Tolerance to Lecanicillium Fungicola and Yield of Agaricus Bisporus

Scientia Horticulturae 190 (2015) 117–122

Contents lists available at ScienceDirect

Scientia Horticulturae

journal homepage: www.elsevier.com/locate/scihorti

Tolerance to Lecanicillium fungicola and yield of Agaricus bisporus

strains used in Brazil

a,∗ b c

Diego Cunha Zied , Janaira Santana Nunes , Vinicius Franco Nicolini ,

d e b

Arturo Pardo Gimenez , Danny Lee Rinker , Eustáquio Souza Dias

Universidade Estadual Paulista (UNESP), Câmpus de Dracena, 17900-000 Dracena, São Paulo, Brazil

Universidade Federal de Lavras, Departamento de Biologia, 37200-000 Lavras, Minas Gerais, Brazil

Faculdades Integradas de Bauru (FIB), 17056-100 Bauru, São Paulo, Brazil

Centro de Investigación, Experimentación y Servicios del Champi˜nón (CIES), 16220 Quintanar del Rey, Cuenca, Spain

Department of Plant Agriculture, University of Guelph, 4890 Guelph, Ontario, Canada

r t i c l e i n f o a b s t r a c t

Article history: Dry bubble disease is a major problem in the commercial cultivation of Agaricus bisporus. In Brazil, there

Received 26 September 2014

are no fungicides registered by the Ministry of Agriculture for control of disease in the cultivation of A.

Received in revised form 7 April 2015

bisporus, nevertheless growers use daily fungicide on crops. The selection of strains more adapted to rustic

Accepted 13 April 2015

conditions and tolerant to Lecanicillium fungicola is another alternative to avoid yield losses. Thereunto

four experiments were conducted. Two cropping trials were set up in mushroom growing rooms (with

Keywords:

and without the presence of the pathogen), and two in vitro trials with varying dilutions and active

White button

ingredient (iprodione and difenoconazole) of products were performed in lab. Our results suggest that

Dry bubble

Yield some commercial strains of A. bisporus are more tolerant to the pathogen than others. Up to 76.5% yield

loss may be caused by the pathogen under experimental conditions. Among the 15 strains studied, only

Fungicides action

Electron micrographs one strain, ABI 11/16 was the more productive in the presence of the pathogen, even with unmarketable

diseased mushrooms. The only strain that showed “in vitro” mycelial growth similar to L. fungicola was

ABI 09/10; however, its yield is not high and the experimental conditions resulted in a 64% reduction in

yield due to the presence of the pathogen. Difenoconazole more strongly inhibited the mycelial growth

of L. fungicola than did iprodione; however, neither fungicide was selective against L. fungicola.

1. Introduction detrimental diseases that affects production of A. bisporus (Lange)

Imbach worldwide (Foulongne-Oriol et al., 2012). Undifferentiated

In Americas the white button mushroom (Agaricus bisporus) spherical masses (dry bubble), differential growth of the stipe and

began to be cultivated in the USA in 1865 and was introduced subsequent rupturing, and circular lesions on the pileus surface

to Brazil in 1953, when Japanese, Chinese, and Italian immigrants are characteristic symptoms of L. fungicola infection, all leading to

moved to Mogi das Cruzes and Atibaia, in the State of São Paulo severe economic losses (North and Wuest, 1993).

(Dias et al., 2008). Original production was under very rustic con- Control of L. fungicola relies on prophylactic measures and the

ditions where the growing rooms consisted of primitive chambers use of fungicides (Gea et al., 2005; Fletcher and Yarham, 1976;

with shelves of bamboo and wood, dirt ﬂoors, and little environ- Wuest et al., 1974). Strict speciﬁcations for the application of chem-

mental controls. Despite the advances of technology with modern icals for pest and disease control in mushroom farms, together with

facilities and environmental management, the crops are commonly the emergence of fungicide tolerance, have led to the consideration

affected by pests and diseases, including Lecanicillium fungicola. of mushroom breeding for resistance to disease as an environmen-

Dry bubble, caused by the fungal pathogen L. fungicola (Preuss) tally sustainable and effective way to limit pathogen epidemics

Zare and Gams (formerly Verticillium fungicola), is one of the most (Largeteau and Savoie, 2010).

Currently, one of the greatest challenges in Brazilian A. bisporus

production is the number of not well characterized strains. All

strains cultivated in Brazil were brought from different countries

∗

Corresponding author at: Rod. Cmte João Ribeiro de Barros, km 651, Bairro das

and have been successively sub-cultured for many years, so that

Antes, 17900-000 Dracena, SP, Brazil. Tel.: +55 18 3821 8200;

most of them have lost their original genetic quality. Often, the

fax: +55 18 3821 8208.

E-mail address: [email protected] (D.C. Zied). origin of the lab from which the strains have been acquired is

http://dx.doi.org/10.1016/j.scienta.2015.04.021

118 D.C. Zied et al. / Scientia Horticulturae 190 (2015) 117–122

Table 1

Additional information for each experiment.

