Chemical and Biological Control of Wet Bubble Disease (Hypomyces perniciosus) in Mushroom Crops

Abstract

Wet bubble disease (WBD) is globally becoming a major problem in mushroom crops. The effectiveness of the application of different chemical (chlorothalonil, metrafenone and prochloraz-Mn) and Bacillus (B. subtilis and B. amyloliquefaciens strains) -based products for the control of WBD in artificially inoculated mushroom crops was studied. Six trials were carried out, with three different inoculum rates. The effect of fungicides on mushroom productivity and disease incidence was assessed. The effectiveness of the treatments is statistically related to the harshness of the disease. The greatest reductions in disease incidence were achieved after treatments with chemical fungicides, notably prochloraz-Mn. When the outbreak was strong, the low calculated incidence reduction values for metrafenone treatments advised against their application. The results also indicated the low effect of both bio-fungicides, at the doses and timing evaluated, for the control of this mushroom disease.

1. Introduction

Wet bubble is a globally distributed disease of the white button mushroom (Agaricus bisporus (Lange) Imbach) that generally does not cause major crop losses [1]. Traditionally, the mycoparasite Hypomyces perniciosus Magnus (formerly Mycogone perniciosa (Magnus) Delacr.) has been considered the causal agent of wet bubble, but, recently, it has been found that other pathogens, such as M. rosea Link and M. xinjiangensis Y.X. Du and N.N. Shi, can infect mushrooms and cause similar symptoms [2,3]. The main source of inoculum is the casing material. The mycoparasite can be spread by splashing water, flies, air, and operators (tools, hands, clothes, etc.). Therefore, strict attention to hygiene is necessary to avoid outbreaks and dispersion of wet bubble [1,4,5].

For many years, outbreaks of WBD have been sporadic, but, recently, this mycoparasite is expanding, particularly in China, where WBD can cause yield losses of around 15–30% [6,7,8,9]. In Spain, WBD outbreaks have been occasional for many years and disease incidence is generally low on well-managed farms, but wet bubble poses a potential threat to mushroom crops [5,10]. A taxonomic and morphological description of the causal agent and symptoms of WBD can be found in the literature [1,4].

The control of WBD is based on integrated disease management (IDM) programs, matching fungicides and correct agronomical management. In this sense, preventive measures applied through efficient cultural practices, such as controlling the levels of moisture of the casing materials, are a tool for decreasing the incidence of mushroom diseases [11]. The naturally established casing microbiota can help to reduce disease development when inoculum levels are low, but they are not effective in suppressing it when disease pressure is high, as is the case when fungal inoculum is artificially introduced [12]. Therefore, in practice, fungicide application remains the main tool for WBD management in mushroom farms. In general, only a few fungicides have been allowed for use in the mushroom industry, including prochloraz, chlorothalonil, and metrafenone in Europe, as well as chlorothalonil, thiabendazole, and metrafenone in North America [13,14]. However, prochloraz has recently been withdrawn in Europe, although it is still permitted in China [9] and Australia [15]. For now, prochloraz-Mn, chlorothalonil, and thiabendazole have been reported to be the most effective fungicides against WBD [5,9,10]. To date, no strong evidence of resistance among H. perniciosus strains has been reported, although their different sensitivity to fungicides has been related to the high variability in the morphology of isolates [6].

Safer and eco-friendly alternatives to chemicals have also been studied for mushroom disease control: essential oils from aromatic plants [16,17,18,19,20], biocontrol agents [21,22], and water-based composts from agricultural wastes (compost teas), especially spent mushroom substrate [23,24]. Bacterial agents are an alternative to chemicals that could be used to control fungal problems without harming the environment, food safety, or human health. Thermophilic bacteria with high antagonistic capacity against pathogenic microorganisms, used as bio-pesticides, belong mostly to the Bacillus species [25]. They can antagonise pathogens by producing specific substances (antibiotics and/or volatile compounds) that affect pathogens, and also as competitors of pathogenic fungi for nutrient sources and space. Several bacterial strains, natural inhabitants of the casing layer in mushroom crops, have been described as conditioning yield performance and disease occurrence [12,26,27,28,29]. However, they sometimes only minimize disease symptoms at low disease pressure [30]. There are some commercial bio-fungicides, based on Bacillus subtilis (Ehrenberg) Cohn and on B. amyloliquefaciens (Fukumoto), which are registered for use against many plant pathogens, and have also been reported as inhibitory treatments against the growth of Trichoderma aggressivum, Cladobotryum mycophilum, and Lecanicillium fungicola, without negative impact on Agaricus bisporus mycelia [31,32,33,34].

