FUNGICIDE SENSITIVITY OF SELECTED VERTICILLIUM FUNGICOLA ISOLATES FROM AGARICUS BISPORUS FARMS

Five isolates of Verticillium fungicola, isolated from diseased fruiting bodies of Agaricus bisporus collected from mushroom farms in Serbia during 2002-2003, were studied. By observing their colony morphology under different growth conditions and their pathogenic characteristics, the isolates were identified as V. fungicola var. fungicola. the peat/ lime casing was the primary source of infection. testing of sensitivity to selected fungicides showed that all isolates were highly resistant to benomyl (Ec50 values were higher than 200.00 mg/l), moderately sensitive to iprodione (Ec50 values were between 11.93 and 22.80 mg/l), and highly sensitive to prochloraz-Mn (Ec50 values were less than 3.00 mg/l).


IntroductIon
Agaricus bisporus (Lange) Imbach is the most commonly cultivated mushroom species (r o y s e , 1996). the production of fruiting bodies is severely afflicted by fungal, bacterial, and viral pathogens that can cause diseases which have an effect on yield and quality.Verticillium fungicola (Preuss) Hassebrauk, with two varieties, fungicola, widespread in Europe, and aleophilum, widespread in north America, is a major A. bisporus pathogen and a causal agent of the disease commonly known as "dry bubble".A. bisporus -V.fungicola interaction is of an invasive necrotrophic nature.the disease severity and symptoms depend on the stage of mushroom development at the time of infection (n o r t h and Wu e s t , 1993, G r o g a n et al., 2000), and the symptoms are necrotic lesions with brown colored spots or streaks, stipe blow-out, and undifferentiated structures containing mycelia of both host and pathogen (S a v o i e and L a r g e t e a u , 2004).

Isolates and growth conditions
Isolates of V. fungicola obtained from diseased fruiting bodies of A. bisporus collected during 2002-2003 in Serbia are shown in table 1. Isolation was done by taking small pieces (2 x 2 x 5 mm) of fruiting bodies with "dry bubble" symptoms, immersing them a 1% sodium hypochlorite solution (for 1 min), and placing them on Potato dextrose agar medium (PdA).the isolates are maintained on PdA at 5 o c in the culture collection of the Pesticide and Environmental research center's Phytopathology Laboratory in Belgrade.Verticillium fungicola var.fungicola 182 (obtained from the collection of Horticulture research International, Wellesbourne, uK) and V. fungicola var.aleophilum dc-170 (obtained from the Plant Pathology department, Penn State university, uSA) were used as controls.
the morphology of colonies of all V. fungicola isolates was studied by observing their growth on PdA, malt agar medium (MA), mushroom dextrose agar medium (MdA), water agar medium (WA), and czapek agar medium (czA) for 7 days at 20 o c. the dimensions of 30 conidia per isolate formed on PdA were measured and compared.temperature influence was investigated on V. fungicola isolates cultivated on the optimal medium for 7 days at 20, 27 and 30 o c. three replicates per treatment and per isolate were used for statistical analysis.

Pathogenicity test
Spawn-run compost (A.bisporus Sylvan A 15), produced by uca & co., Vranovo, Serbia, was used for the pathogenicity test.compost bags were encased with a 40 -50-mm layer of black peat/lime casing ("Makadam" co., Belgrade), which was artificially inoculated with the studied isolates of V. fungicola.the casing inoculation was done by spore suspension spraying (approximately 10 6 conidia/ml) 3 days after encasement.Bags were incubated at 25 o c during spawn-running of the casing (for 7 days), after which temperature was lowered to 18 o c. the inoculated black peat/lime casing was removed and replaced with new sterile one 30 days after the first encasement.

Testing of sensitivity to selected fungicides
Sensitivity analysis was done with isolates grown on PdA amended with the following fungicides: benomyl (Benfungin WP, 50%, Galenika-Fitofarmacija); iprodione (Kidan Ec, 25.5%, Bayer); and prochloraz-Mn (octave WP, 50%, Bayer).the preliminary concentrations of all selected fungicides were 0.01, 0.10, 1.00, 10.00, 100.00, and 1000.00 mg/l.testing of sensitivity to iprodione and prochloraz-Mn was repeated with concentrations of 0.1, 0.5, 1.5, 10.0, 50.0, and 100.0 mg/l; and 0.078, 0.156, 0.312, 0.625, 1.250, 2.500, and 5.000 mg/l, respectively.the plates were inoculated with an inverted mycelial agar disk (10 mm) taken from the edge of 14-day-old culture of V. fungicola var.fungicola isolates, placed centrally on fungicide-amended and fungicide-free medium (control) and incubated at 20 o c. three replicates per treatment and per isolate were done.colony diameter was measured after 7 days of growth.Growth of colonies on the fungi-  cide-amended medium was given as a percentage of the control.Ec 50 and Ec 90 (fungicide concentrations which inhibit mycelial growth by 50 and 90%, respectively) were determined for each isolate by interpolation from computer-generated log-probit plots of fungicide concentration and relative inhibition (Leroux and Gredt, 1972).the effect of fungicides was studied by analyzing means and variance of Ec by the multiple range test (Finney, 1964).rESuLtS