Experiments Treatments Variables analyzed

1st – cropping trial 15 different strains (without conidial suspension) Mushroom yield, number, and weight

2nd – cropping trial 15 different strains (with conidial suspension) Mushroom yield and percentage reduction in yield

3rd – in vitro trial 15 different strains + 1 pure culture of L. fungicola a.i. Mycelial growth and percentage inhibition of

iprodione, and control mycelium growth

4th – in vitro trial 15 different strains + 1 pure culture of L. fungicola a.i. Mycelial growth and percentage inhibition of

difenoconazol, and control mycelium growth

Agaricus bisporus; information related to these strains are described in Table 2.

unknown, and their genetic and physical characteristics (size of followed by proliferation on triticale grain according to Zied et al.

mushroom, color, and ﬁrmness), production parameters, and sus- (2010a). These 15 strains were cultivated in two different con-

ceptibility to diseases such as L. fungicola are undocumented. trolled chambers (with and without conidial suspension) and also

Another fact that should be mentioned is that in Brazil, there used in vitro trials for fungicide application. Table 2 presents the

are currently no products registered by the Ministry of Agriculture main characteristics of the strains.

for control of disease in the commercial cultivation of A. bisporus, Infected mushrooms were collected from the growers listed in

nevertheless growers use daily fungicide on crops. For Brazilian Table 2. The L. fungicola was isolated from necrotic lesions on the

mushroom growers and for those from other developing countries, pilieus surface and was cultured on potato dextrose agar (PDA;

◦

modern mushroom facilities that enable and facilitate strict san- Difco, Lawrence, USA) at 25 C.

itation and hygiene are expensive, thus restricting investment

by Brazilian mushroom growers and those in other developing 2.3. Cropping trials

countries.

Therefore the selection of strains more adapted to rustic condi- Pasteurized and conditioned compost was provided by “Gildo

tions and tolerant to L. fungicola is highly desirable. These strains Saito”, Mogi das Cruzes, in the state of São Paulo. The same compost

would be valuable in the development of newer disease-tolerant batch was used in experiment 1 and 2. Eight kg of compost was

strains. In the present study we evaluated the yield of different A. inoculated with 80 g of the respective spawn strains of A. bisporus

bisporus strains used in Brazil in the presence of L. fungicola. We (prepared as in Zied et al., 2010a), placed into a plastic box and

◦

also performed an in vitro study on the use of fungicides to inhibit pressed. The compost was incubated at 25 C for 15 days. At the

L. fungicola mycelium growth and the respective consequences on end of Phase II, the compost exhibited 68% humidity, following the

A. bisporus growth. quality indicators noted by Zied et al. (2011).

A loamy oxisol-alﬁsol was used as the casing soil. The soil pH

−3

2. Materials and methods was corrected to 7.0 by adding calcium carbonate (10 kg m ) 20

days before casing. After this period, the soil moisture was adjusted

Four experiments were conducted. Two cropping trials were set to 30% and 20% (V/V) wood charcoal (1–2 cm diameter) was mixed

◦

up in mushroom growing rooms, and two in vitro trials with varying into it. The mixture was then pasteurization at 62 C for 4 h. The

dilutions and active ingredients of products were performed in lab casing layer was added on top of the colonized compost until it

(see Table 1). reached 3 cm high.

To determine the effect of the dry bubble disease agent, L. fungi-

2.1. Experimental design cola, on mushroom cultivation, an irrigation suspension of conidia

8 −2

at a concentration of 2.4 × 10 conidia m was applied in the boxes

Two experiments were conducted simultaneously under con- used for the 2nd experiment, 3 days after the addition of the casing

trolled conditions. Each experiment used 15 Agaricus bisporus layer, following the methodology described by Gea et al. (2013).

strains and 8 repetitions of each treatment. Mushrooms were The boxes of the 1st experiment did not receive the suspension

grown in plastic boxes containing 8 kg fresh compost. The 1st of conidia. Both crops were grown for 40 days (spawning to crop

experiment was conducted to verify the production parameters termination), with 3 ﬂushes of harvest.

(mushroom yield, number, and weight) of the 15 commercial During the ﬁrst 8 days after casing, the compost temperature

◦

strains used in Brazil, and the 2nd experiment, used the same was maintained at 25 C, the atmospheric CO2 content less than

strains as experiment 1 with the addition of a conidial suspension 5000 ppm and the relative humidity at 95%. To initiate primordia,

◦

to determine the tolerance of these strains to the pathogen L. fungi- the temperature, CO2 content and humidity were lowered to 19 C,

cola. Experiments 1 and 2 were not performed in duplicate, i.e. not 1000 ppm, and 87%, respectively.

had two crop cycles. The mushrooms were harvested every day at their optimal com-

The other two experiments were in vitro assays to determine the mercial stage of development, corresponding to morphogenetic

resistance of L. fungicola and the A. bisporus strains to two fungicides stages 2, 3, and 4 of the classiﬁcation established by Hammond

applied off-label in mushroom cultivation in Brazil. The experi- and Nichols (1976). Mushroom weight after stipe trimming (cut to

ments consisted of a factorial double (16 strains 4 dilutions), each remove the casing, approximately 1.5 cm) and the total number of

with 5 repetitions performed in petri dishes. The 3rd experiment mushrooms picked from each box was recorded daily.

used the active ingredients iprodione (chemical group dicarbox- Mushroom yield and number of mushrooms per box were con-

imide), and the 4th used the active ingredients difenoconazole verted to yield (fresh weight of mushrooms divided by the fresh

