Elsevier

Microbial Pathogenesis

Volume 149, December 2020, 104509
Microbial Pathogenesis

Antifungal evaluation of fengycin isoforms isolated from Bacillus amyloliquefaciens PPL against Fusarium oxysporum f. sp. lycopersici

https://doi.org/10.1016/j.micpath.2020.104509Get rights and content

Highlights

  • The culture medium upon lecithin supplementation improved the biomass and functions of B. amyloliquefaciens PPL.

  • Bacillus amyloliquefaciens PPL produced the three types of cyclic lipopeptides.

  • The purified iturin A suppressed the development of pepper anthracnose disease.

  • Bioactive peptides of fengycin contributed to the improved antifungal potential against tomato Fusarium wilt.

Abstract

Bacillus amyloliquefaciens PPL is known to have a broad spectrum antifungal activity against plant pathogenic fungi. We focused on the cyclic lipopeptides (CLPs) extracted from the culture broth that are known to promote the ability and the efficiency of B. amyloliquefaciens PPL to control fungal diseases in pepper and tomato. In this study, the PPL strain exhibited enhanced culture yield and increased production of fengycin lipopeptides upon lecithin supplementation. The purified iturin A fraction from strain PPL exhibited higher antifungal activity (73 – 80%) against pepper anthracnose than fengycin fraction in vitro and in vivo. However, the control of tomato Fusarium wilt by the PPL strain was mainly attributed to fengycin lipopeptides. A comparison of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS analysis of the filtrate, we found that the antifungal compounds against Fusarium wilt present in the strain PPL culture filtrate were a series of isoforms of fengycin (type F1, F2, and F3). The purified fengycin F1 type showed better antifungal activity against Fusarium wilt compared the other isoforms. To the best of our knowledge, this is the first study to report the antifungal activity of fengycin isoform types in the context of Fusarium wilt. The CLPs produced by the PPL strain are potential candidates for controlling fungal disease in tomato and pepper plants.

Introduction

Pathogens of the genus Fusarium and Colletotrichum cause the most severe plant diseases once they infect the field because they cause serious yield loss. These pathogens primarily penetrate plant tissues, including flowers, fruits, stems, and roots, and cause a variety of symptoms, eventually resulting in defoliation and loss of yield and quality. Management of plant diseases mainly includes the application of chemical fungicides; however, because these compounds can damage the environment and human health, alternatives and more sustainable strategies are required. Although the development of fungicides synthesized using newer chemicals is necessary, their introduction into the cropping systems requires a long period of application, and resistant field isolates of the pathogen usually appear very quickly, even within a few seasons of the introduction of a new fungicide [1,2].

Bacillus strains are known as primarily beneficial microorganisms, which are often developed as biopesticides to control plant diseases [3,4]. Their protective effects may depend on various mechanisms of direct antagonism of plant-pathogen growth. This antagonistic activity results from the production of antibiotics and has been well documented as an important mechanism that can suppress the growth of pathogens within host plants [5,6]. In particular, Bacillus spp. produce a variety of potent antifungal metabolites; among them are cyclic lipopeptides (CLPs) that include members of the iturin, surfactin, and fengycin families [7]. These CLPs are a combination of variable types of amino acids, and these often include unusual amino acids. Additionally, the length and composition of the fatty acid side chains provide significant structural diversity, which affects the physicochemical properties and biological activities of the CLPs [8]. Different homologous compounds in each CLP family are thus often co-produced [9]. CLPs play a crucial role in antagonizing plant pathogens and are more effective than antibiotics as they are associated with features, such as low toxicity, wide antimicrobial spectrum, and biosurfactant activity. Iturins and fengycins display strong antifungal activity and inhibit the growth of a wide range of plant pathogens [7]. Surfactins are not fungitoxic by themselves but can work synergistically with the antifungal activity of iturin A [10]. It has been suggested that antibiotic production by Bacillus spp. plays an important role in suppressing plant diseases as these strains can synthesize a wide variety of potent antifungal metabolites.

Most previous studies have suggested that the carbon source is the main factor responsible for enhancing fermentation and biosurfactant production by microorganisms [[11], [12], [13], [14]]. However, few studies have reported the effects of the nutritional substrate on the type and level of CLPs produced during microbial growth [[15], [16], [17]]. The mechanism of how the interaction between biocontrol metabolites affects cell growth have not been extensively studied. Such studies are important for extending the biocontrol spectrum of microorganisms to plant fungal pathogens. Recently, we had demonstrated the effect of B. amyloliquefaciens PPL from supplementation of lecithin substrates on bacterial cell growth and production of important biocontrol metabolites. However, the metabolites underlying the activity of strain PPL against fungal pathogen has not been elucidated. Thus, there is a need to elucidate these mechanisms and identify the active metabolites. In this study, we describe the effects of the nutrient source and CLPs type—from the strain PPL—on the culture yield. First, we optimized the cultures for cell growth and the production of biocontrol metabolites. Additionally, the antifungal compounds produced by strain PPL that showed strong in vitro and in vivo inhibition of phytopathogenic fungi were identified the iturin A and fengycins. We further show that a series of isoforms of fengycins affect antifungal activity in the context of Fusarium wilt. These experiments have revealed previously unknown mechanisms of fungal cell death mediated by fengycin isoform types. This study is expected to aid the development of additional strains for the production of metabolites associated with the biocontrol of plant diseases.

Section snippets

Strains and culture conditions

Bacillus amyloliquefaciens PPL KACC 92167P [13] was grown in solid or liquid tryptic soy broth (TSB; Becton Dickinson GmbH, Heidelberg, Germany), potato dextrose broth (PDB; Becton Dickinson GmbH), and mung bean and egg yolk (MBE) broth. The latter contained 10 g fined milled mung beans, 0.8 g lecithin of egg yolk, 3% (v/v) canola oil, 3 g mannitol, 2 g peptone, and 1 g NaCl per liter (pH 7.0). Cultures were incubated by shaking (120 rpm) at 28°C. Vegetative cell growth was measured as optical

Culture nutritional source on cell growth and biosurfactant production

We determined the ability of the PPL strain to improve CLP production under a more effective nutritional context by increasing fungal inhibition using MBE cultures. The substrate of lecithin in MBE media was used as an appropriate culture source while maintaining the growth stage of the strain. The final cell densities were similar for growth on MBE and TSB at 3 d but lower than those on PDB (Table 1). Nutritional sources namely mannitol, mung bean, and lecithin supported good cell growth as

Discussion

Recently, biological control has become important to reduce the hazards associated with the intensive use of chemicals for plant disease management in agriculture. Many studies have been performed to explore non-hazardous alternative biocontrol agents using various microorganisms [21,22]. Among these, Bacillus spp. have been considered potential biocontrol agents for the control of phytopathogens because they have various antifungal activities and are safe to use [[23], [24], [25]]. The genus

Funding

This work was supported by “Agriculture and Livestock Machinery/Equipment industry Technology Development Program (118025-3)”, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry and “Basic Science Research Program (2018R1D1A3B07051266)”, the National Research Foundation of Korea.

Authors contributions

The manuscript was written through contributions of all authors. All authors read and approved the final manuscript. Kang, BR: Investigation, Methodology, Project administration, Resources, Writing - review & editing. Funding acquisition. Park, JS: Formal analysis, Investigation, Methodology, Jung, W-J: Data curation, Validation, Writing - review & editing.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Declaration of competing interest

The authors declare that they have no conflicts of interest.

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