Microbiological Research 223–225 (2019) 129–136 Contents lists available at ScienceDirect Microbiological Research journal homepage: www.elsevier.com/locate/micres Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides T ⁎ Muhammad Azeema,b, ,1, Marina Barba-Aliagac,1, Anna Karin Borg-Karlsona,d, Olle Tereniuse,f, ⁎⁎ Anders Brobergg, Gunaratna Kuttuva Rajaraoc, a Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden b Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan c Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden d Division of Organic Chemistry, Institute of Technology, Tartu University, Tartu 50411, Estonia e Department of Ecology, Swedish University of Agricultural Sciences (SLU), Box 7044, SE-750 07 Uppsala, Sweden f Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24, Uppsala, Sweden g Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences (SLU), Box 7015, SE-750 07 Uppsala, Sweden ARTICLE INFO ABSTRACT Keywords: Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus Anti-fungal compounds reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these Inhibition two Heterobasidion species was investigated. Five different culture media with different culture age were in- Conifers vestigated to study the effect of substrate composition and culture age for metabolite production. Bacterial Lipopeptides cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was ana- Biocontrol lysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the anti- Co-culturing fungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps. 1. Introduction cause severe damage to heartwood and reduce the economic value of timber. The annual economic loss to the European forest industry re- Conifers constitute the dominant group of plants in the Northern- lated to Heterobasidion damage has been estimated to about 800 million hemisphere boreal forests. They play an important role in the economy euro (Asiegbu et al., 2005; Zeng et al., 2018). of many countries by providing raw materials for a number of in- The most common source of Heterobasidion infection for healthy dustries. Being long-lived organisms, conifers are attacked by a number trees is through root connection with infected trees or stumps (Asiegbu of pests including pathogenic fungi; however, they possess powerful et al., 2005). Heterobasidion species produce basidiospores that infect dynamic defence mechanisms against herbivores, insects and pathogens freshly cut stumps, colonize, establish and further infect healthy trees by producing a wide variety of defense compounds such as phenolics, through root interactions (Morrison and Redfern, 1994; Asiegbu et al., tannins and terpenoids (Franceschi et al., 2005). Heterobasidion species 2005). These fungal species can cause root rot in conifers without belong to the most important fungal pathogens due to their ability to showing any apparent symptom of infection for many years (Rönnberg ⁎ Corresponding author at: Department of Industrial Biotechnology, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (M. Azeem), [email protected] (G.K. Rajarao). 1 Equal contribution. https://doi.org/10.1016/j.micres.2019.04.006 Received 6 November 2018; Received in revised form 14 April 2019; Accepted 20 April 2019 Available online 22 April 2019 0944-5013/ © 2019 Elsevier GmbH. All rights reserved. M. Azeem, et al. Microbiological Research 223–225 (2019) 129–136 and Vollbrecht, 1999). The ability to actively spread itself without 2.2. Bacterial isolate identification being detected makes Heterobasidion one of the most important patho- genic agents of economically important conifers. The pure colony of the isolate A18 was mixed in 100 μL of water and Suggested preventive measures include winter cuttings and mixed incubated at 100 °C for 5 min before being vortexed. From this solution, stands; however, these methods may not be feasible due to the industry 1 μL was used for PCR in 25 μL reactions with AccuPrime SuperMixII supply demands, hence finding alternate methods are necessary to (Invitrogen). For 16S rRNA gene amplification, we used the primers 8f control Heterobasidion infections (Oliva et al., 2008). A number of (5′AGAGTTTGATIITGGCTCAG- 3′) and 1501 r (5′-CGGITACCTTGTTA- methods, such as chemical treatment, silvicultural practices and bio- CGAC- 3′) and the program 94 °C for 3 min, 30 cycles of [94 °C for 30 s, control have been adopted to prevent Heterobasidion infection of fresh 58 °C-48 °C for 30 s (the temperature was decreased by 1 °C every cycle stumps during the thinning process of managed forests. The chemical for 10 cycles and then held at 48 °C for 20 cycles), 72 °C for 1 min 30 s], method involves treating stumps or wounds with urea or borate solu- followed by a final extension step at 72 °C for 10 min (Lindh et al., tions, which protects them from pathogenic fungi belonging to Zygo- 2005). mycetes and Basidiomycetes (Vasiliauskas et al., 2004). However, bryo- The 16S rRNA showed that A18 belonged to the Bacillus subtilis phytes and some vascular plants located close to the stumps are group and for subspecies determination to B. subtilis subtilis the phoR adversely affected (Westlund and Nohrstedt, 2000) leading to ecolo- gene sequence was amplified according to Guo et al. (2012) with the gical disturbance in the forest surroundings. primers Bacillus-phoR-F 5′-TTY ARY TCA TGR GAV ACA TT-3′ and Presently, the fungus Phlebiopsis gigantea is used as a biocontrol Bacillus-phoR-R 5′-GGI TAY AAA IAR GAG GAG CC-3′. agent that functions through competing with Heterobasidion species The PCR products were sequenced by Macrogen, South Korea and (Korhonen et al., 1994). For complete prevention from Heterobasidion analyzed with BLASTN (http://www.ncbi.nlm.nih.gov/BLAST/) and infection, full coverage of stumps with P. gigantea suspension is neces- the sequences for isolate A18 were deposited with the GenBank ac- sary as this fungus does not have any antagonistic activity against cession numbers KU729674 (16S rRNA) and KU729673 (phoR). Heterobasidion (Thor and Stenlid, 2005). Other fungal strains having antagonistic activity or resource-competition ability are Bjerkandera 2.3. Fungal culture media and growth conditions adusta (Kallio and Hallarsela, 1979), Penicillium adametzii (Szwajkowska-Michałek et al., 2012), Resinicium bicolor (Holmer and The fungal cultures of Heterobasidion annosum and H. parviporum Stenlid, 1997) and Trichoderma sp. (Nicolotti and Varese, 1996). Re- used in this study were obtained from Department of Forest Mycology cently, Terhonen et al. (2016) reported the antagonistic activity of the and Plant Pathology, Swedish University of Agricultural Sciences, endophytic fungus Cryptosporiopsis sp. against H. parviporum. Many Uppsala, Sweden. Fungi were grown for 10 days and maintained at investigations have been reported where fungal strains were studied for room temperature (22 ± 2 °C) in yeast extract peptone dextrose agar the management of Heterobasidion species. However, there are only a (YEPD) medium. H. parviporum had a higher growth rate than H. an- few reports where bacterial strains were studied for their antagonistic nosum. Fully grown fungal cultures were stored at 4 °C. or competitive activity (Hallaksela, 1993; Murray and Woodward, 2003; Lehr et al., 2007; Mesanza et al., 2016). 2.4. Co-culturing fungal-inhibition bioassay Among the biocontrol agents, Bacillus species are well known bio- logical control agents and are used to suppress soil borne pathogens. Antagonistic activity of B. subtilis A18 was tested against both fungal Their biocontrol mechanisms are competition for space and nutrients, species through co-culturing on PDA plates by modifying and com- induction of systemic resistance and production of antibiotics (Li et al., bining methods described by (Barbieri et al., 2005; Leelasuphakul et al., 2014). Yamamoto et al. (2015) reported that Bacillus amyloliquefaciens 2008). Briefly, a 50 μL bacterial suspension with 108 CFU/mL was strain S13-3 has an antagonistic activity against Colletotrichum gloeos- evenly applied on a small portion