The activity of native Bacillus subtilis strains in control of green mould disease of oyster mushroom ( Pleurotus spp.)

SUMMARY The study aimed to isolate potential biocontrol agents from mushroom substrate that could serve as an alternative to toxic chemicals commonly used for disease control in mushroom production. The antagonistic potential of ten native Bacillus subtilis strains against the causal agents of green mould disease of oyster mushroom, Trichoderma pleuroti and Trichoderma pleuroticola , was evaluated. The antagonistic potential of Bacillus spp. strains was quantified in vitro based on dual cultivation with the pathogen. Growth inhibition of T. pleuroti ranged from 54.44% to 62.22% and no significant differences in antagonistic activity were found between the tested B. subtilis strains. Inhibition of T. pleuroticola was slightly higher, ranging from 55.56% to 69.62% and B. subtilis strain B-358 induced the highest growth inhibition. This research confirmed mushroom substrate to be a good source of antagonistic microorganisms with potentials for use in biological control of green mould in oyster mushroom production.

The activity of native Bacillus subtilis strains in control of green mould disease of oyster mushroom (Pleurotus spp.) INTRODUCTION Oyster mushrooms (Pleurotus spp.) are the third most commonly cultivated basidiomycetes worldwide. Their production can be negatively affected by different fungi, bacteria, viruses, nematodes and arthropods, but the most severe crop losses have been caused by green moulds. Initially, severe green mould infections of oyster mushroom, caused by Trichoderma spp., have been found in South Korea (Park et al., 2004(Park et al., , 2006. Subsequent reports of green mould outbreaks from other countries, including Italy (Woo et al., 2004), Hungary (Hatvani et al., , 2008 and Romania (Kredics et al., 2006), indicated a worldwide threat. Trichoderma species isolated from substrates used for the cultivation of Pleurotus spp. differed from those found on Agaricus bisporus (Lange) Imbach, and they have been described as Trichoderma pleurotum S.H. Yu & M.S. Park (currently accepted, correct name: Trichoderma pleuroti) and Trichoderma pleuroticola S.H. Yu & M.S. Park (Park et al., 2006). T. pleuroticola, closely related to T. aggressivum in damaging A. bisporus, is the most serious threat to oyster mushroom cultivation and has been found widely in soils or on decaying wood. Furthermore, it is more efficient in causing inhibitory effects against P. ostreatus in the substrate, and in utilizing carbon sources, compared to T. pleuroti. So far, T. pleuroti has not been found in natural environments, only in association with cultivated oyster mushroom. Nevertheless, T. pleuroticola is dominant in Italian Pleurotus farms, while the majoritiy of isolates from Hungary belong to the species T. pleuroti (Komoń-Zelazowska et al., 2007). Green mould infection of P. ostreatus is supposed to be transmitted via the substratum for cultivation (cereal straw, sawdust, waste cotton, etc.).
The usual method of controlling mould diseases in mushroom farms worldwide is based on the use of fungicides (Milijašević-Marčić et al., 2017). However, the development of pathogen resistance to fungicides after frequent applications, and the sensitivity of edible mushrooms to treatment compounds are serious problems. Due to resistance incidence to benzimidazoles in mycopathogenic fungi, and evidence of mutagenic effects of this group of fungicides to mammals, the number of available fungicides has decreased. Prochloraz has been selected as the most effective compound for the control of green mould disease on mushroom beds, although its high concentrations have shown harmful effects on mycelial growth and fruiting body development of Pleurotus spp. (Hatvani et al., 2008). The effects of some antagonistic microorganisms have been tested as part of efforts to improve crop protection options. Biopesticides are environment-friendly, have high specificity against target pathogens, and operate through various modes of action. Bacillus species are extremely competitive and produce a variety of secondary metabolites: antibiotics, antifungal volatile substances and cellwall degrading enzymes, which contribute to their antagonistic potential. Additionally, they are safe for the environment, harmless to human health and are generally recognized as safe (GRAS) organisms (Gajbhiye & Kapadnis, 2016). Earlier studies have shown that strains of B. subtilis and B. licheniformis efficiently inhibited the growth of T. harzianum, T. viride and T. pseudokoningii in the cultivation of P. sajor-caju and Lentinula edodes (Chittihunsa et al., 2007). Strains of these two species from mushroom compost were also found effective against T. pleuroti on oyster mushroom (Nagy et al., 2012). Remarkable antagonistic activities of Bacillus spp. originating from livestock manure and cotton-waste composts were also detected against T. harzianum, T. koningii and T. viridescens in P. ostreatus cultivation (Kim et al., 2008).
The objective of this study was to determine and compare the antagonistic activity of Bacillus spp. strains isolated from substrates for oyster and button mushroom cultivation (straw, manure, mushroom compost, black peat) against T. pleuroti and T. pleuroticola, the causal agents of oyster mushroom green mould disease.

