidiceler (3.78%). 3.2. Diffusible antifungal metabolite production In relation to the production of diffusible antifungal metabolites, Fig. 2 shows the fungus growth (cm) from the center of the graph to the end in the direction of each antagonistic microorganism (▲) and in the opposite direction to the antagonistic microorganism (●). Thus, we observed that there was inhibition of the growth of S. sclerotiorum colonies in the direction of B. amyloliquefaciens, suggesting the production of compounds by the bacteria that inhibit the development of the colony. This effect was more pronounced for B. amyloliquefaciens than that observed for other microorganisms. The yeast C. labiduridarum and the bacteria B. macauenses and B. pumilus also showed antifungal metabolite production, which caused less inhibition of phytopathogen growth when compared to that of B. amyloliquefaciens. The bacterium B. acidiceler and the yeast P. kudriavzevii showed no inhibition of colony growth, suggesting the lack of production of antifungal compounds or a low efficiency of the compounds produced (Fig. 2). No inhibition of fungal colony growth was observed in the opposite direction to the antagonist microorganism (Fig. 2, circles). The
2.5. Scanning electron microscopy At the end of the experiment described above, garlic clove fragments from each treatment were collected to evaluate the interaction of phytopathogens with microorganisms by scanning electron microscopy imaging. The fragments collected were immediately fixed in modified
Fig. 1. Growth inhibition (%) of Sclerotinia sclerotiorum in response to volatile and diffusible antifungal metabolite production. * Bars with capital (volatile metabolites) and lower case (diffusible metabolites) letters do not differ according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
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Fig. 2. Development of S. sclerotiorum colonies in Petri dishes together with the antagonistic microorganisms. (A) Growth of the phytopathogen (cm) to the microorganisms’ side (▲) and growth of the phytopathogen (cm) to the opposite side from the microorganisms (●). * Lower case letters (▲) and upper case letters (●) do not differ from each other according to Tukey’s test (p < 0.05). (B) Scheme of diffusible antifungal metabolite production experiment, showing the directions of the phytopathogen growth measures carried out to the microorganisms' side and to the opposite side. This experiment was repeated twice with similar results.
Fig. 3. Illustration of antifungal metabolite production experiments for in vitro control of Sclerotinia sclerotiorum. A - Bacillus amyloliquefaciens; B - Bacillus macauenses; C - Candida labiduridarum; D - Pichia kudriavzevii; E - Bacillus pumilus; F - Bacillus acidiceler.
differences in inhibition of fungus growth between treatments in the test of antifungal metabolite production can be observed in Fig. 3. The percentage of fungal growth inhibition caused by B. amyloliquefaciens was greater than 80% (Fig. 1), showing that its use in the biological control of Sclerotinia sclerotiorum may be promising.
the control garlic cloves (Table 2). The highest phytopathogen control was occasioned by treatment with B. pumilus, where the greatest reduction in area affected by the disease was recorded (86.8%) when compared to that of the other treatments, ranging from 30.18% for B. amyloliquefaciens to 61.47% for C. labiduridarum. The lesions caused by S. sclerotiorum on A. sativum cloves and the reduction in area affected by the disease in the different treatments can be observed in Fig. 5. Scanning electron microscopy images showed the different microorganisms colonizing superficial layers of the garlic cloves, as well as their interaction with the phytopathogen (Fig. 6). The electron micrographs also allow us to note the interaction between the antagonistic microorganisms and the phytopathogen through hyphae colonization, which may cause changes in the morphology of S.