The aim of this work was to study the effectiveness of different chemical and Bacillus-based products for WBD control in commercial mushroom farms artificially inoculated, at different inoculum rates, with the mycoparasite Hypomyces perniciosus. The results will help to develop a suitable IDM program against wet bubble disease.

2. Materials and Methods

2.1. Fungicides and Biological Control Agents

The fungicides tested were the commercial formulations of chlorothalonil 50% CS (Daconil^® 50 SC, Comercial Química Massó SA, Barcelona, Spain), metrafenone 50% CS (Vivando^®, Basf Crop Protection Spain, Barcelona, Spain), and prochloraz-Mn 46% WP (Sporgon^®, Basf Española, Barcelona, Spain). The biological control agents tested were the commercial formulations of Bacillus subtilis 1.34% CS strain QST 713 (Serenade^®, Bayer Crop Science, Valencia, Spain) and Bacillus amyloliquefaciens subsp. plantarum 25% WG strain D747 (Amylo-X^®, Certis Europe, Alicante, Spain). In all trials, the fungicide formulations were diluted in tap water to the required concentration and added as single drench applications to the blocks at a rate of 150 mL per block. The biological and chemical treatments were applied on day one and day five after casing, respectively. Untreated blocks were drenched with tap water (150 mL per block) and used as control (

Table 1. Treatments applied in the six trials.

Commercial Mark	Formulation	Code	Dose
Daconil®	Chlorothalonil 50% CS	(CTL)	3 mL L−1 m−2
Vivando®	Metrafenone 50% CS	(MTF)	1 mL L−1 m−2
Sporgon®	Prochloraz-Mn 46% WP	(PCL)	0.5 g m−2
Serenade®	B. subtilis 1.34% CS strain QST 713	(Bs)	3 g m−2
Amylo-X®	B. amyloliquefaciens subsp. plantarum 25% WG strain D747 (Ba)	(Ba)	3 g m−2

).

2.2. Trial Conditions

Six cropping trials were established following standard practices used in Spanish commercial mushroom farms. The trials were carried out in 20 m³ experimental mushroom growing rooms equipped with a humidification system, a heating/cooling system, internal air circulation/external ventilation, and hermetically sealed doors. This allowed automatic control of temperature, relative humidity, and carbon dioxide. A randomized block design was employed on shelves at three different height levels. In each trial, experimental trays filled with 10 kg of phase III incubated compost (0.15 m² in area), spawned at 1% with Amycel^® XXX (trials 1, 2, 6) and A-15 (trials 3, 4, 5) (Amycel Inc., San Juan Bautista, CA, USA), and cased with peat-based casing material (70% Infertosa^®, Valencia, Spain and 30% Harte Peat LTD, Monaghan, Ireland), 35 mm layer thick (5.5 L per block), were cultivated. The casing, almost saturated with water, was applied on day zero of the cultivation cycle. The environmental conditions were compost temperature of approximately 26 °C, relative humidity (RH) 95–98%, and carbon dioxide (CO₂) > 2000 ppm. Seven to eight days after casing, the growing rooms were ventilated to stimulate the production of mushroom fruiting bodies by gradually reducing the temperature and humidity levels over a 3-day period to 18 °C and approximately 90% RH, together with the introduction of fresh filtered air. Temperature of 18.5 ºC and RH of 85–90% were maintained throughout the cropping. Irrigation of the experimental trays was carried out after the end of harvest of each flush.

