Monitoring of bacterial diseases of Agaricus bisporus in Serbia

Monitoring of button mushroom bacterial diseases was conducted to estimate the presence and identity of mycopathogenic bacteria and to determine the predominant bacterial pathogen in Serbia. Samples were collected from mushroom farms during 20062010 and also from fresh markets during 2014-2015. The collected samples showed either symptoms of brown blotch or different degrees of brown discoloration on caps and stalks of Agaricus bisporus resembling bacterial infection. The presence of bacterial droplets on gills was not recorded. The isolated bacteria were Gram-negative and fluorescent on King’s medium B. In pathogenicity tests, most bacterial isolates induced superficial or sunken brown lesions with differences in the level of discoloration on A. bisporus tissue blocks after artificial inoculation. Based on LOPAT characteristics, the isolates were divided into two groups, showing characteristics of either the LOPAT group Va or group III. Based on these features and other differential biochemical characteristics, the presumptive Pseudomonas tolaasii isolates were confirmed by specific PCR. The other group of isolates was subjected to sequencing of the 16S rDNA. Based on these sequences most isolates were identified as Pseudomonas agarici, while two strains belonged to Pseudomonas fluorescens. The survey resulted in detection and identification of P. tolaasii in 11 locations and P. agarici in 7 locations in Serbian mushroom farms. Most samples from fresh markets were infected with P. tolaasii, suggesting that this pathogen has been the predominant cause of bacterial diseases in Serbian mushroom-growing facilities over the past 10 years.


IntroductIon
Button mushroom (Agaricus bisporus (Lange) Imbach) is one of the most common cultivated edible mushrooms in Serbia with an annual yield of around 9,000 t.Its production is mostly intended for fresh market, but although market demands are increasing, it is still at the level of small-scale growers (Milijašević-Marčić et al., 2012).A variety of harmful organisms may influence the yield and quality of this crop.Among them, mycopathogenic bacteria are considered less important in economic terms than fungi or viruses, but they are still a concern because the means of their control are limited.There are several fluorescent pseudomonads with an ability to cause diseases of A. bisporus (Godfrey et al., 2001;Munsch et al., 2002;Iacobellis, 2011).Among them, Pseudomonas tolaasii (Paine, 1919;Tolaas, 1915) is the most common bacterial pathogen inducing darkbrown, sunken lesions on caps and stalks, and a resulting decrease in yield and quality, and shortened shelf life.Moreover, brown blotch symptoms on A. bisporus appearing as light-brown discoloration may also be caused by P. reactans (Wells et al., 1996;Iacobellis & Lo Cantore, 1997;2003;Munch et al., 2002), Pseudomonas sp.strain NZ17 apparently related to P. syringae (Godfrey et al., 2001) and P. costantinii sp.nov.(Munch et al., 2002).The ginger blotch disease of A. bisporus is caused by P. gingeri (Wong & Preece, 1979) and the drippy gill of A. bisporus is caused by P. agarici (Young, 1970).In addition, Godfrey et al. (2001) and Iacobellis (2011) reported that a number of diverse pseudomonad species other than the known 'blotch-causing organisms' (P.tolaasii, P. gingeri and P. reactans) also have the ability to cause blotch diseases with various discolorations, and appear to participate in the expression of disease symptoms.
Unlike fungal pathogens, which have been thoroughly studied in Serbian mushroom farms (Potočnik et al. 2008(Potočnik et al. , 2009(Potočnik et al. , 2010(Potočnik et al. , 2015;;Kosanović et al., 2013), bacterial pathogens in A. bisporus had not been studied before 2006, when an infection with Pseudomonas agarici was reported for the first time in cultivated mushrooms in Serbia (Obradovic et al., 2006).Subsequently, symptoms resembling brown blotch were observed on several mushroom farms and Pseudomonas tolaasii was reported for the first time by Milijašević-Marčić et al. (2012).After these findings, an extensive survey of button mushroom bacterial diseases both in farms and on fresh markets was conducted to estimate the presence and identity of mycopathogenic bacteria and to identify the predominant bacterial pathogen in Serbia.

MaterIalS and MethodS
Samples of diseased button mushroom were collected from mushroom farms and fresh markets in 18 locations within a ten-year period (Table 1).Bacteria were isolated from A. bisporus fruiting bodies with bacterial blotch symptoms or different degrees of brown discoloration on caps and stalks following the method of Lelliott and Stead (1987).Presumptive colonies which produced a green fluorescent pigment were further purified by subculturing.For short term use, all strains were maintained on King's medium B (KB) slopes at 4°C, and for long term storage they were preserved at −80°C in KB liquid medium with a final concentration of 20% (v/v) glycerol in a culture collection of the Institute of Pesticides and Environmental Protection, Belgrade.
Pathogenicity tests were carried out using healthy, oneday-old A. bisporus whole sporophores and cubes of cap tissue excised with a sterile scalpel.For both trials the tissue was placed into sterile Petri dishes containing filter paper dampened with sterile distilled water.Bacterial isolates and reference strains of P. tolaasii (CFBP 2068 T ) and P. agarici (CFBP 2063 T ) were cultured on solid KB medium for 24 hours and then suspended in sterile distilled water to reach the density of approximately 1×10 8 CFU ml −1 .Negative control sporophores/cubes were treated with sterile distilled water only (Munsch et al. 2000;Lo Cantore & Iacobellis, 2004).The inoculated mushroom tissue was incubated in a humid chamber at room temperature.Tissue degradation and discoloration of the cubes were recorded after 72 h.All bioassays were repeated in triplicate.
Molecular identification was conducted to confirm the identification of P. tolaasii isolates according to a PCR protocol described by Lee et al. (2002) using the primer pair Pt-1A, Pt-1D1 specific for its detection.The reference strain of P. tolaasii CFBP 2068 T was used as positive control.Another group of isolates was identified based on analysing the 16S rDNA sequence.A PCR assay with the universal primers 27F and 1541R was performed for amplification of the bacterial 16S rDNA (Edwards et al., 1989;Lane, 1991).Positive amplicons were sequenced in forward direction with the 27F primer.Sequencing was performed by a commercial service (Macrogen Inc., S. Korea).The obtained sequences were assembled using Pregap4 from the Staden program package (Staden et al., 2000).