3.3. Evaluation of yeasts and Bacillus spp. on S. sclerotiorum control in Allium sativum A reduction in the symptoms of S. sclerotiorum disease in garlic cloves treated with the microorganisms was also observed (Table 2 and Fig. 4). Smaller lesion diameters were observed in garlic cloves treated with B. pumilus (0.69 mm) and Candida labiduridarum (2.02 mm) than in 4
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volatile compounds since there was no contact between the phytopathogen and the antagonist and emphasizing the simplicity and efficiency of the evaluation of production of these compounds. Although the effect of volatile compounds produced by bacteria can be considered low (15 to 26%) to moderate (28 to 53%) in phytopathogenic fungi control, some species produce effective compounds that act to decrease mycelial growth and conidial germination of phytopathogens (Campos et al., 2010). The results obtained in this study showed variations between 45.4 and 74.61% inhibition of mycelial growth of S. sclerotiorum by the volatile compounds produced by the yeasts Pichia kudriavzevii and Candida labiduridarum, respectively, and between 3.78 and 87.61% by Bacillus acidiceler and B. pumilus, respectively (Fig. 1). Fernando et al. (2005) also verified the production of volatile organic compounds with antifungal activity in bacteria isolated from canola and soybean plants, which acted to inhibit the formation of sclerotia and the germination of ascospores, in addition to limiting the mycelial growth of Sclerotinia sclerotiorum in vitro and in soil tests, reducing levels of disease. Fialho et al. (2011) verified the control of S. sclerotiorum in vitro and the reduction in disease incidence in bean seeds through volatile organic compounds produced by the yeast Saccharomyces cerevisiae. Among the bacteria identified by Zou et al. (2007), which produce volatile compounds with antifungal activity, two strains of Bacillus pumilus showed inhibition in the mycelial growth of Paecilomyces lilacinus ranging from 65.2 to 100%. Campos et al. (2010), in their review, observed isolates of Bacillus amyloliquefaciens and Bacillus pumilus with
Table 2 Lesion diameter (mm) of Sclerotinia sclerotiorum in garlic cloves treated with different microorganisms. Microorganisms
Lesion diameter (mm)
Pichia kudriavzevii Candida labiduridarum Bacillus acidiceler Bacillus macauenses Bacillus amyloliquefaciens Bacillus pumilus Control with phytopathogen
3,13 2,02 2,53 3,53 3,66 0,69 5,24
bcz ab bc c c a d
z Means followed by the same letter did not differ from each other according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
sclerotiorum hyphae and help to contain infection by them, as observed for the yeast Pichia kudriavzevii and for the bacteria Bacillus acidiceler and B. amyloliquefaciens (Fig. 6). 4. Discussion In vitro tests have been efficient methods to identify volatile compound production by microorganisms that have the potential to be used in biological control of primary phytopathogens. Four microorganisms evaluated for volatile antifungal metabolite production were efficient in inhibiting phytopathogen colony growth, indicating the production of
Fig. 4. Percentage reduction in area affected by the disease caused by Sclerotinia sclerotiorum in Allium sativum cloves treated with different antagonistic microorganisms. * Bars with equal letters do not differ from each other according to Tukey’s test (p < 0.05). This experiment was repeated twice with similar results.
Fig. 5. Illustration of the effect of microorganisms on Sclerotinia sclerotiorum control in garlic. 5
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Fig. 6. Scanning electron micrographs of the interaction of yeasts and Bacillus with Sclerotinia sclerotiorum on garlic cloves. A - Phytopathogen colonizing garlic without inoculation of potential biocontrol agents; B - Pichia kudriavzevii colonizing the fungal hyphae (arrow); C - Candida labiduridarum and bacteria on the garlic surface (arrow indicating the yeast); D - Bacillus acidiceler with the arrow indicating its colonization in the phytopathogenic hyphae; E - Bacillus macauenses colonizing the garlic surface; F - Interaction between Bacillus amyloliquefaciens and the phytopathogen; G - Surface colonization of the garlic clove by Bacillus pumilus.
