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Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields

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Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields
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  Development of a selective myclobutanil agar(MBA) medium for the isolation of  Fusarium  species from asparagus fields Vladimir Vujanovic, Chantal Hamel, Suha Jabaji-Hare, and Marc St-Arnaud Abstract : A new selective myclobutanil agar medium for the detection of   Fusarium  species is proposed. Ten media for-mulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, malachite green, sodiumhypochlorite, captan, benomyl, chlorotalonil, myclobutanil, thiram, and cupric sulfate) were compared. First, myceliumgrowth and colony appearance of   Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides ,  Epicoccumnigrum ,  Fusarium  sp.,  Fusarium solani ,  Fusarium moniliforme ,  Fusarium oxysporum  f.sp.  dianthi ,  Penicillium  sp., and Trichoderma viride  isolates were compared. Second, the ability of the different media to isolate and enumerate fusariafrom asparagus fields was evaluated. The myclobutanil-based medium showed the highest selectivity to  Fusarium spp. growth but required a slightly longer incubation time (>5 d) than peptone–pentachloronitrobenzene-based agar (PPA)(< 5 d). PPA allowed a faster fusaria growth but also permited the growth of other moulds. The other media were less se-lective and did not allow to isolate fusaria or to differenciate them from other growing fungi. Key words : selective medium, myclobutanil,  Fusarium , soil,  Asparagus . Vujanovic et al. Résumé  : Nous proposons un nouveau milieu sélectif au myclobutanil (MBA) pour la détection des espèces de Fusarium . Dix formulations basées sur différents ingrédients sélectifs (pentachloronitrobenzene (PCNB), Rose Bengal,vert de malachite, hypochlorite de sodium, captan, benomyl, chlorotalonil, myclobutanil, thiram et sulfate de cuivre)ont été évaluées. Premièrement, la croissance du mycélium et l’apparence des colonies d’isolats d’  Alternaria alternata ,  Aspergillus flavus ,  Cladosporium cladosporioides ,  Epicoccum nigrum ,  Fusarium  sp.,  Fusarium solani ,  Fusariummoniliforme ,  Fusarium oxysporum  f.sp.  dianthi ,  Penicillium  sp. et de  Trichoderma viride  ont été comparées.Deuxièmement, la capacité des différents milieux à isoler et permettre le dénombrement des  Fusarium  spp. à partir dechamps d’asperges a été évaluée. Le milieu à base de myclobutanil a montré la plus grande sélectivité pour lacroissance des  Fusarium  mais il nécessitait une période d’incubation légèrement plus longue (>5 j) que le milieugélosé à base de pentachloronitrobenzène et de peptone (PPA) (<5 j). Par contre, le PPA a permis une croissance des Fusarium  plus rapide mais il a permis aussi la croissance d’autres champignons. Les autres milieux se sont avérésmoins efficaces pour isoler sélectivement les  Fusarium  et permettre leur distinction des autres espèces de champignonscroissant simultanément.  Mots clés  : milieu sélectif, myclobutanil,  Fusarium , sol,  Asparagus . 847 Introduction Asparagus (  Asparagus officinalis  L.) is a high-value andlong-term perennial crop with a production cycle of up to15–25 years and a field’s peak production after 5–8 years.The soilborne  Fusarium  spp. are among the most economi-cally important pathogenic fungi (Nelson et al. 1983) andcausal agents of the most serious asparagus diseases harm-ing plantations in North America (Elmer et al. 1996). In Canada, the asparagus crop suffers from a severe Fusariumcrown and root rot that frequently compromises the produc-tion profitability. The interest in investigating abundanceand population structure of   Fusarium  species increased inNorth America in the last decade (Elmer 2001), as thisknowledge is essential to develop biologically based controlmeasures and sustainable cultural practices for asparagusproduction.At least 12 different fusaria have been isolated from as-paragus, but only  Fusarium oxysporum  f.sp.  asparagi , Fusarium proliferatum , and  Fusarium culmorum  have been Can. J. Microbiol.  