(chemical group triazole). weight of compost and multiplied by 100), and individual average

mushroom weight (fresh weight of mushrooms divided by the

2.2. Fungal strains number of mushrooms), following the methodology adopted by

Zied et al. (2010b). Yield in 2nd experiment was calculated through

A total of 15 strains of Agaricus biporus were collected from all biomass produced at the surface of the casing layer, including

growers in Brazil, producing at least 35,000 kg of fresh mushrooms healthy mushrooms, bubbles and other mushrooms deformed due

each month. Mushroom tissue was used to generate mycelium, to the action of L. fungicola. Percent reduction in yield was

D.C. Zied et al. / Scientia Horticulturae 190 (2015) 117–122 119

Table 2

Main characteristics and observed details of each strain used in the experiments, including the city and state where the strains were isolated and the origin of the spawn’s

company.

Strain number City and state of Origin Characteristics

isolation

ABI 11/14 Mogi das Cruzes, São Italy Mushrooms with high texture, white color, smooth, high yield, resulting from

Paulo the basidiocarp with high weight

ABI 11/20 Mogi das Cruzes, São Japan Mushrooms with high texture, white color, smooth, regular yield, and the

Paulo lamellae not break easily

ABI 11/17 Mogi das Cruzes, São Japan Mushrooms with high texture, white color, smooth, and low yield, resulting

Paulo from basidiocarp with high mass

ABI 11/16 Botucatu, São Paulo USA “Triple-X” hybrid produced by Amycel, mushrooms with intermediate texture,

high yield, and tolerance to L. fungicola, but the lamellae break easily

PB 01/01 Botucatu, São Paulo USA Mushrooms with highly texture, cream color, scales, distributed in pileus, and

low yield

PB 06/02 Itu, São Paulo USA Mushrooms with high texture, cream color, with a small amount of scales

distributed in pileus, and low yield

ABI 06/05 Mogi das Cruzes, São ? Mushrooms with high texture, white color, smooth, high yield and the

Paulo lamellae not break easily

ABI 04/02 Suzano, São Paulo ? Mushrooms with high texture, white color, smooth, tolerance to L. fungicola,

low yield, and the lamellae not break easily

ABI 11/15 Botucatu, São Paulo Spain Commercial variety Gurelan 45, mushrooms with high texture, white color,

smooth, resistance to L. fungicola, high yield, and intermediate weight

ABI 05/03 Cabreuva, São Paulo Europe Mushrooms with high texture, white color, smooth, low yield, high weight,

resistance to L. fungicola, the lamellae not break easily

ABI 11/21 Castro, Parana Europe Mushrooms with high texture, white color, smooth, low yield, and the

lamellae not break easily

ABI 06/04 Itu, São Paulo Europe Mushrooms with high texture, white color, smooth, intermediate yield,

resistance to L. fungicola, and interestingly, the basidiocarp do not reach the

stages of development (morphogenesis) 5, 6, and 7

ABI 07/06 Piedade, São Paulo Europe Mushrooms with high texture, white color, smooth, high yield, and the

lamellae not break easily

ABI 09/10 Botucatu, São Paulo China Strain sent by Jun Cao Institute, characteristic for producing mushrooms with

high texture, white color, smooth, and high weight

ABI 11/19 Mogi das Cruzes, São China Mushrooms with high texture, white color, smooth, and low yield

Paulo

calculated as follows: [(Yexp1 − Yexp2)/Yexp1] × 100 (where mycelial growth in control; Rt = average radial mycelial growth in

Yexp1 = yield obtained in 1st experiment; Yexp2 = yield obtained fungicide treatment). The analysis of variance for mycelial growth

−1 −1

in 2nd experiment). The analysis of variance for yield, number, (␮m h and mm 12d ) and percentage inhibition of mycelium

and weight of mushrooms and percent reduction in yield was growth were determined using Tukey’s test (P < 0.05) with SAS soft-

performed using Tukey’s test (P < 0.05) with SAS software (Inc., ware.

Cary, NC).

2.5. Microscopy of the interaction

2.4. In vitro trials

Some healthy mushrooms (strain ABI 06/05) from the 1st

Two experiments were in vitro assays to determine the resis- experiment were inoculated in the laboratory with 20 ␮l of the

tance of L. fungicola and the A. bisporus strains to two fungicides concentrated suspension of 1 × 10 conidia of L. fungicola per ml

applied off-label in mushroom cultivation in Brazil. to the surface of the pileus. The inoculated fruiting bodies were

◦

The starting fungicidal solution used the rates applied by grow- kept in a chamber at 80% humidity and 22 ± 0.5 C. Samples of

2 2

ers: 1.0 g of iprodione per m of casing surface (3rd experiment) 1.5 cm were collected at 15, 25, and 50 h after inoculation. The col-

and 0.4 g of difenoconazole per m of casing surface (4th experi- lected fragments were ﬁxed in modiﬁed Karnovsky solution (2.5%

ment). Iprodione and difenoconazole were diluted in 10 ml and 4 ml glutaraldehyde, formaldehyde 2.5% in sodium cacodylate buffer

of autoclaved distilled water, respectively, to obtain dilutions of 0.05 M, pH 7.2, CaCl2 0.001 M) for 24 h. After ﬁxation, the samples

−1 −2

10 . These solutions were then further diluted to obtain 10 and were examined using the standard protocol for scanning electron

−3

10 . Finally, doses of 0.1 ml iprodione and 0.04 ml difenoconazole microscopy described by Schweikert et al. (2013). The generated

−1 −2 −3

were taken from the 10 , 10 and 10 dilutions and transferred images were recorded and examined in the Photo paint Software

to petri dishes (90 mm in diam.) with the PDA culture medium, package Corel Draw 4.

according to methodology presented by Chrysayi-Tokousbalides

et al. (2007).