Test organisms and culture conditions
The native isolates of Bacillus spp. from the culture collection of the Institute of Pesticides and Environmental Protection which were used in the present study had been isolated from chicken manure samples and compost at various stages of composting, obtained from the composting facility Uča & Co, Vranovo, Serbia (Table 1). The antagonistic Bacillus spp. isolates used in the study had been identified previously, based on the partial sequence of their 16S rRNA gene (Stanojević et al., 2019). The isolates were stored in Luria-Bertani broth with 30% glycerol at -20 ºC until further use. The pathogenic fungal isolates T. pleuroti SZMC 12454 and T. pleuroticola SZMC 23033 used as indicator strains for testing bacterial antagonism were obtained from the culture collection of the Department of Microbiology, University of Szeged, Hungary (Szeged Microbiology Collection, http://szmc.hu/). Identification of these isolates was performed based on sequence analysis of the ribosomal RNA internal transcribed spacer (ITS) region using the primer set ITS1/ITS4, as described by Andersson et al. (2009). A stock culture of each pathogen was maintained on potato dextrose agar (PDA) (Lab M Limeted, Lancashire, UK) at 4ºC. The working culture was prepared by transferring stock agar plugs containing the mycelium onto PDA plates and incubated for 3 days at 22ºC.

In vitro antagonistic activity
The in vitro method for quatification of antagonistic potential of Bacillus spp. strains was based on dual cultivation with the pathogen, and was carried out in three replicates. Agar discs with fresh mycelia of each pathogenic test fungus were transferred to PDA media, while bacterial cultures were streaked at 3 cm distance. Plates inoculated only with the fungus served as control plates. After incubation for 72 h at 25 o C, the size of each inhibition zone was measured, along with mycelial growth both in confronted (R1) and control plates (CR). The percentage of growth inhibition (PGI) of the pathogen was calculated according to the formula:

Statistical analyses
The basic statistical parameters were calculated and the obtained information was presented in histograms as means of mycelial growth inhibition percentage. The data were examined using one-way analysis of variance (ANOVA). The test was used to compare the inhibition of mycelial growth of two fungal pathogens caused by the activity of 10 antagonistic bacterial strains. The mean separation of percentages of mycelial growth inhibition was achieved by Tukey's HSD (honest significance difference) test. The significance of differences was evaluated at P<0.05 for all tests.

In vitro antagonistic activity
Two hundred and twelve bacterial isolates were obtained from substrates used for growing mushrooms. After preliminary screening, ten native Bacillus isolates were chosen for in vitro tests of their antagonistic potential against two causal agents of green mould disease of oyster mushroom (T. pleuroti and T. pleuroticola). The results of the inhibition potential of these isolates in dual culture are shown in Table 2. The Bacillus isolates demonstrated different levels of antagonistic potential against the tested Trichoderma spp. Percentage growth inhibition (PGI) means of the test pathogens are shown in Figures 1 and 2. Based on a comparison of PGI values, T. pleuroticola was shown to be more sensitive to the test bacteria than T. pleuroti. Growth inhibition of T. pleuroti ranged from 54.44% to 62.22% and no significant differences were found among the PGI values of ten Bacillus isolates. The PGI of T. pleuroticola was slightly higher, ranging from 55.56% to 69.62%, depending on the isolates. Strain B-358 induced the highest inhibition of T. pleuroticola, while strain B-309 had the least effect on growth of this mushroom mould. The ANOVA results confirmed statistically significant differences in the antagonistic activity of B. subtilis strains only against T. pleuroticola.

DISCUSSION
It is well-known that microbial inoculants with antagonistic properties towards pathogens of agricultural crops have the potential to replace chemical pesticides which can be harmful to the environment and human health (Solanki et al., 2015). As reported before (Walker et al. 1998, Yoshida et al., 2001Stanojević et al., 2016), the technique of dual culture on agar plates has proved to be an efficient and easy way to select antagonistic bacteria from a random group of bacterial isolates based on their ability to inhibit fungal growth. Several previous studies had shown that Bacillus spp. recovered from mushroom compost exhibited strong activity against causal agents   (Stanojević et al., 2016, Milijašević-Marčić et al., 2017. Additionally, Kim et al. (2008) tested 20 Bacillus spp. from livestock manure and cotton-waste composts. They reported inhibition zones of 9, 12 and 6 mm against T. harzianum, T. koningii and T. viridescens, respectively, similar to the effect that B. subtilis strains had in the present study against T. pleuroti (10 to 12 mm) and T. pleuroticola (10 to 14.5 mm). Furthermore, an evaluation of biocontrol agents against three Trichoderma species (T. harzianum, T. viride and T. pseudokoningii) occurring in P. sajor-caju cultivation showed that the best percentage of inhibition was produced by three Bacillus spp. (35.1%-51.1%) (Shah & Nasreen, 2011). The bacterial strain CNU LI-1 and a range of Bacillus spp. were able to inhibit the mycelial growth of Trichoderma and Hypocrea species in vitro, and their inoculation into pre-sterile substrate was effective for preventing the occurence of green mould without significantly affecting Pleurotus (Hatvani et al., 2008). Nagy et al. (2012) found that B. subitilis and B. amyloliquefaciens strains were very effective against T. pleuroti in vivo and improved crop yield by 10%. These authors found that treatment with B. amyloliquefaciens strain 27 in the amount of 10 7 bacteria g -1 substrate proved to be the most efficient, increasing crop yield by 15-21%.
In the current study, the highest growth inhibition of T. pleuroti induced by B. subtilis strains was 62.22%, while the highest PGI of T. pleuroticola was 69.62%. Strain B-358 induced the highest inhibition of T. pleuroticola among the B. subtillis strains tested in the study, while all examined strains had good antifungal effect against T. pleuroti. This research also confirmed composting material as a good source of antagonistic microorganisms with a potential for use in biological control of green mould in oyster mushroom production. However, to find an adequate biocontrol candidate, it is necessary to test the sensitivity of P. ostreatus to B. subtilis strains, and to conduct in vivo trials under growing conditions.