2.3. Preparation of the Inoculum of Hypomyces perniciosus

Different isolates of H. perniciosus were obtained from A. bisporus mushroom farms located in Castilla-La Mancha (Spain), maintained on potato dextrose agar medium (PDA; Oxoid, Basingstoke, England) at 22 °C in the dark, and morphologically characterized [4]. The H. perniciosus conidial suspensions were prepared on the day of inoculation and consisted of conidia from 2-week-old pure cultures (PDA), washed with sterile distilled water before filtering through a polypropylene net filter [25 µm pore Ø, Millipore^® (Merck KGaA, Darmstadt, Germany)] to remove mycelial fragments. The concentration of each conidial solution was determined using a haemocytometer and diluted in sterile distilled water to the desired concentrations. Blocks were artificially inoculated after casing (day 8) with 20 mL block⁻¹ of the conidial solution watering the surface of the casing, and obtaining a final rate of 10⁶ conidia m⁻² (trial 1: isolate MP19-7; trial 2: MP20-3), 10⁵ conidia m⁻² (trial 3: MP20-1; trial 4: MP21-1), and 10³ conidia m⁻² (trial 5: MP21-4: trial 6: MP20-3). Control (non-inoculated) blocks received 20 mL of sterile distilled water.

2.4. Data Analysis

Mushrooms were hand-picked daily throughout the first (F1), second (F2), and third (F3) flushes. The number and total weight of the fruit bodies were recorded for each treatment. The harvested mushrooms were classified as healthy or infected by H. perniciosus and the average yield (kg m⁻²) of both types was calculated. The effect of fungicides on mushroom productivity was assessed by yield and the biological efficiency (BE), calculated as the ratio between fresh weight of the total yield of harvested mushrooms (healthy and diseased) and the weight of dry substrate at spawning, and expressing the fraction as kg/100 kg compost. Disease incidence (DI) was recorded as a percentage value, based on the ratio between number of diseased sporophores and the total number of mushrooms harvested (healthy and diseased). Fungicide effectiveness was calculated using Abbott’s formula (Abbott, Chicago, IL, USA, 1925): % effectiveness = [(Ic − It)/Ic] × 100 (where Ic = disease incidence of inoculated control; It = disease incidence of treatment) [10].

The experimental design was a randomized complete block design with six replicates per treatment (n = 42). Statistical analysis of yield, biological efficiency, disease incidence, and effectiveness data were performed independently for each trial.

The effectiveness data were also analysed statistically by considering the results of each two trials performed with the same inoculum rate together. Data were analysed by ANOVA, using pertinent previous transformation when necessary. Statistical analyses were performed using Statgraphics Centurion XV (Statistical Graphics Corp., Princeton, NJ, USA).

3. Results

3.1. Yield

The total yield of healthy mushrooms harvested in the non-inoculated control treatment (C) in the six trials ranged from 18.1 to 28 kg m⁻² (Figure 1), while the yield of diseased mushrooms was almost negligible for each flush in each trial (Figure 2). For the inoculated treatments (IC), the yield of healthy mushrooms was statistically lower than described above for the non-inoculated control (C) for trials 1–4, while there were no statistical differences between the C and IC treatments in trials 5 and 6, trials with the lowest inoculum rate (F_6,41 = 0.61; p = 0.7169 for trial 5, and F_6,41 = 1.99, p = 0.0931 for trial 6). The low yield of diseased mushrooms for trial 6 discouraged graphic inclusion in Figure 2 and Figure 3. The yield of diseased mushrooms registered for the IC treatments was related to the inoculation rate, with mean values of 19.4 and 17.6 kg m⁻² for trials 1 and 2, 5.6 and 13.2 kg m⁻² for trials 3 and 4, and 0.94 and 0.12 kg m⁻² of diseased mushrooms in trials 5 and 6. In fact, the yield losses, by weight, reported for the IC treatments ranged between 80–91% for trials 1 and 2, 21–46% for trials 3 and 4, and less than 4% for trials 5 and 6. The same behaviour could be described for both biological treatments (IBa and IBs), which showed no statistical differences in terms of yield of healthy and diseased mushrooms with each other, neither with the inoculated control treatment (IC). The chemical fungicide treatments (ICTL, IMTF, IPCL) were halfway. In the trials with the highest inoculum rate, trials 1 and 2, statistical differences with respect to diseased yield (Figure 2), between IPCL and the other two chemical treatments could be reported (F_6,41 = 92.43; p = 0.0000 for the trial 1, and F_6,41 = 81.23, p = 0.0000 for trial 2). Yield losses were up to 20% for the IPCL treatment, while they were up to 40 and 50% for the ICTL and IMTF treatments, respectively. In the trials with intermediate (trials 3 and 4) and low (trials 5 and 6) inoculum rates, data regarding diseased yields for the chemical treatments were statistically similar.