Discs (0.5 cm in diam.) containing inoculum of fungi (A. bis- 3. Results

porus strains and L. fungicola) were transferred to the centers of

petri dishes containing culture medium and dilutions of the fungi- 3.1. Cropping trials

cide doses. The control consisted of mycelium discs placed on PDA

without fungicide. The dishes were incubated under controlled The 15 strains in the absence of L. fungicola varied substantially

◦

conditions at 24 C. The radial mycelium growth was measured in yield. Strains ABI 11/14, ABI 11/20, ABI 11/16, ABI 06/05, ABI

every 24 h (4 equally spaced measurements) until the fungal colony 11/15, and ABI 07/06 had the highest yields (14.97–21%) (Table 3),

reached the edges of the petri dish. while strains ABI 11/17, PB 01/01, PB 06/02, ABI 04/02, ABI 05/03,

Percentage inhibition of mycelium growth using the fungi- ABI 11/21, ABI 06/04, ABI 09/10, and ABI 11/19 produced the lowest

cides was calculated: [(Rc – Rt)/Rc] × 100 (where Rc = average radial yields, ranging from 8.08 to 13.58%.

120 D.C. Zied et al. / Scientia Horticulturae 190 (2015) 117–122

Table 3

Cropping trial data from the 1st (without conidia suspension) and 2nd experiments (with conidia suspension).

Strain number 1st experiment 2nd experiment

Yield (%) Number (units) Weight (g) Yield (%) Reduction in yield (%)

ABI 11/14 21.00 a 76.00 abcd 28.66 ab 9.36 bcd 45.25 bcde

ABI 11/20 14.97 abcde 49.14 bcdef 25.98 ab 5.01 cd 35.23 bcde

ABI 11/17 11.8 def 34.50 ef 21.60 ab 4.04 cd 34.02 bcde

ABI 11/16 19.88 ab 95.50 a 16.75 b 17.94 a 8.74 abc

PB 01/01 10.81 def 36.00 def 25.25 ab 3.87 cd 64.90 cde

PB 06/02 13.58 bcdef 48.42 bcdef 23.14 ab 7.10 bcd 47.60 cde

ABI 06/05 18.00 abc 69.87 abc 20.62 ab 9.00 bcd 50.00 cde

ABI 04/02 12.47 cdef 44.25 cde 25.50 ab 13.35 ab −7.05 abc

ABI 11/15 17.18 abcd 81.00 ab 16.71 b 13.81 ab 19.61 abcd

ABI 05/03 10.35 ef 30.40 ef 33.80 ab 8.25 bcd 20.48 abcd

ABI 11/21 12.93 cdef 45.57 cdef 23.25 ab 4.60 cd 64.42 cde

ABI 06/04 12.44 cdef 52.57 bcdef 19.71 ab 15.18 ab −22.02 a

ABI 07/06 19.63 abc 59.83 bcde 26.16 ab 11.71 abc 40.01 bcde

ABI 09/10 13.59 bcdef 49.00 bcde 33.71 ab 4.85 cd 63.87 cde

ABI 11/19 8.08 f 23.33 f 39.33 a 1.90 d 76.48 e

Mean 14.2 53.73 24.62 8.64 68.60

CV 25.91 36.62 51.3 54.87 31.48

LSD 6.45 34.49 22.07 8.31 51.98

Values followed by a different letter are signiﬁcantly different at a 5% level according to Tukey’s HSD test.

LSD, least signiﬁcant difference; CV, coefﬁcient of variation.

The strains that achieved higher yields resulted in greater num- it still grew 7% slower than L. fungicola. The mycelial growth rate of

bers of mushrooms harvested. With respect to the weights of the L. fungicola was higher in 4th experiment than in 3rd experiment.

mushrooms, variations from 16.71 to 39.33 g were observed. The Due to the higher mycelial growth of L. fungicola in cul-

strain ABI 11/19 showed a greater mass compared to ABI 11/15 ture medium, the mycelial growth (average of 3rd and 4th

−1

and ABI 11/16, while the rest of strains did not showed statistically experiment = 163 ␮m h ) was examined to better understand the

signiﬁcant differences. process of infection and the aggressiveness of L. fungicola “in vivo”.