48 : 841–847 (2002) DOI: 10.1139/W02-082 © 2002 NRC Canada 841 Received 8 May 2002. Revision received 3 September 2002. Accepted 5 September 2002. Published on the NRC Research PressWeb site at http://cjm.nrc.ca on 7 October 2002. V. Vujanovic 1 and M. St-Arnaud.  Institut de recherche en biologie végétale, Département de sciences biologiques, Université deMontréal and Jardin botanique de Montréal, 4101 East, Sherbrooke Street, Montréal, QC H1X 2B2, Canada. C. Hamel.  McGill University, Department of Natural Resource Sciences, 21111 Lakeshore Road, Ste-Anne-de-Bellevue,QC H9X 3V9, Canada. S. Jabaji-Hare.  McGill University, Department of Plant Science, 21111 Lakeshore Road, Montréal, QC H9X 3V9, Canada. 1 Corresponding author (e-mail: vujanovv@magellan.umontreal.ca).  recognized as causal agents of several symptoms, such asdwarf asparagus, wilt and root rot, seedling blight, foot rot,and stem and crown rot (Elmer 2001). In Canada, the Fusarium  complex involved in the asparagus diseases hasnot been well characterized. Hence, a study of diversity of  Fusarium  populations in asparagus fields within the Quebecgrowing areas in eastern Canada has been undertaken(Vujanovic et al. 2001). For this purpose, fusaria isolation,enumeration from soil and plant parts, and verification of inoculum in field soil became essential.A number of culturing techniques have been developed forthe detection and enumeration of   Fusarium  spp. in plants, soil,and agricultural products (Bullerman and West 1992). Themost commonly used media to isolate  Fusarium  from agricul-tural fields and food are the peptone–pentachloronitrobenzene(PCNB)-based agar (PPA) and the Komada – Rose Bengal-based medium, respectively (Denis et al. 1966; Thrane1999). Recently, the malachite green agar (MGA) was addedto the list of general, selective media to isolate fusaria(Castellá et al. 1997 a ). Although these selective media aresuccessful in isolating  Fusarium  species, there are certaindrawbacks, such as the carcinogenic nature of PCNB (Sweet1986; Castellá et al. 1997 b ) and the fungicidal properties of malachite green and Rose Bengal when exposed to light(Thrane 1996) that affect the growth of   Fusarium  species.Consequently, it was considered important to screen for vari-ous antimicrobial compounds to develop efficient selectivemedia for colony enumeration of   Fusarium  spp.The objectives of the present work were ( i ) to compareseveral ingredients under standardized conditions for theirselectivity for a representative range of fungi frequently iso-lated from soil, including  Fusarium  and ( ii ) to evaluate theireffectiveness in the isolation and distinction of   Fusarium taxa from other soil-inhabiting fungi encountered in aspara-gus fields. Materials and methods Fungal strains and media Initial selections to test fungal tolerance towards anti-microbial agents included the following four  Fusarium  spe-cies (three from the Herbier Marie-Victorin (MT) fungalcollection and one from the American Type Culture Collec-tion (ATCC) fungal collection): Fs,  Fusarium solani  (Mart.)Sacc. (MT 11583); Fm,  Fusarium moniliforme  J. Sheld. (MT11021); Fod,  F. oxysporum  Schlechtend.: Fr. f.sp.  dianthi (Prill. & Declacr.) W.C. Snyder & H.N Hans (ATCC 66048);Fsp,  Fusarium  sp. (MT 11832). Also included were the fol-lowing six mitosporic fungi isolated from asparagus fields inQuébec, eastern Canada: Aa,  Alternaria alternata  (Fr.:Fr.)Keissler; Af,  Aspergillus flavus  Link:Fr.; Cc,  Cladosporiumcladosporioides  (Fresen.) G.A. De Vries; En,  Epicoccumnigrum  Link.; Psp,  Penicillium  sp.; and Tv,  Trichodermaviride  Pers (MT:12345–12351).Ten different selective medium formulations were screenedin initial trials. The basic formula used to prepare the mediawas the Nash and Snyder (1962) medium base (NSMB:peptone 20 g, KH 2 PO 4  1 g, MgSO 4 ·7H 2 O 0.5 g, agar 20 g,distilled water 1 L) supplemented with antibiotics (100 mg/Lstreptomycin sulphate and 12 mg/L neomycin sulphate;Sigma, St. Louis, Mo. U.S.A.). Each of the ten specificantimicrobial agents tested was added to a separate 1 Lbatch of the basic formula, as described by Burgess et al.