Prvi izvještaj o bolesti zelene plijesni u Hrvatskoj

Green mould disease, caused by Trichoderma species, is a severe problem for mushroom growers worldwide, including Croatia. Trichoderma strains were isolated from green mould-affected Agaricus bisporus (button or common mushroom) compost and Pleurotus ostreatus (oyster mushroom) substrate samples collected from Croatian mushroom farms. The causal agents of green mould disease in the oyster mushroom were T. pleurotum and T. pleuroticola , similar to other countries. At the same time, the pathogen of A. bisporus was exclusively the species T. harzianum , which is different from earlier ﬁ ndings and indicates that the range of mushroom pathogens is widening. The temperature proﬁ les of the isolates and their hosts overlapped, thus no range was found that would allow optimal growth of the mushrooms without mould contamination. Ferulic acid and certain phenolic compounds, such as thymol showed remarkable fungistatic effect on the Trichoderma isolates, but inhibited the host mushrooms as well. However, commercial fungicides prochloraz and carbendazim were effective agents for pest management. This is the ﬁ rst report on green mould disease of cultivated mushrooms in Croatia.


Mushroom green mould disease
Mushrooms dominating commercial cultivation worldwide are Agaricus bisporus (button or common mushroom), Lentinula edodes (shiitake), and Pleurotus ostreatus (oyster mushroom) (1).Conditions under which these mushrooms are cultivated favour fast mould growth.Moulds compete for space and nutrients more effectively than the mushrooms and can produce secondary toxic metabolites, extracellular enzymes, as well as various volatile organic compounds (2,3), that can substantially lower or even entirely block commercial production.

Agaricus bisporus
The fi rst green mould epidemic was reported in Northern Ireland in 1985, quickly followed by outbreaks in several European countries (4)(5)(6)(7).In the early 1990s, a similar disease appeared in mushroom crops in the United States and Canada (8)(9)(10).Aggressive biotypes had originally been identifi ed as Trichoderma harzianum Th2 and Th4, but later they were re-described on the basis of morphological characteristics and of the phylogenetic analyses of the internal transcribed spacer (ITS) 1 region and the translation elongation factor 1-alpha (tef1) gene as new species T. aggressivum f. europaeum and T. aggressivum f. aggressivum, respectively (11).Agaricus green mould reported in Hungary seems to have been caused by Trichoderma aggressivum f. europaeum (12).

Status in Croatia
Similar to the neighbouring countries (Hungary, Serbia), the production of both A. bisporus and P. ostreatus in Croatia is affected by green mould infections.Though the disease generates serious production problems with significant economic consequences, its background has not been studied in Croatia until now.

Isolation of fungal strains
Trichoderma strains were isolated from green mould-affected samples.Compost and substrate samples were obtained from a farm in north-western Croatia growing button mushroom and a farm in central Croatia growing oyster mushroom.Button mushroom compost samples were collected in the summer and oyster mushroom in both the summer and the winter growing seasons.Two green mould-affected and two healthy samples were collected each time.Trichoderma infections appeared sporadically at both farms at the time of sampling.Trichoderma isolates were deposited in the culture collection of the Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb under MFBF codes.Host mushrooms (button and oyster) were isolated from healthy samples collected together with the infected ones.All strains were isolated and maintained according to Hatvani et al. (12).

Species identifi cation
DNA extraction, PCR-amplifi cation of the internal transcribed spacer (ITS) region, DNA sequencing, and sequence analysis were performed as described by Kredics et al. (16).

In vitro confrontation assays
The aggressiveness of the isolates towards their corresponding host mushrooms was tested in vitro in dual-plate assays according to Szekeres et al. (21).

Characterisation of the isolates
The effect of temperature, natural compounds, and commercial fungicides on the mycelial growth of two isolates from each species in comparison with their host mushrooms were tested on solid YEG medium (12).The fungi were inoculated onto the solid surface as mycelial plugs (4 mm diameter) taken from the actively growing edge of young colonies.Trichoderma, P. ostreatus, and A. bisporus colony diameters were measured after two days, one week, and two weeks, respectively.All experiments were repeated three times.

Isolation of fungal strains
Twenty Trichoderma strains were isolated from green mould-affected oyster mushroom substrate and twenty from button mushroom compost samples.

Species identifi cation
DNA was extracted from the isolates, and the ITS regions were amplifi ed by PCR.The amplicons were subjected to automatic sequencing (external service), and the DNA sequences analysed by TrichOKEY v. 2.0 software (22, www.isth.info).All the 20 isolates collected from the button mushroom compost were identifi ed as T. harzianum, while strains isolated from the oyster mushroom substrate included T. pleurotum and T. pleuroticola (9 and 11 isolates, respectively).Alignments (20) revealed that the ITS sequences of the isolates belonging to the same species did not share the same pattern.The differences suggested that they belonged to different strains within the species, but this did not affect the results of identifi cation.

Dual-plate assays
At the sampling sites we observed heavy colonisation of the button mushroom compost (Figure 1) and the oyster mushroom substrate by Trichoderma.The in vitro confrontation assays performed between the isolates and the colonies of button and oyster mushroom also revealed high aggressiveness of the Trichoderma strains towards their hosts.No signifi cant difference was found between the antagonistic activity of the isolates obtained from the same samples; all of them overgrew the mushroom colonies completely after four days of incubation and produced conidia on their surface.Figure 2 shows button mushroom inoculated as a single colony (A) and in confrontation with Trichoderma harzianum MFBF 10389 (B).

