The effects of casing soil treatment with Bacillus subtilis Ch-13 biofungicide on green mould control and mushroom yield

SUMMARY The impact of a biofungicide based on Bacillus subtilis Ch-13 on mushroom yield and efficacy in suppression of Trichoderma aggressivum f. europaeum T77 from Serbia was estimated in comparision with a similar microbial fungicide, Bacillus velezensis QST713, and the chemical fungicide prochloraz manganese. The biofungicide B. velezensis QST713 is registered for treatments of mushrooms and other crops in many countries but it is not currently available on the Serbian market. The tested B. subtilis Ch-13 fungicide enhanced mushroom yield 12%, compared with an uninoculated control, and notably more than B. velezensis QST713 applied at its higher test concentrations. Regarding the efficacy of the biofungicides in control of the compost pathogen T. aggressivum f. europaeum , B. subtilis Ch-13 applied in concentration of 3 × 10 8 CFU per m 2 showed higher efficacy than the higher concentrations (5 × 10 9 and 1 × 10 10 CFU per m 2 ) of B. velezensis QST713. The biofungicide based on B. subtilis Ch-13 should be further investigated regarding its different modes of application to ensure better efficacy in disease control as it showed beneficial features in both promoting A. bisporus production and suppressing the growth of the aggressive compost pathogen T. aggressivum , the causal agent of devastating green mould disease.


INTRODUCTION
Green mould, caused by compost-inhabiting Trichoderma aggressivum Samuels & W. Gams (Seaby, 1996;Samuels et al., 2002), is the most serious fungal disease of cultivated mushroom (Agaricus bisporus L.). Serious outbreaks of disease result in great yield losses. In the 1990s, the aggressive species appeared simultaneously in the British Isles and North America (Doyle, 1991;Romaine et al., 1996) and rapidly spread to other European countries, including Serbia, and to other continents (Kosanović et al., 2013). Its two forms, Trichoderma aggressivum f. aggresivum Samuels & W. Gams in North America and T. aggressivum f. europaeum Samuels & W. Gams in Europe, are phylogenetically closely related to T. harzianum Rifai. They emerged by population adaptation to their respective environmental conditions in mushroom-growing facilities and have never been found in the wild (Kredics et al., 2010).
Only a few fungicides have been registered and officially recommended for mushroom cultivation worldwide, i.e. prochloraz and metraphenone, while chlorothalonil, and the benzimidazoles thiabendazole and thiophanate-methyl are still in use in North America (Romaine et al., 1996;Grogan & Gaze, 2000). Over time, Trichoderma species have developed resistance to benzimidazoles in cultivated mushroom farms (Grogan & Fletcher, 1993;Grogan et al., 1996;Romaine et al., 2005;Grogan, 2008). The fungicide prochloraz has been shown to be very susceptible to degradation in soils.  reported a decrease in its concentration to less than 25% in casing soil, following two split applications. Also, the authors noted the rate of disappearance of the fungicide to be faster after the second spray (one week later) compared to the first application (after 18 days), which suggests microbial degradation.
Fungicides usually reduce the mycelial growth of A. bisporus to some extent, and their application must always be a balance between the benefit from disease control and reduced vigor of mushroom crop. Many chemicals have been withdrawn from the market over the past two decades. Moreover, there is an increased demand for biorational measures to control outbreaks rather than resorting to chemicals (Grogan, 2008). An alternative to chemical control of Trichoderma green mould is the application of beneficial microorganisms, mainly Bacillus species as biocontrol agents (Savoie et al. 2001;Védie & Rousseau, 2008). The most studied Bacillus subtilis (Ehrenberg) Cohn strain used as the biofungicide QST713 has been recently renamed to Bacillus velezensis (Pandin et al., 2018a,b). However, it is not registered in Serbia. The biocontrol strain B. velezensis QST713 was chosen for a correlation test with B. subtilis Ch-13, the newly introduced strain with antifungal and phytostimulating characteristics, which has been registered as a microbiological fertilizer, fungicide and wheat seed disinfectant in the Russian Federation, Kazakhstan and Moldova (Chebotar et al., 2009;Kayin et al., 2015). Both Bacillus strains were examined for their impact on mushroom yield and efficacy against T. aggressivum f. europaeum from Serbia, when applied to mushroom casing soil and in comparison with the fungicide prochloraz manganese in a mushroom growing room.