(1994), who proposed the PPA medium containing NSMBplus 1 g/L PCNB. Media were prepared by adding to theNSMB one active ingredient in the following concentrationsper litre of media: ( i ) 25 mg malachite green (Sigma),( ii ) 50 mg Rose Bengal (Sigma), ( iii ) 1.2 mg sodiumhypochlorite, ( iv ) 2.5 mg chlorothalonil (Daconil 2787 ® ,Zenca Agro Inc., Calgary, Alta.), ( v ) 2 g captan (Maestro75 ® , Tomen Agro Inc., Toronto, Ont.), ( vi ) 160 mg benomyl(Benlate 50WP ® , DuPont Canada Inc., Mississauga, Ont.),( vii ) 200 mg myclobutanil (Nova 40WP ® , Rohm and HassCanada, Brossard, Qué.), ( viii ) 1 g thiram (Thiram 80WP ® ,Plant Products Co. Ltd., Brampton, Ont.), ( ix ) 2 g cupric sul-fate (CuSO 4 ·5H 2 O), or (  x ) 1 g PCNB. The concentrationused for all active ingredients correspond with the proposeddoses labeled on each of the fungicidal products or has beenchosen according to published data (Papavizas 1967; Tsao1970; Dhingra and Sinclair 1994). A 2% potato dextroseagar (PDA, Becton Dickinson Canada Inc., Mississauga,Ont.) was used as the control. All prepared media weremixed thoroughly and poured into petri dishes (20 mL/9-cmplate). Fungal tolerance to antimicrobial agents in pureculture Culture media were inoculated with each of the fourstrains of   Fusarium  and six other mitosporic fungi isolatedfrom asparagus fields. Petri dishes were inoculated with 3-mm discs taken from the periphery of actively growing 8-day-old fungal cultures growing on PDA (Papavizas 1967)and were incubated at 20°C in the dark. The colony diame-ters were measured after 7, 10, and 14 days of inoculation.The growth of each fungal strain was expressed as the pro-portion of growth on each tested media compared withgrowth on the control PDA. The experiment was replicatedfive times with three plates per treatment in each repetition.  Fusarium  isolation from seeded or natural soil samples Three different soil types representing the gradient of soilprofile encountered in asparagus production in Quebec werecollected from commercial asparagus fields (Table 1). Threesoil subsamples (3 × 100 g) in each soil type were collectedwithin a triangular area of 43.3 m 2 (10 m × 10 m × 10 m)using a standard soil probe. Then, the subsamples were cou-pled and mixed thoroughly to obtain one representative soilsample of 300 g for each soil type analysis. Soil texture wasdetermined according to the method of Sheldrick and Wang(1993), and soil nitrate and ammonium concentration was de-termined with a Lachat flow injection autoanalyzer (LachatQuickChem, Milwaukee, Wis.) after KCl extraction (Maynardand Kalra 1993). Soil-available nutrients were extracted withthe Mehlich-3 solution (Sen Tran and Simard 1993) andmeasured with a Lachat flow injection autoanalyzer (P andK) or by atomic absorption spectrometry (Perkin-Elmer5380, Norwalk, Conn.). Soil microbial biomass C and Nwere determined by the fumigation–extraction method(Voroney et al. 1993). Soil 1 was a sandy soil harvested atl’Assomption (Table 1), soil 2 was a sandy loam from Trois-Rivières, and soil 3 was a loam from the Joliette area, classi-fied according to Tissen and Moir (1993). Additional con- © 2002 NRC Canada 842 Can. J. Microbiol. Vol. 48, 2002  trols were prepared. Soil 4 was sandy soil (300 g)autoclaved for 15 min at 121°C (pH adjusted to 6.3 with0.01 M NaOH after sterilization) and divided into threesubsamples (3 × 100 g) to obtain an identical number of soilreplicates as compared with the natural soils. Then, thesewere seeded with the previously listed fungal species as fol-lows: 1 mL suspension containing 10 3 conidia of each fun-gal species was added to a 100-g soil subsample. Theconidial suspensions were prepared by harvesting theconidia with sterile, distilled water from 8-day-old culturesof each species growing on PDA at 20°C in the dark. Thesoil sample was then thoroughly mixed and incubated for48 h at 22°C in the dark. Subsequently, three subsamples of 100 g were tied and mixed thoroughly to obtain one repre-sentative sample of 300 g for artificially infested soil.Dilution series were prepared from 3-g subsamples of each of the three nonsterilized soils (1–3) and from theseeded sandy soil (4). One millilitre from the 10 –2 dilutionwas applied on each of the 10 selective media describedabove. The experiment was replicated five times with threeplates per treatment in each repetition. The plates were incu-bated at 22°C in the dark, and observations were made after7 days. The total fungal colony-forming units (CFU) werecounted for each