Characterisation of the isolates
As all Trichoderma isolates showed similarly high antagonistic activity towards their hosts, two strains of each species with different ITS types (see section "Species identifi cation" in results) were selected for further characterisation in comparison with the button and oyster mushroom, namely T. harzianum MFBF 10389 and 10390, T. pleuroticola MFBF 10383 and 10387, and T. pleurotum MFBF 10386 and 10388.
The temperature growth profi les of the Trichoderma isolates and their hosts were found to be highly overlapping, with optima between 25 °C and 30 °C. Figure 3 shows the temperature growth profi les of the button mushroom (A) and oyster mushroom pathogenic Trichoderma isolates (B) in comparison with their host mushrooms.
The effect of the 10 natural compounds on the mycelial growth of the Trichoderma isolates and the mushrooms was also tested.Thymol, ferulic acid, (+)-menthol, and (-)-menthol showed remarkable inhibitory effect on the Trichoderma isolates at low concentrations (between 0.08 mg mL -1 and 1.25 mg mL -1 , Table 1).However, at these concentrations they entirely blocked the growth of the mushroom strains as well.
Among the commercial fungicides tested, tebuconazol was more inhibitory to mushrooms than to the Trichoderma isolates.Prochloraz and carbendazim showed promising features for controlling green mould disease in cultivated button and oyster mushrooms, as they inhibited the growth of the examined Trichoderma strains even at low concentrations without affecting their hosts (Table 2).Based on the higher MIC, T. harzianum isolates were more tolerant to all fungicides tested than the Pleurotus-pathogenic species (T.pleurotum and T. pleuroticola).

DISCUSSION
In this study, the causal agents and potential means of disease control of mushroom green mould were examined based on Croatian samples.Oyster mushroom green mould disease was caused by T. pleurotum and T. pleuroticola, which is in accordance with fi ndings from other countries (16)(17)(18)(19)(20).At the same time, in the cultivation of button mushroom the sole isolated pathogen was T. harzianum.These results add a new name to the list of the potential mushroompathogenic Trichoderma species, as earlier studies from other countries identifi ed only T. aggressivum as the button mushroom pathogen (11,12).This fi nding suggests a continuous evolving of green mould disease in cultivated mushrooms and underlines the importance of monitoring these infections.
In order to fi nd proper means of disease control, we investigated the effects of temperature, natural compounds, and commercial fungicides on pathogen and host mycelial growth.Similar to an earlier report by Woo et al. (15), the temperature profi les of the pathogens and their hosts highly overlapped (Figure 3), showing no room for effective disease control.Green mould isolates tolerated most of the natural compounds even at concentrations above 10 mg mL -1 .However, thymol, ferulic acid, (+)-menthol, and (-)-menthol inhibited their growth at concentrations  as low as 0.08 mg mL -1 to 1.25 mg mL -1 (Table 1).These results show that phenols have a remarkable inhibitory effect on the growth of green mould isolates.Notable differences were observed between species and isolates in their susceptibility to ferulic acid and thymol.Šegvić Klarić et al. (23) showed the inhibitory effect of thymol on the growth of moulds, including Trichoderma spp., even at very low concentrations (MIC 1.6 μg mL -1 to 6.72 μg mL -1 ).In contrast, our Trichoderma isolates tolerated this compound at much higher concentrations (0.08 mg mL -1 to 0.32 mg mL -1 ).However, as thymol, ferulic acid, and menthol blocked the growth of the host mushroom as well, their use in pest management is not possible.
Of the commercial fungicides tested, prochloraz and carbendazim proved to be effi cient in inhibiting the green mould isolates at very low concentrations (0.63 μg mL -1 to 5 μg mL -1 ) and did not infl uence the growth of their host mushrooms (Table 2), which is similar to fi ndings reported by Woo et al. (15) in oyster mushroom.Therefore they might be considered as potential chemical control agents to prevent or stop the spreading of mushroom green mould disease.

Figure 2
Figure 2 Button mushroom inoculated as a single colony (A) and in confrontation with Trichoderma harzianum MFBF 10389 (B)

Figure 3
Figure 3 Temperature growth profi les of button mushroom (A) and oyster mushroom pathogenic Trichoderma isolates (B) and their hosts (colony diameters in mm)

Table 1 Minimal inhibitory concentrations (MIC, mg mL -1 ) of natural compounds on the Trichoderma pathogens in comparison with control oyster and button mushrooms Compound Nature T. pleuroticola MFBF 10383 T. pleuroticola MFBF 10387 T. pleurotum MFBF 10386 T. pleurotum MFBF 10388 T. harzianum MFBF 10389 T. harzianum MFBF 10390 Oyster mushroom Button mushroom
PP: polyphenol, PA: phenolic acid, F: fl avonoid, P: phenol, MFBF: the number of strains from the Collection of Microorganisms, Department of Microbiology, Faculty of Pharmacy and Biochemistry University of Zagreb, NT: not tested

Table 2
Minimal inhibitory concentrations (MIC, μg mL -1 ) of commercial fungicides on the Trichoderma pathogens in comparison with control oyster and button mushrooms MFBF: the number of strains from the Collection of Microorganisms, Department of Microbiology, Faculty of Pharmacy and Biochemistry University of Zagreb