antifungal activity against several phytopathogens, including Sclerotinia sclerotiorum. Satisfactory results were observed for these two bacteria in the in vitro assays, with a reduction in the mycelial growth of S. sclerotiorum by 87.61 and 80.75% by volatile antifungal metabolites produced by B. pumilus and B. amyloliquefaciens, respectively, and 82.35%
by diffusible antifungal metabolites produced by B. amyloliquefaciens. The diffusible antifungal metabolite test identified the bacterium B. amyloliquefaciens as highly efficient (above 80%) in inhibiting the hyphae growth of S. sclerotiorum and identified two more bacteria (Bacillus pumilus and B. macauenses) and one yeast (Candida labiduridarum) with 6
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moderate potential (31.37–37.25 %) in phytopathogen control. The production of secondary metabolites by microorganisms has also been extensively studied for application in the biological control of major plant diseases. It has been proven that some Bacillus amyloliquefaciens strains produce lipopeptides such as C17 bacillomycin D, anteiso-C13 and iso-C13 [Ile7] surfactins with an potent antifungal action, being proven to control Botrytis cinerea in tomato and cucumber (Masmoudi et al., 2017; Tanaka et al., 2015). Metabolite production may explain the high efficiency of this bacterium in inhibiting the growth of S. sclerotiorum colonies in vitro. No inhibition of fungal colony growth was observed in the opposite direction of antagonistic microorganisms (Fig. 2, circles), suggesting that the inhibition of phytopathogen development is associated with its proximity to the evaluated microorganisms and reinforcing the possibility of production of inhibitory compounds by bacteria and yeasts. Suneeta et al. (2016) observed control of Sclerotium rolfsii by Bacillus isolates through antibiosis, with the action of these microorganisms in the field occurring through the release of extracellular compounds, antimicrobial substances and plant hormones in the rhizosphere, causing a reduction in disease development, in addition to helping in plant growth and helping in the absorption of nutrients present in the soil. The behavior of B. amyloliquefaciens was satisfactory in both experiments with production of volatile and diffusible antifungal metabolites on in vitro evaluations, demonstrating its potential for application in phytopathogen control. This microorganism has already been used as an alternative to chemicals in control of brown rot, caused by Monilinia laxa and Monilinia fructicola, reducing more than 60.0 and 75.5% of disease incidence and severity in peach fruits under laboratory conditions, using a concentration of 107 CFU ml−1 (Gotor-Vila et al., 2017). In studies by Vinodkumar et al. (2017), Bacillus amyloliquefaciens inhibited 45% of the mycelial growth of S. sclerotiorum and 100% of its sclerotia production on in vitro tests. This bacterium also reduced the incidence of white mold (4.6%), in addition to promoting the growth of clove plants (Dianthus caryophyllus) that were immersed in bacterial suspension for in vivo tests (Vinodkumar et al., 2017). The application of B. amyloliquefaciens at a concentration of 108 cells ml−1 for Sclerotinia sclerotiorum control in Allium sativum cloves presented a 31.29% reduction in the area affected by white mold (Fig. 4), and in vitro, it showed an inhibition of 82.35% of the development of the pathogen (Fig. 1), again demonstrating the biological control potential of this bacterium. Antifungal action against Aspergillus parasiticus has been observed through low molecular weight extracellular compounds produced by the bacterium Bacillus pumilus, which act to inhibit spore germination and hyphae development as well as to inhibit the production of aflatoxins (Bottone and Peluso, 2003; Munimbazi and Bullerman, 1997). The application of B. pumilus to garlic presented satisfactory results in S. sclerotiorum biological control, which may be related to the production of antifungal metabolites. In addition, the Bacillus pumilus result in the in vivo assay with Allium sativum was more expressive than that in the in vitro parity tests. This result may be related to the induction of resistance caused by this bacterium in garlic, increasing the efficiency of the control of phytopathogens, and further studies are necessary to confirm this activity. The phytopathogen Sclerotinia sclerotiorum is difficult to control, and the lack of genetic resistance against this pathogen led Bochalya et al. (2016) to study the induction of systemic resistance in Indian mustard (Brassica juncea L.) through treatment with rhizobacteria, observing high rates of disease control with the application of Pseudomonas and Bacillus related to the high total phenolic and polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), peroxidase (PO) and chitinase activity. The inhibitory action caused by microorganisms on conidial germination and phytopathogen colony development is one of the main
