A large-scale study on the effectiveness of a Bacillus subtilis Ch-13-based biofungicide against green mould disease and mushroom yield improvement

SUMMARY The aim of this study was to test a biofungicide based on Bacillus subtilis Ch-13 and its effectiveness in the control of green mould disease of cultivated mushroom in comparison with the fungicide prochloraz. Biofungicide effectiveness in disease control and impact on yield were evaluated on Agaricus bisporus after its natural infection with Trichoderma aggressivum in a commercial mushroom growing facility. An assay for testing the microbial efficacy of the biofungicide was conducted in two different procedures involving either three or two split doses. The highest statistically significant effectiveness in green mould control was shown by the fungicide prochloraz (71.43%), followed by the biofungicide applied in tree split doses (53.57%), and finally its two doses (45.46%). The biofungicide significantly improved yield in comparison with an untreated control and the fungicide prochloraz. Three split applications of B. subtilis strain Ch-13 enhanced mushroom yield to a larger extent than its two split doses, although the same final amount was used in both procedures. Biofungicide application in three split doses increased the total mass of harvested mushrooms 8.41% compared to the untreated control, and 10.53% compared to the fungicide prochloraz. These results implied that the biofungicide should be applied in three split applications: 30 ml (second day after casing) + 15 ml (two weeks after casing) + 15 ml (after first flush, 20-25 days after casing). The biofungicide B. subtilis Ch-13 should be further investigated regarding its joint usage with chemical fungicides in different application procedures, as it showed remarkable characteristics both in terms of promoting mushroom yield and inhibiting the spread of mycopathogenic T. aggressivum.


INTRODUCTION
The most devastating pathogen of cultivated mushroom (Agaricus bisporus L.) is Trichoderma aggressivum Samuels & W. Gams (Samuels et al., 2002) and, unlike other casing mycopathogens, it colonizes the substrate of A. bisporus and causes crop losses of between 60 and 100% (O'Brien et al., 2017). Kosanović et al. (2020) revealed that the concentration of T. aggressivum conidial suspension of 10 -4 conidia per ml decreased mushroom yield 29-56%, while an inoculum of 10 -3 conidia per ml caused 68-100% yield decrease. The fungicides prochloraz and metrafenone are allowed to be used in edible mushroom cultivation in the EU (Carrasco et al., 2017). These two fungicides have been registered in Serbia for other crops but not yet approved for use in mushrooms cultivation (Team of editors, 2020). Furthermore, prochloraz decomposes due to microbial degradation in casing soil, and its effectiveness in disease control is so rapidly lost after application (Grogan et al., 2000).
A good alternative to chemical control of mushroom diseases is the application of antagonistic microorganisms, primarily Bacillus species (Savoie et al. 2001). A biofungicide based on the most frequently used Canadian strain of Bacillus velezensis (Ehrenberg) Cohn, QST713, registered against many plant pathogens and mycopathogens (Védie & Rousseau, 2008;Pandin et al., 2018;Potočnik et al., 2018), is not available on the Serbian market at present. The Russian strain Bacillus subtilis (Ehrenberg) Cohn Ch-13 (Chebotar et al., 2009;Kayin et al., 2015), which has recently become available in Serbia, was compared with the chemical fungicide prochloraz and B. velezensis QST713 in a recent small-scale study in the experimental mushroom growing room (Potočnik et al., 2019). The biofungicide B. subtilis Ch-13 showed higher effectiveness against the compost pathogen T. aggressivum, and also increased mushroom yield more with its lower concentration than B. velezensis QST713 (Potočnik et al., 2019). Large-scale experiments with edible mushroom disease control are rather scarce. One of the few was conducted by Regnier and Combrinck (2010), establishing a suitable application regime (40 µl l -1 ) for non-formulated essential oils of lemon, verbena, thyme and lemongrass, as well as two of their main components (nerol and thymol), against M. perniciosa in commercial growing facility under conditions of natural infection.
Based on the promising results of the previous small-scale experiment (Potočnik et al., 2019), the aim of this study was to compare the biofungicide based on B. subtilis Ch-13 and the fungicide prochloraz regarding green mould disease control under conditions of natural infection. The impact on mushroom yield was also estimated during this large-scale experiment in a commercial mushroom growing facility.

Antifungal agents
The biofungicide Ekstrasol F SC (BioGenesis d.o.o., Belgrade, Serbia), based on Bacillus subtilis Ch-13 (1 × 10 8 CFU ml -1 ), was tested as a potential antifungal agent for the control of T. aggressivum in natural infections of casing soil. The experiment was conducted in B8 growing chamber of the mushroom production facility of Delta Danube d.o.o., Kovin, Serbia. The biological efficacy and effectiveness of the biofungicide was evaluated by comparing it with the commercial fungicide prochloraz (Mirage® EC, ADAMA Agricultural Solutions UK Ltd., UK; content of a.i. 450 ml l -1 ).