soil sample. The number of   Fusarium spp. CFU were also counted on PDA and on the media con-taining myclobutanil (MBA), PCNB (PPA), and MGA. Fusarium  colonies were examined microscopically to con-firm the species using standard literature (Burgess et al.1994; Gerlach and Nirenberg 1982; Nelson et al. 1983). Effect of PPA and MBA media on the growth of fusaria from asparagus natural field soils The two media that appeared the most effective in enu-merating fusaria from asparagus fields were compared withPDA with respect to growth inhibition. Seven  Fusarium  spe-cies isolated during this study from various asparagus fields(Québec, Canada) were inoculated on PPA, MBA, and PDA,and colony diameter was measured on two axes after 96 h of incubation at 20°C in the dark. This experiment was repli-cated three times with five plates per treatment. Statistical methods Statistical analyses were carried out using the ANOVAprocedure of the SAS software (SAS Institute Inc., Cary,N.C., U.S.A.). Differences between treatments were deter-mined by Tukey’s Studentized range tests and were consid-ered significant at  P  < 0.05. Results Tolerance of   Fusarium  and mitosporic fungi toantimicrobial agents While Rose Bengal, sodium hypochlorite, captan, andthiram varied in their respective effect on the growth of fusaria and other fungal isolates, these additives did notcompletely inhibit any tested fungal isolate (Table 2). Fur-thermore, the weak sodium hypochlorite solution stimulatedthe growth of most fungal isolates, while thiram stimulatedall fusaria to a quicker growth during the first two days afterinoculation (data not shown). 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PCNB, chlorotalonil, myclobutanil, malachitegreen, and cupric sulphate all completely inhibited variousisolates, but malachite green and cupric sulfate also pre-vented the growth of one  Fusarium  ( Fusarium  sp.) isolate.Fungal colonies were not very clear on chlorothalonil(2.5 mg/L), and it was difficult to distinguish  Fusarium spp. Therefore, this antifungal agent was eliminated in fur-ther trials.PCNB and myclobutanil considerably reduced but did notprevent the fusaria growth. The latter was also the most ef-fective to prevent the growth of other fungal isolates, whilethe former was completely ineffective on  Penicillium  sp. andalso allowed a slow growth of   Epiccocum nigrum . Myclo-butanil and malachite green were highly restrictive tosaprophytic fungal growth, but both required a longer incu-bation time (>5 d) for visible colony formation than PCNB(data not shown). Malachite green also allowed the germina-tion and initial hyphal growth of   Trichoderma  propagulesbut reduced considerably further mycelial development.  Fusarium  isolation from naturally or artificiallyinfested soils Apart from the PDA control medium, PPA containingPCNB yielded the highest total fungal CFU numbers in allsoil types, while myclobutanil, malachite green, cupric sul-fate, and benomyl generated lower number of fungal colonies(Fig. 1). PPA permitted the isolation of as many fusarium col-onies (99%) as on PDA (Fig. 2), showing also a low restric-tion to some  Aspergillus  (79%),  Penicillium  (21%),  Rhizopus (81%), and  Trichoderma  (96%), but otherwise highly inhib-iting other saprobes. In contrast, sodium hypochlorite, RoseBengal, cupric sulfate, and captan inhibited fusaria at leastin the same proportion as the other fungi, while benomyl © 2002 NRC Canada 844 Can. J. Microbiol. Vol. 48, 2002 Growth ratio (%)*Common name Concentration (mg/L) Fs Fm Fod Fsp Aa Af Cc En Psp TvPCNB 1000 54 33 53 21 0 0 0 26 99 0Rose Bengal 50 87 57 96 65 86 44 19 74 83 35Malachite green 25 23 10 37 0 18 0 0 0 9 29Sodium hypochlorite 1.2 135 83 136 109 115 74 124 101 97 41Chlorothalonil 2.5 † 35 29 42 25 51 0 0 0 53 26Captan 2000 † 96 61 99 53 15.7 6.7 48 34 107 64Benomyl 160 † 0 0 0 0 0 69 0 0 0 0Myclobutanil 200 † 36 22 21 3 0 0 0 0 9 0Thiram 1000 † 17 71 100 46 75 52 100 31 96 49Cupric sulfate 2000 78 0 33 31 57 49 0 28 63 10 Note:  Fs,  Fusarium solani ; Fm,  F. moniliforme ; Fod,  F. oxysporum  f.sp.  dianthi † ; Fsp,  Fusarium  sp.; Aa,  Alternaria alternata ; Af,  Aspergillus flavus ;Cc,  Cladosporium cladosporioides ; En,  Epicoccum nigrum ; Psp,  Penicillium  sp; Tv,  Trichoderma viride ; PCNB, pentachloronitrobenzene.