reSultS and dIScuSSIon
Our monitoring of bacterial diseases in mushroom farms and fresh markets in Serbia revealed the presence of symptoms of brown blotch or different degrees of brown discoloration on fruiting bodies of A. bisporus (Figures 1  and 2).Over a hundred bacterial isolates were obtained after isolation from symptomatic samples.Forty-five green fluorescent, Gram negative isolates from different sources and locations were selected for pathogenicity tests.The bacterial isolates showed different degrees of discoloration and tissue degradation on the mushroom cubes, varying from light to dark brown (Figure 3).Thirty-six bacterial isolates caused sunken brown lesions on A. bisporus cap tissue blocks after 72 h, consistent with those caused by the reference strain of P. tolaasii.The remaining isolates showed light brown superficial discoloration both on tissue blocks and sporophores similar to the P. agarici reference strain.Based on the studied biochemical properties, all tested bacterial isolates could also be divided into two groups.Those that showed LOPAT characteristics of the group Va also had the following biochemical properties: catalase positive, aesculin hydrolysis negative, Tween 80 hydrolysis positive, casein hydrolysis positive; gelatine hydrolysis and nitrate reduction negative; they used mannitol, erythritol, sorbitol, inositol and trechalose as carbon sources, but did not use sucrose, arabinose or D(-)tartrate, showing characteristics of P. tolaasii (Table 2).Their identity was confirmed in PCR tests.The other group of isolates (7), as well as the reference strain of P. agarici, showed LOPAT characteristics of the group III, and did not utilize sorbitol, erythritol, L-arabinose, L-rhamnose, L-arabitol, 2-ketogluconate or D-tartrate (Table 2).The identity of these seven isolates was confirmed based on analyses of the 16S rDNA sequence as P. agarici (deposited in the NCBI GenBank under the accession numbers: KX261220; KX261221; KX261222; KX261223; KX261224; KX261225 and KX261226 for strains Poz 8/7; Jak 4; OP-6; Pad-4; Veg2; Vg 1 and Vin 6, respectively).However, the remaining two bacterial isolates showed 99 or 100% similarity with P. fluorescens.Generally, the results of this survey showed that P. tolaasii was the predominant bacterium associated with brown discoloration symptoms on A. bisporus, which is not unexpected considering that P. tolaasii has been recognized as the most common button mushroom pathogen inducing considerable losses in the cultivation process.However, recent studies have pointed out that brown blotch of A. bisporus, caused by P. tolaasii, is a complex disease since some other pseudomonad species also have the ability to cause blotch symptoms with various levels of discolorations (Godfrey et al., 2001;Iacobellis, 2011).Moreover, the pathogenicity tests in our study also showed differences in the level of discoloration of the inoculated mushroom cap tissue, implying that some other fluorescent pseudomonads participated in the expression of disease symptoms.On the other hand, P. agarici was detected and identified in seven different locations from 2006-2010.Further monitoring in fresh markets did not show repeated isolation of this bacterium.It is also noteworthy that typical drippy gill symptoms were not observed in samples infected with P. agarici.The same observation had been made by Geels et al. (1994) and Lo Cantore and Iacobellis (2004), suggesting a pathogen ability to induce different symptoms in various environmental conditions.
In regard to the economic impact of P. tolaasii as the predominant bacterial pathogen in Serbian mushroom farms, it mostly depends on the phytosanitary measures conducted in growing facilities.Although small growers are still prevailing, they are willing to introduce and apply strict sanitary measures and novelties in mushroom growing techniques.

concluSIonS
The survey resulted in the detection and identification of P. tolaasii in 11 locations and P. agarici in seven locations of Serbian mushroom farms.Most samples from fresh markets were infected with P. tolaasii, suggesting that this pathogen has been the predominant cause of bacterial diseases in Serbian mushroom-growing facilities over the past 10 years.(Schaad et al., 2001;Lo Cantore and Iacobellis, 2004).

acknowledgeMent
This study was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia as part of grant TR 31043.

Figure 1 .
Figure 1.Sunken brown lesions on A. bisporus caps induced by P. tolaasii

Figure 3 .
Figure 3. Superficial and sunken brown lesions with differences in the level of discoloration on A. bisporus tissue blocks after artificial inoculation-pathogenicity tests

Table 1 .
Results of monitoring of bacterial pathogens in A. bisporus in Serbia