Fungal species and culture conditions
The pathogenic fungus T. aggressivum f. europaeum T77, originally isolated from mushroom compost in a Serbian mushroom farm at Barajevo-Lisovići in 2010, was taken from the culture collection of the Institute of Pesticides and Environmental Protection (Belgrade, Serbia). The strain was then identified based on morphophysiological characteristics and ITS1/ITS4 sequence analyses (Kosanović et al., 2013). The fungus was maintained on potato dextrose agar (PDA) medium (fresh-peeled potatoes; dextrose, Torlak, Serbia; agar, Torlak, Serbia) at 4°C. For inoculum preparation, agar discs with the fungal isolates were inoculated onto PDA medium and the plates were incubated for three days at 22°C. For in vivo trials, conidia from three-day old cultures were flooded with 10 ml of sterile distilled water and Tween 20 (v/v 0.01%), and filtered through double layers of cheesecloth.

Tests in mushroom growing room
Mushroom substrate was provided by the compost producer »Uča & Co.« Vranovo, Smederevo, Serbia. Plastic boxes sized 0.340 × 0.215 × 0.130 m (l × w × h) were filled with 1.5 kg of compost mixed with 15 g of grain spawn of A.bisporus A15 (Sylvan, Hungária zRt) to prepare 1% spawned substrate. Twelve plastic boxes were used in calculations as 1 m 2 of casing surface for treatment. Inoculation of T. aggressivum f. europaeum T77 was performed with the culture grown on PDA at 25 o C for three days. Mycelia of the pathogen was scraped from the surface of PDA plates, mixed with water and Tween 20 (v/v 0.01%) (REANAL Finomvegyszergyár Rt., Hungary, No.: 805383) and filtered through sterile gauze. Spore concentration was determined by counting on a hemocytometer and the suspension was diluted to achieve the final concentration of 10 6 conidia ml −1 . Inoculation of T. aggressivum f. europaeum T77 was performed two days after spawned compost was placed into boxes, by pipetting spore suspension (10 6 conidia per m 2 ) down the inner walls of each box. The boxes were incubated at 25 o C (spawn-run) for 18 days. Compost was cased with 1.3 kg of black peat casing soil Terahum (Treset d.o.o., Veliko Gradište, Serbia), amended with limestone (1.4%, Tara, Dobanovci, Serbia) and disinfected with peracetic acid 0.02% (Peral-S 15%, Vetprom, Belgrade, Serbia), 90 ml per m 2 of casing. Soil was cased in a 50 mm layer and incubated at 22 o C for 8 days (case-run). The day of casing was regarded as day one. The next seven days air temeperature was reduced in stages to 17 o C. The fungicide prochloraz manganese was applied at the standard product application rate of 0.6 g of active ingredient (a.i.) in 1.8 l H 2 O per 1 m 2 of casing surface on the fourth day after casing. The biofungicide B. subtilis Ch-13 was used in three different doses: 10 ml (1 × 10 8 CFU), 20 ml (2 × 10 8 CFU) and 30 ml (3 × 10 8 CFU), each volume diluted in 1 l of water and applied per m 2 of casing surface. The biofungicide B. velezenzis QST713 was also used in three different doses: 0.1 g (5.13 × 10 9 CFU), 0.2 g (1.03 × 10 10 CFU), and 3 g (1.5 × 10 11 CFU), each volume diluted in 1 l of water and applied per m 2 of casing surface. Application doses of the two biofungicides were chosen based on their recomended doses. Also, it was not possible to apply the same dose of each biofungicide due to their different product formulation. Both biofungicides based on Bacillus spp. were applied on the second day after casing. Treatments with both biofungicides and the fungicide prochloraz manganese were repeated after the first flush, approximately 22 days after casing. All treatments were applied by spraying corresponding water suspensions on mushroom bed areas prepared for six plots, i.e. a total area of 0.5 m 2 . The trial consisted of two groups, uninoculated plots and those inoculated with T. aggressivum f. europaeum T77. Control plots within both groups were sprayed with tap water.
The plots were arranged in a completely random design with six replicates per treatment. The experiment was repeated twice and average values from both repetitions were computed. 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. with and without symptoms of green mould disease. The effect of fungicides on mushroom productivity was evaluated by calculating biological efficacy (BE) as the ratio of fresh weight of total fruiting body yield and weight of dry spawned substrate, according to Chrysayi-Tokousbalides et al. (2007), and expressed as %: BE = (fresh total fruiting body yield/dry spawned substrate mass) × 100 Fungicide effectiveness was calculated by Abbott's formula (Abbott, 1925): where Ic -disease incidence in inoculated control; It -disease incidence in treated samples (Gea et al., 2010). Disease incidence was recorded as a percentage of fruiting bodies with symptoms compared with those without symptoms.