Among the 15 strains, 6 (ABI 11/20, ABI 06/05, ABI 04/02, ABI The growth of the germ tube (after 15 h), the formation of spores

05/03, ABI 11/21 and ABI 07/06) deserve attention because their veil and the verticillate (=whorled) arrangement of the phialides (after

did not break easily (Table 2); producers in Brazil grow these strains 25 h), and the complete colonization of the mushroom tissue, along

in rustic chambers, where the temperature remains between 21 with the appearance of brown, depressed necrotic spots (after 50 h),

◦

and 24 C. Another strain used in Brazil during the summer is ABI were observed (Fig. 1). Thus, depending on the amount of inocu-

06/04, which does not reach the stages of maturity 5, 6, and 7. A few lum in a growth chamber, after approximately 2 days, the ﬁrst

mushrooms were intentionally not collected, and although they symptoms appear in infected mushrooms.

went into senescence, the veil of lamellae did not break. Iprodione was not effective against L. fungicola. The lower dilu-

−1

The harvest in the 2nd experiment with L. fungicola began 16 tion (10 ) inhibited the mycelial growth of the pathogen by only

days after casing, 3 days before the harvest in the 1st experiment. 15% (Table 4). However, the same dose inhibited 100% of A. bisporus

−2

Strains ABI 04/02 and ABI 06/04 showed increased yields (com- mycelial growth in the evaluated strains. The 10 dilution showed

pared to the 1st experiment) on the order of 7.05 and 22.02%, an intermediate effect, where the mycelial growth was stimulated

suggesting that these strains are more tolerant (not negatively in some strains and inhibited in others.

affected in the yield) to L. fungicola, which could make them impor- Difenoconazole showed the highest percentage inhibition of

−1 −2

tant in a breeding program. Note that although not reduce the yield; mycelial growth in the treatments. The dilutions of 10 and 10

the mushrooms had symptoms of pathogen infection. inhibited the mycelial growth of the pathogen by 38–76%, which

Some strains with the highest yield in the 1st experiment is superior to the results of the iprodione treatment. However,

showed drastic yield reduction (ABI 11/14, ABI 06/05, ABI 11/21, both dilutions completely inhibited the mycelial growth of the A.

−3

ABI 07/06), in presence of L. fungicola (Table 3). Even some strains bisporus strains. The 10 dilution provided a stimulus in some

with intermediary yield in the 1st experiment showed drastic Agaricus strains and also inhibited others.

yield reduction in the 2nd experiment. In the 2nd experiment,

mushroom numbers and weights were not assessed due to the

deformation of the mushrooms infected by the pathogen for the 4. Discussion

most of the strains. The clustering of deformed primordia (undiffer-

entiated spherical masses) also made it difﬁcult to count individual Two crops were cultivated in experimental growth chambers to

mushrooms. determine the production response of 15 commonly used Brazilian

strains of A. bisporus and the inﬂuence of the pathogen L. fungicola

3.2. In vitro trials on the these selected isolates. In the chamber without addition

of the pathogen, the highest yield was 21%, which is lower than

The 3rd and 4th experiments were designed to compare the rate the yields obtained in Europe by Straatsma et al. (2013), Pardo-

of mycelial growth of L. fungicola compared to the 15 strains of A. Giménez et al. (2010) and Zied et al. (2010b), which range from 12

bisporus (Table 4) in the presence of iprodione and difenoconazol. to 43%, with growing cycles of approximately 32–45 days of har-

In the 3rd experiment, strains of A. bisporus that were closer to the vest. Strains ABI 11/24, ABI 11/16, ABI 06/05, ABI 11/15 and ABI

rate of L. fungicola mycelial growth were ABI 11/17, ABI 06/04, ABI 07/06 may be considered the most appropriate for the cultivation

09/10, and ABI 11/19, growing 28, 27, 21, and 18% more slowly than in a controlled environment, because they had the highest yields

L. fungicola. In the 4th experiment, the only A. bisporus strain with (17.18–21%). However, for a mushroom facility without control of

mycelium growth that was equal to L. fungicola was ABI 09/10, and L. fungicola, the strain ABI 11/16 seems to be the better, since it

D.C. Zied et al. / Scientia Horticulturae 190 (2015) 117–122 121

Table 4

In vitro trial data from the 3rd (iprodione) and 4th (difenoconazol) experiments with the effect of fungicides applied on 15 strains of A. bisporus and 1 isolated of L. fungicola.

Mycelial growth was used as control, without addition of the fungicides.

Strain 3rd experiment (iprodione) 4th experiment (dinenoconazol)

number

Mycelial Mycelial Inhibition of mycelium Mycelial Mycelial Inhibition of mycelium

growth growth growth (%) growth growth growth (%)

−1 −1 −1 −1

(␮m h ) (mm 12d ) (␮m h ) (mm 12d )

10−3 10−2 10−1 10−3 10−2 10−1

* ** * **

ABI 11/14 74 defg 21.26 cde −126 d C 6 abc B 100 a A 118 b 34.66 b 27 a B 100 a A 100 a A

ABI 11/20 78 cde 22.47 cd −55 bc C 0 abc B 100 a A 110 b 32.48 b 7 ab B 100 a A 100 a A

ABI 11/17 100 bc 29.06 b −14 ab B 10 abc B 100 a A 104 bcd 29.81 bc 31 a B 100 a A 100 a A

ABI 11/16 78 cde 22.38 cd −45 bc C 1 abc B 100 a A 99 bcd 28.21 bc 11 abc B 100 a A 100 a A

−

PB 01/01 52 g 14.75 f 44 bc C6abc B 100 a A50e 13.82 d 8 abc B 100 a A 100 a A

PB 06/02 54 fg 15.48 ef −87 cd C −18 bc B 100 a A 110 b 31.44 b −1 bc B 100 a A 100 a A

