Bacillus subtilisandTrichoderma harzianumcontrol postharvest pathogens of strawberry fruits in vitro (original) (raw)
Bacillus subtilis and Trichoderma harzianum control postharvest pathogens of strawberry fruits in vitro
F.C. Ayduki, L.C. Rozwalka, M.A.C. Zawadneak and F.L. Cuquel
Universidade Federal do Paraná, Curitiba, Brazil.
Abstract
This research assessed the efficacy of Bacillus subtilis (Bs) and Trichoderma harzianum (Th) (antibiosis, parasitism and volatile compounds production) for the control of important postharvest strawberry (Fragaria ×\times ananassa) pathogens, including Rhizopus stolonifer ( RsR s ) (soft rot), Botrytis cinerea (Bc) (gray mold), and Colletotrichum spp. (Col) (anthracnose), in paired cultures. Those are etiologic agents of major strawberry (Fragaria ×\times ananassa) postharvest diseases. Commercial available antagonists, and pathogens isolated from infected strawberry fruit were used. Pathogens were paired in petri dishes containing potato dextrose agar (PDA) with Bs aliquots of 80,60,4080,60,40 and 20μ L20 \mu \mathrm{~L} plate −1{ }^{-1} or Th aliquots of 62.50,46.88,31.2562.50,46.88,31.25 and 16.60μ L16.60 \mu \mathrm{~L} plate −1{ }^{-1}. Plates were incubated at 25±1∘C25 \pm 1^{\circ} \mathrm{C}, photoperiod of 12 h . The Bs 80 μL\mu \mathrm{L} aliquot reduced mycelial growth of BcB c and ColC o l at 90 and 98%98 \%, respectively. Th did not inhibit growth of Rs.BsR s . B s and ThT h produced no volatile compounds in petri dishes able to inhibit the pathogens. The inhibition zones and the reduced mycelial growth of pathogens showed that Bs and Th acted by antibiosis. These results indicate the potential of Bs and Th for postharvest strawberry diseases control.
Keywords: biological control, antagonistic microorganisms, Rhizopus stolonifer, Botrytis cinerea, Colletotrichum spp.
INTRODUCTION
Worldwide strawberry (Fragaria ×\times ananassa Duch.) is one of the most popular fruits, appreciated for its unique flavor. But production has its challenges, including the need for control of preharvest and postharvest rots. Some of the most important rots of strawberry in Brazil are Rhizopus rot, grey mould, and Anthracnose fruit rot, caused by Rhizopus stolonifer, Botrytis cinerea, and Colletotrichum spp., respectively (Reis and Costa, 2011). These diseases are a limiting factor for the production and distribution of strawberries, causing the reduction of quality and yield in several other countries.
Although fungicides are considered the primary postharvest disease control measure (Tripathi and Dubey, 2004; Sharma and Tripathi, 2006), losses caused by fungi in storage raise doubts about their efficacy. Continuous and inappropriate use have also caused development of fungicide-resistant strains (Ghini and Kimati, 2000; Reimann and Deising, 2000). Furthermore, concerns about the impact of fungicides on the consumer, worker, and the environment are cause for the development of safer and lower risk alternatives (Unnikrishnan and Nath, 2002).
Pesticide residues on fruits and vegetables worry consumers. A survey of pesticide residues on fresh food in Brazil between 2001-2007, identified strawberry as the one of the crops with the highest percentage of contaminated samples (44.2%) (ANVISA, 2008). Therefore, alternative strategies to manage diseases became essential, urgent and necessary. Biological control may meet some of the requirements but thus far has been not proven a viable alternative (Gouvea et al., 2009).
Several antagonistic microorganisms have been reported to control pathogens in different pathosystems, in vitro or/and in vivo. Among them, Bacillus subtilis and Trichoderma harzianum have been cited for their antifungal activities. B. subtilis showed efficacy in the control of B. cinerea in grapes when combined with Metschnikowia pulcherrima yeast (Mondino et al., 2012). There are reports of antagonistic activity against
C. gloeosporioides isolated from papaya fruit (Hasan et al., 2012) and significant mycelial growth inhibition of R. stolonifer postharvest pathogen of peach fruit, in vitro and in vivo (Wang et al., 2013). T. harzianum suppressed the growth of postharvest pathogens of tomato such as Rhizopus sp., Fusarium sp. and Penicillium stekii (El-Katatny et al., 2011).