Tests in mushroom growing room
Treatments of casing soil in the mushroom growing chamber were carried out according to standard PP 1/270 (1) methodology (EPPO, 2010), using the biofungicide based on B. subtilis Ch-13 and the commercial prochloraz-based chemical fungicide.
Mushroom substrate packed in plastic bags sized 0.4 × 0.6 × 0.25 m (l × w × h), filled with 18 kg of compost and spawned with 0.7% of grain spawn of A. bisporus (Italspwan, Onigo di Pederobba, Italy), was provided by the compost producer Champicomp d.o.o., Pločica, Kovin, Serbia. Five plastic bags provided a casing surface of 1 m 2 which was used for treatment calculation. Compost was cased with 7 kg of black peat casing soil (Pešter peat soil, Dallas Company, Tutin, Serbia), and disinfected with peracetic acid 0.02% (Peral-S 15%, Vetprom, Belgrade, Serbia), 90 ml per m 2 of casing. Casing soil was cased in a 50 mm layer and incubated at 25 o C for 8 days (case-run). The day of casing was regarded as day one. Over the next seven days air temperature was reduced in stages to 17 o C. The fungicide prochloraz and the biofungicide were repeatedly applied using an automatic "fir" sprayer with 10 full cone nozzles. Prochloraz was applied at the standard product application rate registered in the EU in two split applications, each treatment consisting of 1.5 ml in 1.8 l H 2 O per 1 m 2 of casing surface on the fourth day after casing and after the first flush (approximately 20-25 days after casing). The biofungicide B. subtilis Ch-13 was used in two different application procedures in the same total amount of 60 ml per m 2 of casing surface: (1) three times: 30 ml (second day after casing) + 15 ml (two weeks after casing) + 15 ml (after first flush, approximately 20-25 days after casing); and (2) twice: 30 ml (second day after casing) + 30 ml (after first flush, approximately 20-25 days after casing). Each volume was diluted in 1 l of water and applied per m 2 of casing surface. Untreated control plots within groups were sprayed with tap water.
Each treatment and untreated control was repeated twice in a randomized block design experiment with casing area of 56 m 2 per block consisting of 224 bags of mushroom substrate (repetitions). The average values from both trials are presented. The fruiting bodies were hand-picked in two successive production flushes: the first from day 14 to 22 after casing, the second from day 23 to 35. The harvested mushrooms were weighed and divided into two groups based on visual observation, i.e. either with or without symptoms of green mould disease. Fungicide effectiveness was calculated by Abbott's formula (Abbott, 1925): where Ic -disease incidence in inoculated control; Itdisease incidence in treated samples. Disease incidence was recorded as a percentage of fruiting bodies with symptoms compared with those without symptoms.
The effect of fungicides on mushroom productivity was evaluated as biological efficiency (BE), calculated as the ratio of fresh weight of total fruiting body yield and weight of dry spawned substrate, and expressed as percentages (Chrysayi-Tokousbalides et al., 2007) according to formula: BE = (fresh total fruiting body yield/dry spawned substrate mass) × 100.

Statistical analyses
Data were examined using the one-way analysis of variance (ANOVA), including the comparison of means by the F-test. The test was used to compare the significance of differences among data for the average biological efficacy and effectiveness of different bio/ fungicide treatments against T. aggressivum in the mushroom growing chamber. In all analyses, the level of significance was at least P<0.05 (Sokal & Rohlf, 1995). Statistical data analysis was performed using the software Statistica for Windows 6.0 (Stat Soft Italia, 1997).