Statistical analysis
Data were examined using the one-way analysis of variance (ANOVA), including comparison of means by F-test. The test was used to compare the significance  (Sokal & Rohlf, 1995). Statistical data analysis was performed using the software Statistica for Windows 6.0 (Stat Soft Italia, 1997).

RESULTS AND DISCUSSION
Brown spots of a few millimeters were found on A. bisporus fruiting bodies in plots inoculated with T. aggressivum f. europaeum T77 16 days after casing. Larger spots and necrotic lesions of a few centimeters were found three days later. Small emerald green colonies, a few centimeters in diameter, were noted on the casing surface 28 days after casing. A few days later, colonies became larger as reported before (Milijašević-Marčić et al., 2017).
Comparison of the two microbial biofungicides was very difficult because the commercially available products had different formulations and concentrations of active ingredients, i.e. B. subtilis Ch-13 was formulated as a suspension concentrate 1 × 10 7 CFU ml -1 and B. velezensis QST713 was formulated as a wettable powder 5.13 × 10 10 CFU g -1 (Table 1). Also, it was not possible to apply these two biofungicides at the same dose because it would differ considerably from their recomended doses. Therefore, the tested doses of B. subtilis Ch-13 suspension were 10, 20 and 30 ml per m 2 of casing soil, to obtain concentrations of 1, 2, and 3 × 10 8 CFU per m 2 . The first two doses of B. velezensis QST713 were 0.1 and 0.2 g per m 2 to obtain lower concentrations (5 × 10 9 and 1 × 10 10 CFU per m 2 ) for comparison with the other biofungicide as the available product based on B. subtilis Ch-13 could not be more concentrated. The third tested dose of B. velezensis QST713 was 3 g per m 2 of casing soil as its standard application rate analogous to the concentration of 1.5 × 10 11 CFU per m 2 . The impact on yield in all treatment plots is presented in Figure 1. All three B. subtilis Ch-13 treatments showed higher mushroom production in uninoculated plots than in plots inoculated with T. aggressivum. Conversely, inoculated plots treated with prochloraz manganese and B. velezensis QST713 (at all test doses), as well as the untreated inoculated control, produced higher total yields than the corresponding uninoculated plots. This complies with a previous assumption that the pathogen T. aggressivum could enhance the growth and fructification of A. bisporus (Mumpuni et al., 1998). It has been noted that the presence of vegetative mycelium is necessary for intensive sporulation of the pathogen (Mamoun et al., 2000). In addition, Mumpuni et al. (1998) suggested the existence of mutual impact of the pathogen and the host. Particularly, the stimulation of Trichoderma by metabolites produced by A. bisporus and a relatively low level of inhibition of A. bisporus by the pathogen facilitates colonization of compost by both fungi. However, as compost colonization reaches its maximum, a change in the competitive balance in favor of T. aggressivum f. europaeum results in the inhibition of fruiting body production by A. bisporus and supports devastating green mould epidemics affecting mushroom production. It is interesting that only B. subtilis Ch-13 treatments suppressed that effect of T. aggressivum. Hence, the highest yield was found in both inoculated control plots and B. velezensis QST treatment at 1.5 × 10 11 CFU per m 2 (standard product application rate). It is noteworthy that the next highest mushroom production was found in uninoculated plots treated with B. subtilis Ch-13 at 3 × 10 8 CFU per m 2 (30 ml per m 2 ), thus enhancing mushroom yield 12% compared to uninoculated control, although a much lower concentration was applied in that treatment than in B. velezensis QST713 treatment. Uninoculated plots treated with the biofungicide B. subtilis Ch-13 in all tested doses (1, 2, and 3 × 10 8 CFU per m 2 ) had higher yields than the uninoculated untreated control. On the other hand, plots treated with B. velezensis QST713 applied at the least dose of 0.1 g per m 2 (5 × 10 9 CFU per m 2 ) had a significantly lower yield than the uninoculated fungicide efficacy % = [(Ic-It)/Ic] × 100, Ic -disease incidence in inoculated control, It -disease incidence in treated plots; data are means of six replicates in two trials ± SE, standard error of means; SEDs, standard error of differences = 14; df, degree of freedom = 6; F = 1186.1; P-value = 0.001. Values within series marked with same letters are not significantly different according to F test (P<0.05). untreated control. Again, the ability of B. subtilis Ch-13 to enhance the yield of A. bisporus was much better compared to the higher concentration of B. velezensis QST713. Moreover, uninoculated plots treated with all doses of the biofungicide B. subtilis Ch-13 showed higher yields than plots treated with the fungicide prochloraz manganese, while plots treated with B. velezensis QST713 had higher yield than those treated with the chemical fungicide only when the highest dose was applied. All three tested doses of B. subtilis Ch-13 and the two highest of B. velezensis QST713 improved mushroom yield to a level corresponding to yield reported in the previous study of Milijašević-Marčić et al. (2017). The lowest dose of B. velezensis QST713 (5 × 10 9 CFU per m 2 ) did not enhance mushroom production, which is consistent with results reported from a study by Kosanović et al. (2013), where B. velezensis QST713 was tested at a dose of 8 × 10 9 CFU per m 2 . Control plots inoculated with T. aggressivum exhibited higher A. bisporus production than control plots without the pathogen in two trials out of four conducted by Potočnik et al. (2018). In addition, Milijašević-Marčić et al. (2017) reported the highest yield in inoculated plots treated with both QST713 strain and prochloraz manganese in comparison with the matching plots without the pathogen. The highest efficacy against T. aggressivum f. europaeum was achieved using the fungicide prochloraz manganese (76.87%) (Figure 2). Similar efficacy of prochloraz had also been found in a previous study by Potočnik et al. (2018) (70.4%), where T. aggressivum f. europaeum (10 4 conidia per m 2 ) was inoculated 10 days after spawning by pouring conidial suspension in holes in the compost. In addition, the results of this study revealed a higher efficacy of prochloraz manganese compared to the similar study of Milijašević-Marčić et al. (2017) (49.4%). These differences may be attributed to different inoculation timing and application rates of the fungicide. In the previous study conducted by Milijašević-Marčić et al. (2017) T. aggressivum was added to the surface of compost one day after spawning, and the application rate of prochloraz manganese was 2.4 g per m 2 , while inoculation in the current investigation was conducted two days after spawned compost was placed into plastic boxes and the standard application rate of the fungicide was 3 g per m 2 . Those findings have demonstrated that infestation of compost with T. aggressivum f. europaeum at spawning is significantly more devastating than later inoculation. The second highest efficacy was noted for treatment with B. velezensis QST713 at standard application rate (1.5 × 10 11 CFU per m 2 ) (54.08%). It is noteworthy that B. subtilis Ch-13 applied at the concentration of 3 × 10 8 CFU per m 2 showed better efficacy (35.45%) than B. velezensis QST713 (32.05%) applied at the higher concentration (10 10 CFU per m 2 ). Also, B. subtilis Ch-13 applied at the concentration of 2 × 10 8 CFU per m 2 showed a significantly higher efficacy (27.4%) than B. velezensis QST713 at 5 × 10 9 CFU per m 2 (23.03%). The results with B. subtilis Ch-13 tested doses suggested that it could be applied at a much lower concentration than B. velezensis QST713 to achieve satisfactory efficacy against T. aggressivum. An explanation of better B. subtilis Ch-13 characteristics, including both mushroom yield promotion and suppression of the pathogen, compared with B. velezensis QST713, could be in faster activation of useful bacteria in suspension concentrate and/or in their own features. Further investigation of different modes of application of B. subtilis Ch-13 is recommended as it showed beneficial features in both promoting A. bisporus production and suppression of growth of the aggressive compost pathogen T. aggressivum, the causal agent of devastating green mould disease.