Commercial products based on biological agents for plant disease control, such as Bacillus spp. and Trichoderma spp., showed inhibitory effect against several plant pathogens (Bettiol et al., 2012). However, the antagonistic mechanism, or mode of action, is not always known. Furthermore, the inhibition potential of those products can change due to the sensibility of strains isolated from different regions.
The goal of this study was to assess the inhibition potential of B. subtilis and T. harzianum for control of Rhizopus stolonifer, Botrytis cinerea and Colletotrichum species.
MATERIALS AND METHODS
The assays were performed in the Phytopathology Laboratory of the Department of Crop Protection, Federal University of Paraná, Curitiba, Paraná, Brazil. R. stolonifer (Rs), B. cinerea (Bc) and Colletotrichum spp. (Col) were isolated from infected and sporulating strawberry fruits produced in the Metropolitan Region of Curitiba, Paraná State, Brazil. The pathogen cultures were grown on potato-dextrose-agar (PDA) under a photoperiod of 12 h in BOD at 25±1∘C25 \pm 1^{\circ} \mathrm{C}.
The antagonistic microorganisms B. subtillis (Bs) and T. harzianum (Th), from commercial formulations of biofungicides, were respectively used in aliquots of 80,60,4080,60,40 and 20μ L(5,13×1010ufcg−1)20 \mu \mathrm{~L}\left(5,13 \times 10^{10} \mathrm{ufc} \mathrm{g}^{-1}\right) and 62.50,46.88,31.2562.50,46.88,31.25 and 16.60μ L(2×10916.60 \mu \mathrm{~L}\left(2 \times 10^{9}\right. viable conidia mL−1\mathrm{mL}^{-1} ), respectively. Inhibition potential and mode of action of antagonistic microorganisms against Rs,BcR s, B c and ColC o l in vitro, were assessed using parings of antagonistic microorganisms and the plant pathogens in vitro.
Discs of BcB c and ColC o l in aseptic conditions ( 5 and 10 mm diameter, respectively) and suspension of Rs grown in PDA medium were paired with Bs and Th aliquots placed on opposite sides at 1.5 cm from the edges of the petri dishes with PDA medium. In the controls, discs of Rs,BcR s, B c and ColC o l alone and aliquots of BsB s and ThT h (at test concentrations) were placed separately at 1.5 cm from the edges of the petri dishes. The treatments were incubated at 25±1∘C25 \pm 1^{\circ} \mathrm{C} with photoperiod of 12 h in a BOD incubator. Six replications were prepared per each treatment, for a total of 72 replications with Bs and Th aliquots. The mycelial growth was assessed daily measuring the diameter of the pathogen colony along two perpendicular opposite lines, until the pathogen colony reached one of the edges both in the control or in any treatment. The inhibition percentage of the mycelial growth was determined three days after incubation.
RESULTS AND DISCUSSION
Bacillus subtilis inhibited Bc and Col mycelial growth at all concentrations (Table 1). However, this antagonist did not inhibit the mycelial growth of Rs. The rapid development of Th inhibited the mycelial growth of Bc,RsB c, R s and ColC o l in all concentrations.
The sporulation of Rs was inhibited by Th at 62.50,46.88,31.25μ L62.50,46.88,31.25 \mu \mathrm{~L}. The presence of inhibition zones was observed in Bs and Bc,ThB c, T h and RsR s and, ThT h and ColC o l paired cultures, indicating that BsB s and ThT h acted by antibiosis producing antifungal or toxic substances.
Biological control is defined as the control of a microorganism by another organism and the mechanisms of action involved are antibiosis, competition, parasitism, predation, hypovirulence, and the induction of host defense (Bettiol, 1991).