Symptoms of "dry bubble" disease
Single or clusters of undifferentiated fruiting bodies of A. bisporus with symptoms similar to those caused by V. fungicola were observed in screening of mushroom farms in Serbia during 2002-2003.Large brown spots with a fuzzy grayish tint were noticed on fully differentiated fruiting bodies after 12 days (Fig. 1), and undifferentiated fruiting bodies were found 18 days after artificial inoculation of casing with the pathogen (Fig. 2).

Identification of isolates identification
the following morphological characteristics were recorded in the analyzed isolates: dense white aerial mycelia; absence of pigment production; hyaline, erect conidiophores with groups of divergent phialides of verticilliate form; hyaline, cylindrical phialides with slightly inflated base and acute tip; and hyaline, unicellular, ellipsoid to cylindrical conidia produced in a gelatinous matrix.conidia in all studied isolates measured 2.95-7.38 x 1.97-2.46µm.After comparing our isolates with control isolates, we concluded that all Serbian isolates were V. fungicola var.fungicola.
the diameter of colonies of all isolates after 7 days of growth showed significant differences depending on composition of the medium (Fig. 3), ranging between 10.25 mm on MdA (ViV 1 ) and 14.17 mm on PdA (P 2 V 3 ).
Serbian as well as uK V. fungicola isolates grew only at 20 o c; growth was absent at 27 and 30 o c.However, V. fungicola var.aleophilum dc-170 grew at all investigated temperatures, the optimal one being 27 o c.

Pathogenicity test
Fruiting bodies with split stems and grayishbrown spots on the whole surface of the cap were noticed 12 days after inoculation of the casing with isolates VV 2 and P 2 V 3 ; and 13 days after inoculation with isolates ViV 1 , raV 1, and BeV 1 (Fig. 1).undifferentiated masses of fruiting bodies with a dry surface covered with a dusty gray layer of conidia were observed on the 18 th day after inoculation with isolates VV 2 and P 2 V 3 ; on the 19 th day after inoculation with ViV 1 and raV 1 ; and on the 20 th day after inoculation with BeV 1 (Fig. 2).However, fruiting bodies with "dry bubble" symptoms were not recorded after replacing the infected casing with a sterile new one.