RESULTS AND DISCUSSION
Dark green colonies were observed on the sides of compost surface eight days after casing, corresponding to first symptoms of green mould disease caused by T. aggressivum (Milijašević-Marčić et al., 2017).
Suppression of green mould disease incidence by using bio/fungicides is shown in Figure 1. The biofungicide B. subtilis Ch-13 significantly decreased disease incidence after natural T. aggressivum infection of cultivated mushrooms, compared to the chemical fungicide prochloraz and untreated control. The effectiveness of disease control was presented in two ways: in comparison with the standard fungicide prochloraz (E st ) set to 100%, and in relation to untreated control (E k ) ( Table 1). The highest effectiveness in green mould control was shown by the fungicide prochloraz (71.43%), followed by the biofungicide B. subtilis Ch-13 applied in three split doses (53.57%). B. subtilis Ch-13 used in two split applications was the least effective against the pathogen (46.45%). Despite the same final concentration, the effectiveness of B. subtilis Ch-13 in green mould disease control was significantly higher when it was applied three times than in two applications. B. subtilis Ch-13 used in three split applications demonstrated effectiveness which was 17.86% lower than that of the standard chemical fungicide but still exceeded 50% in comparison with untreated control.
In the previous small-scale experiment, Potočnik et al. (2019) reported that B. subtilis Ch-13, applied at the concertation of 10 8 CFU per m 2 , achieved 23% effectiveness when used in the amount of 10 ml m -2 ; 27% in 20 ml m -2 , and 35% in 30 ml m -2 against T. aggressivum. The strain Ch-13 applied at the dose of 2 × 10 8 CFU per m 2 showed better efficacy (27.4%) than B. velezensis QST713 (23%) used at its higher concentration (5 × 10 9 CFU per m 2 ). Prochloraz showed the highest effectiveness in disease control in both our experiments, 71% in the current large-scale study after natural infection, and 77% in the earlier small-scale assay after artificial infection with T. aggressivum 10 6 conidia per m 2 of casing soil (Potočnik et al., 2019).
A statistically significant increase in mushroom yield was noted when the biofungicide B. subtilis Ch-13 was used in two and three split doses, in comparison with the untreated control and prochloraz fungicide (Figure 2). The chemical fungicide (standard) did not significantly improve mushroom yield compared to the untreated control. Furthermore, impact on mushroom yield was shown as a biological efficiency coefficient (BE) when either the impact of untreated control (BE st ) or the standard fungicide prochloraz (BE k ) were set to 100 % ( Table 2). The biofungicide increased mushroom yield more when it was used frequently, i.e. in three split applications, than only twice, although the same final amount was used in both treatments.

Disease incidence (%)
The strain B. subtilis Ch-13 used in three split applications improved the total mass of harvested mushrooms compared both with the untreated control (8.41%) and prochloraz fungicide (10.53%).

Biological efficiency (BE%)
As for the inoculated treatments in the same experiment, the biofungicide B. subtilis Ch-13 used at the concentration of 1 × 10 8 CFU ml -1 increased yield (68%) more than B. velezensis QST713 (63%) used at its higher concentration of 5 × 10 9 CFU ml -1 (Potočnik et al., 2019). In the current large-scale experiment, the biofungicide B. subtilis Ch-13 concentration of 1 × 10 8 CFU ml -1 , and its dose of 60 ml m -2 of casing soil, improved yield 83-85%, while the biofungicide was applied in the small-scale experiment at lower doses and achieved proportionately lower yield: at the concentration of 10 ml m -2 -68%, 20 ml m -2 -72% and at 30 ml m -2 -74% (Potočnik et al., 2019). Similar yields (79%) were obtained in untreated control plots in both experiments, i.e. in the previous smallscale trial (artificial infection) (Potočnik et al., 2019) and the current large-scale trial under conditions of natural infection. The mode of action of Bacillus spp. biofungicides is based on competition for nutrients, substrate colonization (Chen et al., 2013), synthesis of antibiotics, iron chelators, antifungal volatile organic compounds and cell wall degrading enzymes (Manjula & Podile, 2005). Competition could also be responsible for the inhibition of T. aggressivum growth. Furthermore, B. subtilis strains are considered safe for the environment and harmless to human health and are generally recognized as safe (GRAS) organisms (FDA, 2020). Additionally, Bacillus spp. strains form endospores which ensure their survival and persistence in the environment (Cawoy et al., 2011). The current investigation of different procedures for the application of B. subtilis Ch-13 revealed benefits from applying three split doses to suppress the growth of T. aggressivum, an aggressive compost pathogen and causal agent of green mould disease, and to promote A. bisporus production.

CONCLUSION
The biofungicide based on B. subtilis Ch-13 showed better efficacy in green mould disease control and the highest positive impact on mushroom production when it was used in three split applications, rather than two. It suggests that the biofungicide should be applied three times: 30 ml (on the second day after casing) + 15 ml (two weeks after casing,) + 15 ml (after the first flush, approximately 20-25 days after casing). The microbial biofungicide B. subtilis Ch-13, which is harmless to the environment and non-target organisms, should be further investigated regarding its combinations with chemical fungicides in order to achieve better efficacy in disease control as it showed remarkable characteristics both in inhibiting the spread of the mycopathogen T. aggressivum, the causal agent of the most serious mushroom disease, and in promoting mushrooms production.

ACKNOWLEDGEMENT
This large-scale experiment was conducted in the mushroom production and processing plant Delta