ABI 06/05 70 efg 19.74 def −28 ab C 10 abc B 100 a A 86 bcd 25.09 bc 2 abc B 100 a A 100 a A

ABI 04/02 74 defg 21.06 cde −117 d B −104 d B 100 a A 74 cde 20.81 cd −63 d C 100 a A 100 a A

ABI 11/15 60 efg 17.86 def −43 bc C 12 abc B 100 a A 96 bcd 27.20 bc 10 abc B 100 a A 100 a A

ABI 05/03 74 defg 20.95 cde −127 d C −24 bc B 100 a A 108 bc 31.16 b 2 abc B 100 a A 100 a A

ABI 11/21 76 def 22.02 cd −54 bc C 1 abc B 100 a A 104 bcd 30.07 bc 21 abc B 100 a A 100 a A

ABI 06/04 94 bcd 27.06 bc −32 ab C 19 ab B 100 a A 92 bcd 26.26 bc −48 d C 100 a A 100 a A

ABI 07/06 66 efg 18.49 def −44 bc C −23 bc B 100 a A 92 bcd 26.16 bc −40 d C 100 a A 100 a A

−

ABI 09/10 102 b 29.41 b −88 cd C 30 c B 100 a A 182 a 53.15 a −8 bc B 100 a A 100 a A

ABI 11/19 104 b 30.66 b 9 a C 42 a B 100 a A 72 de 20.66 cd 14 ab B 100 a A 100 a A

L. fungicola 130 a 37.29 a −25 ab B −22 bc B 15 b A 196 a 56.80 a 9 abc C 38 b B 76 a A

Mean 80 23.17 9 106 30.60 64

CV 12.42 11.94 228 14.68 14.67 27

* ** * **

LSD 20 6.18 47 32 34 10.04 38 7

Lowercase letters compare results in the same columns within each variable and treatment analyzed. Uppercase letters compare results in the same line within each variable

analyzed.

CV, coefﬁcient of variation.

LSD, least signiﬁcant difference of strain values.

LSD, least signiﬁcant difference of dilution values.

showed a yield of 17.94% in the presence of L. fungicola inoculation, and the time of application (days after casing), as noted by North

even some mushrooms showed disease symptoms. and Wuest (1993). Therefore, further studies must be performed

The yield reductions caused by L. fungicola ranged from 8.74 to to assay different times of applying the conidial suspension to the

76.48%. Best yields under disease pressure were observed in strains casing layer (at 0, 3, 6, 9, 12, and 15 days after casing) for a better

ABI 04/02 and 06/04, suggesting that these strains are more tol- understanding of the interactions that occur in ﬁeld cultivation.

erant to L. fungicola. However, it should be noted that 71% of the In Brazil, preventive measures such as the use of a good cas-

total mushrooms produced were non-standard commercial qual- ing material, hygiene management, and pest control practices are

ity because the mushrooms had symptoms of bubbles. Similar data employed to minimize losses caused by L. fungicola; however,

were observed by Gea et al. (2003), in which the prevalence of fungicides have been frequently applied to reduce the amount of

mushrooms per m with incidences of L. fungicola ranged from 0 damage caused by the pathogen, even being prohibited by the Min-

to 79% and varied according to the casing layer used. Largeteau istry of Agriculture. The majority of widely used products are in

and Savoie (2008), with inoculations a conidial suspension 11 days the dicarboximide chemical group, such as iprodione, or the triaz-

after casing, observed that the total weight of the inoculated crops ole chemical group, such as difenoconazole. Both do not effectively

did not differ signiﬁcantly from of the uninoculated control, but control the disease in vitro, as was observed in this work and other

−1 −2

inoculation with highly aggressive isolates resulted in a signiﬁcant authors (Gandy and Spencer, 1981). Both (10 and 10 ) dilu-

increase in the total number of mushroom. tions of iprodione and difenoconazole were more fungitoxic to the

The pathogen induces various symptoms on the host, includ- strains of A. bisporus than they were to L. fungicola, in accordance

ing undifferentiated spherical masses (bubbles), bent and/or split with the in vitro results obtained. These results should be consid-

stipes (blowout), and lesions on the pileus and stipes (spots) (Bailey ered on a detailed “in vivo” research. A dissimilar situation exists

et al., 2013; Berendsen et al., 2010; Largeteau et al., 2004). All of with prochloraz (Chrysayi-Tokousbalides et al., 2007), which was

these symptoms were observed in the current work, with the pre- shown previously to inhibit the in vitro growth of V. fungicola at very

−1

dominant symptom of undifferentiated spherical masses, which low concentrations (ED50 of 0.002 ␮g ml ) and to be fungitoxic to

−1

were directly related to the amount of conidial suspension applied A. bisporus at much higher levels (ED50 of 5.08 ␮g ml ). Gandy

Fig. 1. Scanning electron micrographs showing fruiting bodies of A. bisporus inoculated with L. fungicola. (A) Germ tubes after 15 h of inoculation (hai) (bar = 2 ␮m), (B)

formation of verticil and spores after 25 h of inoculation (bar = 10 ␮m) and (C) tissue colonization and development of symptoms at 50 h (bar = 10 ␮m).