Microorganisms that act by antibiosis such as bacteria, usually have wide spectrum of action, so that the inhibition of fungi caused by production of toxic substances is more effective than any other mechanism of action involved (Kupper et al., 2003). Basurto-Cadena et al. (2012) attributed the in vitro antagonistic activity of a native Bs strain isolate from strawberry plants in Mexico against strawberry isolates of R. solani and F. verticillioides, to the presence of proteases and bacteriocin-like inhibitor substances. Hang et al. (2005) reported that Bs S1-0210 inhibited BcB c isolate of strawberry and caused an abnormal shape of mycelia due the production of antibiotic substance which is diffused into the agar
Table 1. Inhibition of mycelia growth (%) of Rhizopus stolonifer, Botrytis cinerea and Colletotrichum spp. by Bacillus subtilis and Trichoderma harzianum in potato dextrose agar medium (PDA) three days after incubation.
| Treatments | Inhibition of mycelial growth (%) | ||
|---|---|---|---|
| Botrytis cinerea | Rhizopus stolonifer | Colletotrichum spp. | |
| Control | 0 | 0 | 0 |
| Bs 80.0μ L80.0 \mu \mathrm{~L} | 90 | 0 | 98 |
| Bs 60.0μ L60.0 \mu \mathrm{~L} | 45 | 0 | 53 |
| Bs 40.0μ L40.0 \mu \mathrm{~L} | 38 | 0 | 50 |
| Bs 20.0μ L20.0 \mu \mathrm{~L} | 30 | 0 | 72 |
| Th 62.50μ L62.50 \mu \mathrm{~L} | na1\mathrm{na}^{1} | 55 | 30 |
| Th 46.88μ L46.88 \mu \mathrm{~L} | na | 53 | 25 |
| Th 31.25μ L31.25 \mu \mathrm{~L} | na | 50 | 40 |
| Th 16.60μ L16.60 \mu \mathrm{~L} | na | 0 | 30 |
'na: Botrytis cinerea mycelia growth inhibition by Trichoderma harzianum was not evaluated.
Mycelial growth of pathogens was observed in all petri dishes that also contained antagonists, indicating that the antagonistic microorganisms did not produce inhibitory volatile compounds in the conditions of the assay. However, Bomfim et al. (2010) observed the inhibition of Rs in vitro and on passion fruit with volatile and non-volatile metabolites produced by Th and other species of this antagonistic. The knowledge of these mechanisms or mode of action, antifungal activity and the identification of their secondary biologically active metabolites represent the possibility for the chemical industry to synthetize new fungicides that may be offered as biofungicides or ecofriendly fungicides and to assist in the development of biocontrol strategies.
CONCLUSIONS
- Bacillus subtilis and T. harzianum showed inhibition potential for the control of RR. stolonifer, B. cinerea and Colletotrichum spp.
- The presence of inhibition zones indicated that B. subtilis and T. harzianum acted by antibiosis.
- Trichoderma harzianum inhibited the sporulation of R. stolonifer.
Literature cited
ANVISA (Agência Nacional de Vigilância Sanitária). (2008). www.portal.anvisa.gov.br.
Basurto-Cadena, M.G.L., Vázquez-Arista, M., García-Jiménez, J., Salcedo-Hernández, R., Bideshi, D.K., and BarbozaCorona, J.E. (2012). Isolation of a new Mexican strain of Bacillus subtilis with antifungal and antibacterial activities. Scientific World J. doi:10.1100/2012/384978
Bettiol, W. (1991). Componentes do controle biológico de doenças de plantas. Embrapa Hortaliças. 5, 1-5.
Bettiol, W., Morandi, M.A.B., and Pinto, Z.V., Júnior, T.J. de P., Corrêa, E.B., Moura, A.B., Lucon, C.M.M., Costa, J. de C. do B., and Bezerra, J.L. (2012). Produtos Comerciais à Base de Agentes de Biocontrole de Doenças de Plantas, Doc. 88 (Brazil: Embrapa Meio Ambiente), pp. 155.