3.2. Biological Efficiency

The EB values calculated, in each trial, for all treatments were comparable in almost every case (

Table 2. Biological efficiency (kg healthy and diseased yield 100 kg dried compost) calculated for each of the trials.

Treatment	Trial 1	Trial 2	Trial 3	Trial 4	Trial 5
C *	86 ± 4 a	70 ± 11 a	104 ± 8 a	121 ± 6 a	108 ± 7 a
IC	102 ± 5 bc	74 ± 5 a	104 ± 23 a	120 ± 8 a	104 ± 10 a
ICTL	87 ± 5 a	77 ± 15 a	96 ± 8 a	115 ± 5 a	103 ± 6 a
IMTF	92 ± 8 ab	82 ± 7 a	104 ± 4 a	118 ± 6 a	109 ± 8 a
IPCL	88 ± 5 a	77 ± 4 a	103 ± 8 a	120 ± 4 a	103 ± 15 a
IBa	108 ± 8 c	74 ± 6 a	103 ± 5 a	125 ± 6 a	108 ± 6 a
IBs	102 ± 5 bc	76 ± 7 a	102 ± 10 a	125 ± 12 a	109 ± 6 a
F	12.24	1.08	0.35	1.38	0.66
P	0.0000	0.3912	0.9039	0.2499	0.6811
LSD	11.30	15.44	10.31	13.58	15.78
SED	2.56	3.49	0.23	3.07	3.57

* C: uninoculated control; IC: inoculated control; ICTL: inoculated and chlorothalonil treatment; IMTF: inoculated and metrafenone treatment; IPCL: inoculated and prochloraz-Mn treatment; IBa: inoculated and B. amyloliquefaciens treatment; IBs: inoculated and B. subtilis treatment. Different letters indicate statistical differences between treatments, at 95% of significance (p < 0.05).

). In fact, if some statistical differences were observed, these were due to the increase recorded for the most affected treatments (trial 1: IC, IBA, and IBs treatments).

3.3. Incidence of Wet Bubble Disease

The incidence of wet bubble disease, defined as the percentage of diseased mushrooms vs total (healthy and diseased) harvested in the three flushes, for each of the first five trials is shown in Figure 3. In trials 1 and 2, the levels recorded for IC, IBa, and IBs treatments were above 80% for the first, second, and third flushes. In the case of the chemical fungicide, the incidence reported for ICTL, IMTF, and IPCL treatments was statistically lower for the first and second flushes (F_6,41 = 53.14; p = 0.0000 for the first flush and F_6,41 = 120.60; p = 0.0000 for the second flush in trial 1, and F_6,41 = 131.43; p = 0.0000 for the first flush and F_6,41 = 70.40; p = 0.0000 for the second flush in trial 2) than those reported above, but in the third flush the incidence for the IMTF treatment could be considered similar to the IC treatment (F_6,41 = 64.32; p = 0.0000 for trial 1, and F_6,41 = 44.73; p = 0.0000 for trial 2). When the inoculum rate was moderate, trials 3 and 4, although incidence levels were generally lower, the differences between treatments remained like those described for the previous trials. Incidence levels for IC, IBa, and IBs treatments were below 60% for all flushes in trial 3 and increased from 40% (first flush) to 100% (third flush) in trial 4. For all three chemical treatments, incidence levels were below 20% in all flushes, apart from the 40% recorded in the third flush of trial 4. When the inoculum rate was lowest, trial 5, the incidence for the first two flushes was so low that no statistical difference was recorded between all treatments (F_6,41 = 1.66; p = 0.1604 for the first flush, and F_6,41 = 2.42; p = 0.0565 for the second flush). In the third flush, only IC and IBs treatments showed notable levels of disease incidence (40%), and statistical differences with ICTL and IPCL treatments (F_6,41 = 6.58; p = 0.0001).