CONCLUSION
The biofungicide based on B. subtilis Ch-13 showed the highest positive impact on mushroom production. The highest yield in uninoculated plots was found after treatment with B. subtilis Ch-13 at 10 8 CFU per m 2 , higher than after B. velezensis QST713 treatment with its higher concentration (1.5 × 10 11 CFU per m 2 ). The significantly better ability of B. subtilis Ch-13 (applied at 10 8 CFU per m 2 ) to enhance A. bisporus yield was obvious as it exceeded the yield in uninoculated control, compared to the higher concentration of B. velezensis QST713 of 5 × 10 9 CFU per m 2 , which negatively affected the yield. B. subtilis Ch-13 may be assumed to suppress the effect of T. aggressivum on mushroom yield, and significantly enhance mushroom production (12 %). The fungicide prochloraz manganese may be expected to have the highest efficacy in green mould control, and B. velezensis QST713 to follow it with its standard application concentration of 1.5 × 10 11 CFU per m 2 . However, it is interesting that B. subtilis Ch-13, although applied at lower concentration (3 × 10 8 CFU per m 2 ), demonstrated better efficacy than B. velezensis QST713 applied at concentrations of 5 × 10 9 and 10 10 CFU per m 2 . Different modes and timing of application of the biofungicide based on B. subtilis Ch-13 should be further investigated to obtain both better yield and efficacy of disease control.