Bomfim, M.P., São José, A.R., Rebouças, T.N.H., Almeida, S.S., Souza, I.V.B., and Dias, N.O. (2010). Antagonic effect in vitro and in vivo of Trichoderma spp. to Rhizopus stolonifer in yellow passion fruit. Summa Phytopathol. 36, 6167 http://dx.doi.org/10.1590/S0100-54052010000100011.
El-Katatny, M.H., El-Katatny, M.S., Fadl-Allah, E.M., and Emam, A.S. (2011). Antagonistic effect of two isolates of Trichoderma harzianum against postharvest pathogens of tomato (Lycopersicon esculentum). Arch. Phytopathol. Pflanzenschutz 44 (7), 637-654 http://dx.doi.org/10.1080/03235400903266438.
Ghini, R., and Kimati, H. (2000). Resistência de Fungos a Fungicidas (Embrapa Meio Ambiente), 1, pp. 78.
Gouvea, A., Kuhn, O.J., Mazaro, S.M., May-De Mio, L.L., Deschamps, C., Biasi, L.A., and de C. Fonseca, V. (2009).
Controle de doenças foliares e de flores e qualidade pós-colheita do morangueiro tratado com Saccharomyces cerevisiae. Hortic. Bras. 27 (4), 527-533 http://dx.doi.org/10.1590/S0102-05362009000400020.
Hang, N.T.T., Oh, S., Kim, G.H., Hur, J., and Koh, Y.J. (2005). Bacillus subtilis S1-0210 as a biocontrol agent against Botrytis cinerea in strawberries. Plant Pathol. J. 21 (1), 59-63 http://dx.doi.org/10.5423/PPJ.2005.21.1.059.
Hasan, M.F., Mahmud, T.M.M., Ding, P., and Kadir, J. (2012). Control of postharvest anthracnose disease and quality of papaya using Bacillus subtilis strain B34 enhanced with sodium bicarbonate and Aloe vera gel. Acta Hortic. 1012, 653-660 http://dx.doi.org/10.17660/ActaHortic.2013.1012.88.
Kupper, K.C., Gimenes-Fernandes, N., and de Goes, A. (2003). Controle biológico de Colletotrichum acutatum, agente causal da queda prematura dos frutos cítricos. Fitopatol. Bras. 28 (3), 251-257 http://dx.doi.org/ 10.1590/S0100-41582003000300005.
Mondino, P., Casanova, L., Calero, G., Betancur, O., and Alaniz, S. (2012). Zimevit: a biofungicide that combines the action of one bacteria and one yeast for the control of gray mold of grape caused by Botrytis cinerea. Rev. Bras. de Agroecologia. 7, 127-134.
Reimann, S., and Deising, H.B. (2000). Fungicides: risk of resistance development and search for new targets. Arch. Phytopathol. Pflanzenschutz 33, 329-349 http://dx.doi.org/10.1080/03235400009383354.
Reis, A., and Costa, H. (2011). Principais Doenças do Morangueiro no Brasil e Seu Controle, 96 (Brazil: Embrapa Hortaliças), pp. 9 .
Sharma, N., and Tripathi, A. (2006). Fungitoxicity of the essential oil of Citrus sinensis on post-harvest pathogens. World J. Microbiol. Biotechnol. 22 (6), 587-593 http://dx.doi.org/10.1007/s11274-005-9075-3.
Tripathi, P., and Dubey, N.K. (2004). Exploitation of natural products as an alternative strategy to control postharvest fungal rotting of fruit and vegetables. Postharvest Biol. Technol. 32 (3), 235-245 http://dx.doi.org/ 10.1016/j.postharvbio.2003.11.005.
Unnikrishnan, V., and Nath, B.S. (2002). Hazardous chemicals in foods. Indian J. Dairy Biosciences. 11, 155-158.
Wang, X., Wang, J., Jin, P., and Zheng, Y. (2013). Investigating the efficacy of Bacillus subtilis SM21 on controlling Rhizopus rot in peach fruit. Int. J. Food Microbiol. 164 (2-3), 141-147 http://dx.doi.org/10.1016/j.ijfoodmicro. 2013.04.010. PubMed