3.4. Effectiveness of the Different Biological and Chemical Products

The effectiveness calculated for the two Bacillus treatments in trials 1 and 2, with the highest inoculum rate, were statistically lower than the other treatments, maintaining low values for the three flushes. As for the chemical fungicides, the PCL treatment showed the highest effectiveness, then CTL, and finally the MTF treatment. The effectiveness of these fungicides clearly decreased in the third flush (

Table 3. Effectiveness, for each inoculum rate, of the different assayed treatments reported for each of the three harvested flushes.

Inoculum	Treatment	F1	F2	F3	F2,35	p	LSD	SED
106 CFU m−2	CTL *	56.1 cB **	55.9 cB	20.4 bA	22.38	0.0000	9.94	2.86
	MTF	23.8 bB	39.2 bB	8.3 aA	14.17	0.0000	12.10	3.49
	PCL	83.1 dB	79.2 dB	41.4 cA	27.96	0.0000	9.74	2.80
	Ba	4.6 aA	3.2 aA	0.9 aA	0.76	0.4746	8.33	2.40
	Bs	2.8 aA	5.6 aA	0.9 aA	1.07	0.3546	8.57	2.47
	F4,59	79.30	100.25	26.36
	p	0.0000	0.0000	0.0000
	LSD	11.79	9.89	12.09
	SED	2.96	2.48	3.03
105 CFU m−2	CTL	83.0 bcA	77.8 bA	70.1 bA	0.84	0.4389	18.14	5.23
	MTF	71.9 bA	77.0 bA	74.8 bA	0.05	0.9511	13.56	3.91
	PCL	95.0 cA	92.2 bA	82.0 bA	1.17	0.3233	14.75	4.25
	Ba	14.8 aA	19.0 aA	15.4 aA	0.23	0.7952	18.62	5.36
	Bs	20.7 aA	18.5 aA	17.0 aA	0.09	0.9106	119.83	5.71
	F4,59	35.99	38.52	23.09
	p	0.0000	0.0000	0.0000
	LSD	19.50	17.25	22.07
	SED	4.89	4.32	5.53

* CTL: chlorothalonil treatment; MTF: metrafenone treatment; PCL: prochloraz-Mn treatment; Ba: B. amyloliquefaciens treatment; Bs: B. subtilis treatment. ** In a same column, different lowercase letters indicate statistical differences between treatments, at 95% of significance (p < 0.01). In a same row, different capital letters indicate statistical differences between flushes, at 95% of significance (p < 0.01).

). When the inoculum rate was intermediate, trials 3 and 4, the effectiveness of both Bacillus treatments was again statistically lower than that of the chemical treatments, which were also comparable with each other for the second and the third flush. In the first flush, the effectiveness of PCL was statistically superior to that of the MTF treatment. Moreover, the particular effectiveness for each treatment remained comparable for all three flushes. In trial 5, with the lowest inoculum rate, statistically significant differences were only recorded, for the third flush, between the effectiveness of PCL and CTL (average value > 95%) and the Bs treatment (45%), with intermediate values for MTF (87%) and Ba (64%).

Regarding inoculum rate (

Table 4. Total effectiveness, for each treatment, as a function of the inoculum rate.

Inoculum	CTL *	MTF	PCL	Ba	Bs
105 CFU m−2	75.9 ± 20.4 b **	77.9 ± 9.9 b	90.5 ± 10.2 b	14.5 ± 18.2 b	18.2 ± 17.0 b
106 CFU m−2	43.4 ± 11.6 a	25.6 ± 12.3 a	71.9 ± 9.9 a	1.9 ± 2.5 a	2.9 ± 5.1 a
F1,23	20.97	108.26	19.21	5.91	8.91
p	0.0001	0.0000	0.0002	0.0237	0.0068
LSD	9.97	6.51	8.63	10.33	10.73
SED	3.40	2.22	2.94	3.52	3.66

* CTL: chlorothalonil treatment; MTF: metrafenone treatment; PCL: prochloraz-Mn treatment; Ba: B. amyloliquefaciens treatment; Bs: B. subtilis treatment. ** In the same column, different letters indicate statistical differences between inoculum rates.