122 D.C. Zied et al. / Scientia Horticulturae 190 (2015) 117–122

(1981) observed that iprodione in experimental crops reduced the References

yield of A. bisporus.

Bailey, A.M., Collopy, P.D., Thomas, D.J., Sergeant, M.R., Costa, A.M., Barker, G.L., Mills,

Despite the fact that iprodione does not provide effective

P.R., Challen, M.P., Foster, G., 2013. Transcriptomic analysis of the interactions

pathogen control, it was on the short list of fungicides that demon-

between Agaricus bisporus and Lecanicillium fungicola. Fungal Genet. Biol. 55,

strated activity against L. fungicola (Gandy and Spencer, 1981), and 67–76.

Berendsen, R.L., Kalkhove, S.I., Lugones, L.G., Baars, J.J., Wösten, H.A., Bakker, P.A.,

it is also registered for application in commercial cultivation of A.

2010. Effects of the mushroom-volatile 1-octen-3-ol on dry bubble disease. Appl.

bisporus in several countries, including Spain (De Linan,˜ 1994).

Microbiol. Biotechnol. 97 (12), 5535–5543.

Two modes of action of fungicides were selected, one contact Berendsen, R.L., Kalkhove, S.I., Lugones, L.G., Wösten, H.A., Bakker, P.A., 2012. Germi-

nation of Lecanicillium fungicola in the mycosphere of Agaricus bisporus. Environ.

and one systemic. The contact fungicide works mainly by pre-

Microbiol. Rep. 4, 227–233.

venting the formation of the germ tube of the fungus, thereby

Bernardo, D., Cabo, A.P., Novaes-Ledieu, M., Mendoza, C.G., 2004. Verticillium disease

your action to reduce mycelial growth is low (as studied in this or dry bubble of cultivated mushrooms: the Agaricus bisporus lectin recognizes

research using petri dishes with mycelial growth). This result was and binds the Verticillium fungicola cell wall glucogalactomannan. Can. J. Micro-

biol. 50, 729–735.

evidenced using iprodione with contact action, which reduced only

Calonje, M., Mendoza, C.G., Cabo, A.P., Bernardo, D., Novaes-Ledieu, M., 2000. Interac-

15% of mycelial growth of L. fungicola. Thus we highlight the speciﬁc

tion between the mycoparasite Verticillium fungicola and the vegetative mycelial

question, why iprodione with contact action showed signiﬁcant phase of Agaricus bisporus. Mycol. Res. 104, 988–992.

Chrysayi-Tokousbalides, M., Kastanias, M.A., Philippoussis, A., Diamantopoulou, P.,

reduction in the mycelial growth of all strains of A. bisporus?

2007. Selective fungitoxicity of famoxadone, tebuconazole and triﬂoxystrobin

Already the difenoconazole was more effective than iprodione,

between Verticillium fungicola and Agaricus bisporus. Crop. Prot. 26 (4), 469–475.

showing greater inhibition of L. fungicola growth in the range from Cross, M.J., Jacobs, L., 1968. Some observations on the biology of spores of Verticillium

− −

2 1 2 malthousei. Mushroom Sci. 7, 239–244.

38 to 76% (10 and 10 dilutions of 0.4 g per m ). The difeno-

De Linan,˜ C., 1994. Vademecum de produtos ﬁtossanitários y nutricionales. Ediciones

conazole has systemic action, indicated for use after the formation

Agrotécnicas, Madrid.

of germ tube and the growth of the hyphae of the fungus. Thus it is Dias, E.S., Labory, C.R.G., Herrera, K.M.S., Alves, A.A., Torres, G.A., Rinker, D.L., 2008.

Cytological studies of Agaricus brasiliensis. World J. Microbiol. Biotechnol. 24

supposed that when it is intended to make a preventative treatment

(11), 2473–2479.

should be used iprodione, on the other hand, when it is intended

Fletcher, J.T., Yarham, D.J., 1976. The incidence of benomyl tolerance in Verticillium

to make a curative treatment should use difenoconazole. Note that fungicola, Mycogone perniciosa and Hypomyces rosellus in mushroom crops. Ann.

Appl. Biol. 83, 4898–4903.

in this study we didn’t applied fungicides in cultivation, so this is a

hypothesis. Foulongne-Oriol, M., Rodier, A., Savoie, J.M., 2012. Relationship between yield com-

ponents and partial resistance to Lecanicillium fungicola in the button mushroom,

The strain ABI 09/10 was the only one with a similar rate of Agaricus bisporus, assessed by quantitative trait locus mapping. Appl. Environ.

mycelial growth compared to L. fungicola (Table 4) and could be an Microbiol. 78, 2435–2442.

Gandy, D.G., 1981. Sensivity of mushroom strains to fungicides. Mushroom Sci. 11

interesting strain to be used in a breeding program to obtain new

(2), 473–484.

hybrids to be competitive against L. fungicola. On the other hand,

Gandy, D.G., Spencer, D.M., 1981. Fungicide evaluation for control of dry dubble,

previous studies in the literature report that L. fungicola is unable caused by Verticillium fungicola, on commercial mushroom strains. Sci. Hortic.