mechanisms of action sought by biological control studies involving bacteria and yeasts. This action may be related to antibiosis or parasitism, in which the microorganism colonizes and impairs phytopathogen structures, as observed in some strains of Trichoderma in studies on the control of S. rolfsii (Hirpara et al., 2017). Against the fungus Sclerotinia sclerotiorum, there is a report of a species of Aspergillus (ASP-4 strain) that parasitizes and destroys the sclerotia (Hu et al., 2013), in addition to the species Coniothyrium minitans, which through mycoparasitism and antibiosis is able to degrade oxalic acid, an important virulence factor of this phytopathogen (Zeng et al., 2014). Additionally, actinobacteria of the genus Streptomyces isolated from Brazilian tropical soils were able to irreversibly inhibit the growth of S. sclerotiorum through the production of chitinolytic enzymes, such as exochitinase, endochitinase, glucanase and peptidase, which degrade the cell wall of hyphae and sclerotia of the fungus (Fróes et al., 2012). The colonization of phytopathogen hyphae by biocontrol agents was also observed in this work by the yeast Pichia kudriavzevii and by the bacteria Bacillus acidiceler and B. amyloliquefaciens, indicating a possible occurrence of parasitism in biological control of the fungus. In addition to antibiosis, induction of systemic resistance and mycoparasitism (Chaurasia et al., 2005; Droby et al., 2002; Gond et al., 2015), yeasts and bacteria also act through nutrient competition and niche exclusion since they have the ability to colonize rapidly because they are single-celled organisms with a higher growth rate than filamentous fungi that are multicellular (Sun et al., 2017; Úbeda et al., 2014). These diverse abilities developed by antagonistic microorganisms, such as antibiosis, mycoparasitism, and competition for nutrients and space, among others, play an important role in the biological control of phytopathogens, such as S. sclerotiorum, especially the fact that they can act through more than one of these action mechanisms, increasing their efficiency. In the electron micrographs, it was possible to observe colonization of the Allium sativum surface by the antagonistic microorganisms tested, indicating potential for nutrient and space competition and niche exclusion, as well as for biofilm formation (Fig. 6B–G). Rahman et al. (2016) observed biofilm formation by all Bacillus isolates tested against S. sclerotiorum. The biofilm is an important mechanism for the colonization on the surface of the roots and leaves that helps in diverse phytopathogen prevention. Chi et al. (2015) also verified biofilm formation by the yeast Pichia kudriavzevii, noting that this state, in addition to being involved in biological control activity, is a morphological form that is more resistant to high temperatures and oxidative stress. Kaushal et al. (2017) observed the biological control of white mold caused by Sclerotinia sclerotiorum in cauliflower (Brassica oleracea var. Botrytis L.) by applying a Bacillus pumilus cell suspension (1 × 108 CFU ml−1), suppressing the severity of the disease by 93% and increasing culture productivity by 36%, showing that Bacillus pumilus is a potential bacterium for both biocontrol and plant growth promotion. In the present study, the bacterium B. pumilus showed the greatest reduction in area affected by white mold on garlic cloves (Allium sativum), reaching 86.74% (Fig. 4). More research is needed to verify its effects in the field, both for S. sclerotiorum control and for A. sativum productivity. 5. Conclusion The results present some antagonistic microorganisms with potential for control of Sclerotinia sclerotiorum, especially Bacillus amyloliquefaciens and B. pumilus among the bacteria tested and Candida labiduridarum among the yeasts. Bacillus amyloliquefaciens probably produce compounds that were inhibitory to Sclerotium sclerotiorum development in vitro. Bacillus pumilus and Candida labiduridarum presented the greatest reduction in area affected by the disease in the in vivo test, showing potential for use in biological control of S. sclerotiorum in Allium sativum. These results open a broad future line of work, like the (1) identification of the metabolites produced by these microorganisms, to determine which components are responsible for the antifungal 7
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activity; (2) test of the most efficient population of these microorganisms, as well as their combination, and measure the antifungal activity and the ideal concentration of their metabolites; (3) test of different kinds of products formulation; and (4) perform tests applying these microorganisms, or their metabolites, in greenhouse and in field conditions to make sure that they are efficient biocontrol agents.
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