), statistical analysis for each treatment showed that the effectiveness reported for the intermediate inoculum rate was statistically superior to those for the highest rate.

4. Discussion

Wet bubble disease (WBD) has normally been a sporadic problem in mushroom crops. However, outbreaks have been increasing worldwide and it is now even considered a devastating disease in some growing countries [9]. Chemical fungicides have traditionally been used as preventive treatments to control WBD [5], but bacterial-based products could be an important alternative to them. Bacillus spp. are ubiquitous bacteria found in soils and are widely used as biocontrol agents due to their ability to antagonize crop pathogens and the possibility of developing a stable commercial spore-based product [35]. The potential phytotoxic effect of B. subtilis-based products on the mycelia of commercial fungi has been studied in vitro and in vivo, with favourable results [27,32,33,36,37]. There is also some work related to the in vitro antimicrobial activities of different strains of B. subtilis against mushroom fungal pathogens [27,33,34,38,39], and there are a few studies focused on the antifungal mechanism and application of commercial B. subtilis-based products during mushroom cultivation [27,32]. This paper shows the results of the application of different chemical and Bacillus-based products for the control of WBD in artificially inoculated mushroom crops.

Firstly, it should be noted that no phytotoxic effect of the treatments has been recorded. All calculated BE values calculated were comparable. The result is in agreement with the literature reporting the absence of harmful effects on the white button mushroom mycelia in both in vitro and in vivo trials after different treatments with Bacillus spp. [32,33]. Disease incidence, and consequently yield losses, recorded in the inoculated control (IC) blocks were related to the inoculum rate, being negligible in the trials with the lowest inoculum rate (10³ conidia m⁻²). The greatest reductions in WBD incidence occurred after treatment with fungicides chlorothalonil (ICTL), metrafenone (IMTF), and prochloraz (IPCL). In trials 3 and 4, with an intermediate inoculum rate (10⁵ conidia m⁻²), the reduction of WBD was statistically comparable after the three fungicide treatments. The reported effectiveness was >70% for all three fungicides, and even close to 90% for prochloraz-Mn. However, when the inoculum rate was the highest (10⁶ conidia m⁻²) (trials 1 and 2), differences between the three fungicides were observed, with the prochloraz-Mn treatment standing out with an effectiveness of approximately 80% for the two first flushes. In these trials, the effectiveness of chlorothalonil could be considered acceptable (>50%) for the two first flushes, but the low value calculated for metrafenone advised against its application when a hard outbreak was expected.

Incidence values recorded for the inoculated and Bacillus-treated blocks (IBa and IBs) were statistically similar to inoculated and untreated (IC) blocks in almost all cases. In fact, the estimated effectiveness against WBD for both Bacillus treatments was less than 20% in any of the trials; the higher inoculum rate, the lower the effectiveness. This fact highlighted the poor effect of these bio-fungicides against WBD. The literature reports that the application of some Bacillus spp. strains showed promise for the suppression of compost green mould and dry bubble disease of A. bisporus [27,32,40]. A mixture of fungicide and biocontrol agent (prochloraz:Serenade^®, 80:20) has even been studied against compost green mould, reporting that Serenade^® showed better disease control when applied alone than its mixture with the fungicide, not being as effective as prochloraz-Mn but showing better disease control than a tea tree oil-based biofungicide [41]. In these studies, treatments with the Bacillus biofungicide and the chemical fungicide were repeated after the first flush, approximately 22 days after casing, which doubled the application dose. This could be the reason for the divergence from the calculated effectiveness. Carrasco and Preston [30] reported that some Bacillus treatments could minimize disease symptoms only when disease pressure was low. The harshness of the outbreaks produced in trials 1–4 could explain the divergence on the calculated effectiveness. However, in the case of the lowest inoculum rate (trial 5), when disease incidence was negligible (during the first two flushes), the low effectiveness of Bacillus- treatments against WBD was also remarkable. All this argues against the usefulness of these Bacillus treatments, applied at the doses and timing reported in the paper, against web bubble disease in mushroom crops. More studies looking for alternatives to chemical fungicides must be developed, mainly due to the risk of using them to consumer health.