Testing of sensitivity to selected fungicides
In these in vitro investigations of the sensitivity of V. fungicola var.fungicola isolates to the selected fungicides, all studied isolates showed high resistance to benomyl (Ec 50 values were between 234.55 to 359.95 mg/l); moderate sensitivity to iprodione (Ec 50 values were in the range from 11.93 to 22.80 mg/l); and high sensitivity to prochloraz-Mn (Ec 50 values were in the range from 1.11 to 2.51 mg/l); (table 2).dIScuSSIon to judge from taxonomic criteria based on the optimal growth temperature (G a m s and Va n Z a a y e n , 1982; n a i r and M a c a u l e y, 1987), all V. fungicola isolates from Serbian A. bisporus farms were V. fungicola var.fungicola.G a m s and Va n Z a a y e n (1982) emphasized V. fungicola varieties differ significantly with respect to the optimal temperature for mycelial growth (20-24 0 c for var.fungicola and 30 0 c for var.aleophilum).B o n n e n and H o p k i n s (1997) observed a high level of homogenicity in colony morphology, virulence, and fungicide response among analyzed V. fungicola isolates from north America and placed them in the same rAPd group.But in investigations of hydrolytic enzyme production and genetic variability between two  , 2006).contrary to the situation with var.aleophilum, L a r g e t e a u et al. ( 2006) showed the presence of significant differences of physiological and pathogenicity traits among var.fungicola isolates.
the noted symptoms on A. bisporus fruiting bodies caused by V. fungicola var.fungicola isolates on Sebian farms were similar to ones previously described on farms worldwide (n a i r n and M a c a u l e y, 1987; S t a u n t o n and d u n n e , 1990; n o r t h and Wu e s t , 1993; S a v o i e and L a r g e t e a u , 2004). the results of this study confirm the results of Wo n g and P r e e c e (1987), and n o r t h and Wu e s t (1993), who showed the peat/ lime casing to be the primary source of V. fungicola, and that the earliest possible infection occurred during the encasement period, but not before, because conidia which were present in spawned compost were not able to cause development of the disease.using electron microscopy, d r a g t et al. (1996) showed that V. fungicola grows both outside and inside the hyphae of A. bisporus fruiting bodies, and emphasized that the pathogen penetrates host chitin cell walls by the combined effect of mechanical pressure and wall-lytic enzymes.M i l l s et al. (2000) isolated and identified β-1-6-glucanases, chitinases, serine proteinase, stearase, and esterase from culture filtrates of V. fungicola grown in the presence of A. bisporus cell wall, and A t h e y -P o l l a r d et al. ( 2003) isolated the cap-binding protein (eIF4E) from A. bisporus and V. fungicola and described its gene nucleotide and amino acid composition.
Agaricus species can protect themselves from V. fungicola invasion by production of extracellular phenoloxidases, H 2 o 2 , and antibiotics (L a r g et e a u et al., 2006; S c o r e et al., 1997; S a v o i e et al., 2004), but efficiency of self-defense depends on the level of resistance of Agaricus species (and even strainstrains) to V. fungicola, as well as on sensitivity of the pathogen to host metabolites (S a v o i e and L a r g e t e a u , 2004; d r a g t et al., 1995; J a u r e z d e l c a r m e n et al., 2002).Thus, G e a et al. (2003) noted that 26-47% of fruiting bodies on A. bisporus farms but only 4-12% on A. bitorquis farms were infected, while S a v o i e and L a r g e t e a u (2004) showed that the majority of V. fungicola isolates were susceptible to lower H 2 o 2 concentration.In recent decades, a common method of pathogen control on farms worldwide is application of various fungicides.For improvement of crop protection and reduction of production costs, the effects of some new fungicides are being tested.However, fungicide efficiency depends on frequency of usage (B o n n e n and H o p k i n s , 1997), as well as on the persistence of fungicides in high concentrations in the casing during cultivation (G r o g a n and J e k e s , 2003).
According to the criteria established by G e a et al. (2003,2005), V. fungicola isolates from Serbian A. bisporus farms were highly resistant to benomyl, moderately sensitive to iprodione, and highly sensitive to prochloraz-Mn.B o n n e n and H o p k i n s (1997) also showed absence of benomyl sensitivity in isolates obtained after the year 1979.However, contrary to the situation with Serbian V. fungicola isolates, Spanish isolates were resistant to iprodione with Ec 50 values higher than 50.00 mg/L (G e a et al., 1997).In the case of resistance to prochloraz-Mn, the picture is very different from strain to strain of V. fungicola.to be specific, 70% of pathogen isolates from Great Britain and Spain were moderately sensitive to prochloraz-Mn with Ec 50 values ranging from 5.0 to 8.0 mg/L (G r o g a n et al., 2000; G e a et al., 2003), and some farms reported unsatisfactory levels of control by that fungicide, which was explained by the fact that resistance was developed with the passage of time (G e a et al. 2005).Those authors analyzed 105 V. fungicola var.fungicola isolates from Spanish mushroom crops collected in the period between 1992 and 1999 and demonstrated that their resistance ranged from low (Ec 50 value of 0.8 mg/l) in 1992 to moderate (Ec 50 value of 8.8 mg/l) in 1998.In the case of isolates from 1999, 29.86% were sensitive (Ec 50 value of 5.0 mg/l) and 14% slightly tolerant (Ec 50 values equal to or above 5.0 mg/l), while 60% grew at a fungicide concentration of 50.0 mg/l and 40% at 100.0 mg/l.B e r n a r d o et al. (2002,2004) noted that prochloraz-Mn alters structure of the cell wall, as well as the ratio of cell wall components and their structure in both V. fungicola and A. bisporus, which can be attributed to the fungicide's inhibitory effect on sterol biosynthesis.d i a m a n t o p o u l o u et al. ( 2006) confirmed the adverse effects of fungicides in testing of tebuconazole, a new fungicide, which caused pileus deformations and severe reduction of total yield at a concentration of 0.8 g/m 2 and deviation in sporophore color at a concentration of 1.2 g/m 2 .owing to everything mentioned above and despite the existence of some efficient fungicides for V. fungicola control, special attention is now being paid to genetic reduction of pathogen virulence by generation of mutants with diminished ability to utilize chitin as a carbon source (A m e y et al., 2003), as well as to the possibility of using natural products (such as essential oils of different plants) to inhibit pathogen activity (S o k o v i ć et al., 2006).
ethyl)) carbamoylimiazole) (G e a et al., 1997).However, development of pathogen resistance to fungicides after frequent application (B o n n e n and H o p k i n s , 1997; G e a et al., 1997, 2003; G r o g a n et al., 2000) and host sensitivity to fungicides (d i a m a n t o p o u l o u et al., 2006) are a serious problems.