*Growth ratio was calculated as the percentage of growth on a media containing a specific additive, compared with that on PDA; all media weresupplemented with streptomycin sulfate and neomycin sulfate. † Commercial fungicide formulation. Table 2.  Effect of antimicrobial additives on the growth of four  Fusarium  species and six other species of mitosporic fungi. Fig. 1.  Total fungal CFU isolated from asparagus natural and in-fested soils on media containing various antimicrobial ingredi-ents. Error bars are standard errors of the means. Fig. 2.  Total fusarium CFU isolated from asparagus natural andinfested soils on PDA (potato dextrose agar), PPA (peptone–pentachloronitrobenzene agar), MBA (myclobutanil agar), andMGA (malachite green agar) media. Within each soil type,means with different letters are significantly different ( P  < 0.05)by Tukey’s Studentized range tests.  nearly completely inhibited the growth of all soil microor-ganisms (Fig. 1). PPA and myclobutanil showed the highestdiversity of   Fusarium  species, allowing the recognition of generic characteristics of fusaria in  Liseola  and  Elegans  butnot in the  Discolor  ,  Gibbosum , and  Roseum  sections(Fig. 3), without transferring colonies to a suitable medium.Seven  Fusarium  species were encountered on PCNB andmyclobutanil:  Fusarium avenaceum  (Fr.:Fr.) Sacc. (Section:  Roseum ),  F. culmorum  (Wm.G. Sm.) Sacc. (Section:  Discolor  ),  Fusarium equiseti  (Corda) Sacc. (Section:  Gibossum ), Fusarium graminearum  Schwabe (Section:  Discolor  ), F. oxysporum  Schlechtend.:Fr. f.sp.  asparagi  S.I. Cohen &Heald (Section:  Elegans ),  F. proliferatum  (Matsush.)Nirenberg (Section:  Liseola ), and  Fusarium sambucinum Fuckel (Section:  Discolor  ).Beside  Fusarium -isolated taxa, myclobutanil was restric-tive to isolation of all other soil microorganisms. Contrary tothe effectiveness of PPA and myclobutanil, no appreciablerecovery of   Fusarium  isolates was obtained with malachitegreen, although this medium was free from a majority of saprobes, excluding  Trichoderma , as was myclobutanil. Effect of PPA and MBA media on growth of fusariafrom asparagus fields PPA (PCNB) permitted a similar growth as PDA for six Fusarium  species, with only  F. proliferatum  showing a slightbut significant growth reduction (Figs. 4 and 5). On theother hand, myclobutanil significantly reduced the growth of all species. PPA and PDA allowed the measurement of  Fusarium  growth earlier than MBA, which required a mini-mum of 5 days of incubation under the concentration used(200 mg/L). Besides  F. proliferatum  and  F. oxysporum  f.sp. asparagi , no significant fructification was obtained for othertested fusaria on PPA and MBA. Also, only these speciesabundantly produced conidia-bearing structures on PDA un-der the tested conditions and within the duration of the ex-periment. Discussion Selective media, using many antimicrobial compoundssingly or in combinations, have been used for decades (Tsao1970; Mandeel et al. 1995) with the objective of a higher ef-fectiveness in  Fusarium  spp. isolation and enumeration. Themost widely used is the Nash and Snyder (1962) mediumemploying the common soil fungicide PCNB, which par-tially inhibits many fungal contaminants but allows the nor-mal development of   Fusarium  spp. Other selective agentsused in  Fusarium  isolation media reported are dichloran(Andrews and Pitt 1986), a mixture of dichloran andiprodione (Abildgren et al. 1987), and Rose Bengal,benomyl, and captan (Elad and Chet 1983), but all of themallow the growth of other fungal taxa. Only malachite greenproved to be highly selective for  Fusarium  spp. and restric-tive to other microbiota (Singh and Nene 1965; Castellá etal. 1997 a ), although it did not always give satisfactory re-sults at the proposed concentration (Papavizas 1967). © 2002 NRC Canada Vujanovic et al. 845 Fig. 3.  Colony morphology of   Fusarium equiseti  ( Gibbosum  section) colonies after 1 month of growth on (A) PDA (potato dextroseagar) and (B) MBA (myclobutanil agar) and of   Fusarium avenaceum  (Roseum section) on (C) PDA and (D) PPA (peptone–pentachloronitrobenzene agar) media incubated at 20°C in darkness (bar = 1 cm).
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