14, 107–115.

to infect vegetative A. bisporus mycelium and can only infect the

Gea, F.J.A., Tello, J.C., Navarro, M.J., 2003. Occurrence of Verticillium fungicola var.

fruiting bodies and primordia at the beginning of the differentiation

Fungicola on Agaricus bitorquis Mushroom Crops in Spain. J. Phytopathol. 151,

(Bernardo et al., 2004; Calonje et al., 2000; Cross and Jacobs, 1968), 98–100.

Gea, F.J.A., Navarro, M.J., Tello, J.C., 2005. Reduced sensitivity of the mushroom

which suggests that infection, must take place in the casing layer.

pathogen Verticillium fungicola to prochloraz manganese in vitro. Mycol. Res.

L. fungicola spore germination is inhibited in the casing through

109, 741–745.

antibiotics produced by the casing microbiota (Calonje et al., 2000), Gea, F.J.A., Carrasco, J., Santos, M., Dianez, F., Navarro, M.J., 2013. Incidence of Lecani-

cillium fungicola in white-button mushroom (Agaricus bisporus) cultivated with

and the time point of infection by L. fungicola is therefore linked to

two types of casing soil. J. Plant Pathol. 95 (1), 163–166.

the formation of mushrooms (Berendsen et al., 2012).

Hammond, J.B.W., Nichols, R., 1976. Carbohydrate metabolism in Agaricus bisporus

Mushroom disease control is necessary and the use of fungicides (Lange) Sing.: changes in soluble carbohydrate during growth of mycelium and

sporophore. J. Gen. Microbiol. 93, 309–320.

is a key factor in such control, although it is predicted the integrated

Largeteau, M.L., Mata, G., Savoie, J.M., 2004. Verticillium fungicola var. fungicola

control using chemicals, biological and biotechnology approaches

affects Agaricus bisporus cultivation in Mexico. FEMS Microbiol. Lett. 236,

will begin to dominate. 191–196.

Largeteau, M.L., Savoie, J.M., 2008. Effect of the fungal pathogen Verticillium fungicola

on fruiting initiation of its host, Agaricus bisporus. Mycol. Res. 112, 825–828.

5. Conclusion

Largeteau, M., Savoie, J.M., 2010. Microbially induced diseases of Agaricus bisporus:

biochemical mechanisms and impact on commercial mushroom production.

There are some commercial strains of A. bisporus are more tol- Appl. Microbiol. Biotechnol. 86, 63–73.

North, L.H., Wuest, P.J., 1993. The infection process and symptom expression of

erant to the pathogen than others. Up to 76.5% yield loss may be

Verticillium disease of Agaricus bisporus. Can. J. Plant Pathol. 15, 74–80.

caused by the pathogen under experimental conditions. Among

Pardo-Giménez, A., Zied, D.C., Pardo-González, J.E., 2010. Utilización de compost

the 15 strains studied, only one strain, ABI 11/16 was the more agotado de champinón˜ como capa de coberturas en nuevos ciclos de producción.

Pesq. Agropec. Bras. 45, 1164–1171.

productive in the presence of the pathogen, even with unmar-

Schweikert, N., Hofmann, A., Schulz, M., Scheuermann, M., Boles, S.T., Hanemann, T.,

ketable diseased mushrooms. The only strain that showed “in vitro”

Hahn, H., Indris, S., 2013. Suppressed lithium dendrite growth in lithium batter-

mycelial growth similar to L. fungicola was ABI 09/10; however, its ies using ionic liquid electrolytes: investigation by electrochemical impedance

spectroscopy, scanning electron microscopy, and in situ Li nuclear magnetic

yield is not high and the experimental conditions resulted in a 64%

resonance spectroscopy. J. Power Sources 228, 237–243.

reduction in yield due to the presence of the pathogen. Difenocona-

Straatsma, G., Sonnenberg, A.S., van Griensven, L.J., 2013. Development and growth

zole more strongly inhibited the mycelial growth of L. fungicola than of fruit bodies and crops of the button mushroom, Agaricus bisporus. Fungal Biol.

117 (10), 697–707.

did iprodione; however, neither fungicide was selective against L.

Wuest, P.J., Cole, H., Sanders, P.L., 1974. Tolerance of Verticillium malthousei to beno-

fungicola.

myl. Phytopathology 64, 331–334.

Zied, D.C., Minhoni, M.T.A., Kopytowski Filho, J., Andrade, M.C.N., 2010a. Production

Acknowledgments of Agaricus blazei ss. Heinemann (A. brasiliensis) on different casing layers and

environments. World J. Microbiol. Biotechnol. 26, 1857–1863.

Zied, D.C., Minhoni, M.T.A., Pardo, J.E., Pardo, A., 2010b. A study of compost added

We would like to thank the Foundation for Research Support of to casing technique in Agaricus bisporus cultivation from Phase III bulk compost.

HortScience 45, 1649–1653.

the State of Minas Gerais (FAPEMIG – CAG/BPD 00081-11) and the

Zied, D.C., Pardo-González, J.E., Minhoni, M.T.A., Pardo-Giménez, A., 2011. A reliable

Foundation for Research Support of the State of São Paulo (FAPESP

quality index for mushroom cultivation. J. Agric. Sci. 3 (4), 50–61.

– 2012/14783-4) for ﬁnancial support.