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Applied and Environmental Microbiology, July 2003, p . 4190-4191, Vol . 69, No . 7
A Selective Medium for Quantitative Reisolation of Trichoderma harzianum from Agaricus bisporus Compost
Josie Williams,1 John M . Clarkson,1, Peter R . Mills,2 and Richard M . Cooper1*
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,1
Department of Microbial Biotechnology, Horticultural Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom2
Received 14 April 2003/
Accepted 17 April 2003
We adapted a selective medium, previously developed for reisolation of Trichoderma spp . from soil, for quantitative determination of growth of T . harzianum from commercial Agaricus bisporus composts . This medium enables comparisons of aggressive (sensu inhibition of A . bisporus yield) with nonaggressive T . harzianum groups . The resulting medium contains the antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb and was highly selective, allowing the recovery of T . harzianum, as viable conidia and hyphal fragments, in compact colonies with the absence of visible microbial contaminants .
Isolates of Trichoderma harzianum that attack the commercial mushroom Agaricus bisporus (11) colonize spawned compost and result in substantial yield reductions (9) . In another study (11), we tested the hypothesis that saprophytic growth is a key aspect of aggressiveness, in addition to mycoparasitism, which has usually been considered as the basis of antagonism by Trichoderma spp . (for examples, see references 2 and 11) . This study required reproducible and quantitative comparison of growth in commercial compost of aggressive and nonaggressive isolates of T . harzianum . Such analyses are efficient only if a medium selective for T . harzianum is available .
Several media selective for Trichoderma spp . from soil have been described previously (1, 5, 7, 11) . However, some of the medium components are no longer available or their use is now prohibited . Also, compost harbors an abundant and diverse microbial community, including thermophilic species, which differs from that in soil and probably requires a different range of inhibitors to exclude it (4) . In preliminary tests of the most recently described Trichoderma-selective medium (TSM) (1) for which all components were available, this medium was relatively ineffective for the quantitative determination of CFU of T . harzianum from compost, mainly because of inhibition of conidial germination . Therefore, we evaluated each antimicrobial component of TSM and additional antibiotics against T . harzianum in vitro . Both aggressive and nonaggressive isolates were tested to ensure that they were recovered in a similar manner .
Fungal strains and inoculation of compost.
Fungal strains, growth, and inoculation of commercial mushroom compost with T . harzianum are described by Williams et al . (11) . Group Th1 (nonaggressive) is represented by isolate Th1(c), and group Th2 (aggressive) is represented by isolate T7 .
Development of a T . harzianum-selective medium (THSM).
We adapted the TSM described by Askew and Laing (1) for soil samples for use with mushroom compost . The antimicrobial components of this medium originally contained chloramphenicol, quintozene, captan, and either propamocarb hydrochloride or metalaxyl . In adapting the TSM, consideration was given to the exclusion of fungal and bacterial constituents of mushroom compost . Malt extract agar (MEA) (2% wt/vol) was supplemented with combinations of antibiotics (chloramphenicol, streptomycin sulfate, and nystatin [Sigma, Poole, United Kingdom]) to prevent the growth of bacteria and actinomycetes . To inhibit fungi, we tested the fungicides quintozene (Sigma), captan (Syngenta, Bracknell, United Kingdom), and propamocarb hydrochloride (Levington Horticulture, Ipswich, United Kingdom) in addition to the growth inhibitors rose Bengal and sodium deoxycholate (Sigma) (Tables 1 and 2) . These media were investigated for effects on hyphal growth and conidial germination of T . harzianum, since the propagules in samples extracted from compost (determined as CFU) comprise both hyphal fragments and conidia .
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TABLE 1 . Germination and hyphal growth of T . harzianum strains on media with combinations of antimicrobial compounds
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TABLE 2 . Comparison of effects of fungicidal components on the germination and hyphal growth of T . harzianum strain T7
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The final THSM consisted of a basal medium comprising (all amounts are per liter) 0.2 g of MgSO4 · 7H2O, 0.9 g of K2HPO4, 1.0 g of NH4NO3, 0.15 g of KCl, 0.15 g of rose Bengal, 3 g of glucose, and 20 g of agar in 950 ml of distilled water, which was autoclaved at 121°C for 15 min . The antimicrobial and fungicidal ingredients (all amounts are per liter) were 0.25 g of chloramphenicol, 9.0 ml of streptomycin stock solution (1% wt/vol), 0.2 g of quintozene, and 1.2 ml of propamocarb (772 g of active ingredient per liter), all in 40 ml of sterile distilled water, and the mixture was added to the cooled basal medium .
Quantification of T . harzianum growth in mushroom compost.
The recovery of T . harzianum from compost has been described by Williams et al . (11) . Briefly, compost samples were steeped in 100 mM sodium tetrapyrophosphate and then agitated in a stomacher (3) before 100-µl samples from log dilutions were inoculated onto THSM . Plates were incubated at 25°C for 4 days .
Effect of combinations of antimicrobial compounds on hyphal growth and germination.
The hyphal growth of T . harzianum was significantly (P < 0.05) lower on four media based on MEA containing combinations of antibiotics and fungicides (media 1 to 4) and on Askew and Laing's TSM than was growth on 2% MEA (Table 1) . The reduction of colony diameter was most pronounced with TSM, on which it was typically 9- and 10-fold lower than on MEA for Th1 and Th2, respectively . Conidial germination of both T . harzianum groups was prevented on media 3 and 4 and on TSM, while the antibiotics and sodium deoxycholate (media 1 and 2) had no effect (Table 1) . Nystatin and either captan or propamocarb inhibited germination .
Analysis of individual fungicides.
Individual fungicide ingredients were incorporated into 2% MEA to identify the component(s) of TSM that inhibits germination of T . harzianum spores (MEA controls were not included since the rapid growth rates would not allow the formation of small, compact colonies) (Table 1) . Captan, a component of Askew and Laing's TSM, was inhibitory to conidial germination . Germination occurred on media containing rose Bengal, quintozene, or propamocarb, and the diameter of colonies increased in this order (Table 2) . Similar recoveries occurred on media with these three inhibitors when plates were inoculated with 100 µl of T . harzianum conidial suspensions containing ca . 10 conidia (Table 2) . A combination of quintozene and propamocarb appeared optimal because it allowed high recovery, ca . 13 (standard error, ±0.6) colonies per plate, from conidial suspensions exposed to these compounds and because colonies were generally 10% of the size of those on MEA (Table 2) . This compact growth facilitated the assessment of large numbers of colonies per plate . The growth and germination of isolates from aggressive and nonaggressive groups were not differentiated by any medium, except for colony diameters on medium 3 (Table 1) .
Infested compost samples plated on THSM yielded only T . harzianum colonies, whereas the plates from T . harzianum-free compost were devoid of any visible microbial growth . Typical propagule numbers from infested compost 3 weeks after inoculation ranged from 103 to 105 CFU g-1 (fresh weight) (11) .
Previous analyses of the growth of T . harzianum in compost have been semiquantitative at best and based on visual assessment of mycelial extension (6) . Others have employed biomass-associated metabolic activity as estimated by fluorescein-hydrolyzing activity (8); however, this method identified no growth differences between Th2 and non-Th2 strains in sterilized compost with or without A . bisporus. In this study, we adapted the TSM medium, originally selective for Trichoderma spp . from soil (1), to allow reproducible comparisons of growth of T . harzianum in mushroom compost . Streptomycin was added to reduce the bacterial population, and the captan was removed, since it inhibited germination of T . harzianum conidia . Germination was critical, since many of the CFU counted were recovered as spores . The resulting THSM medium was highly selective for T . harzianum, and no contaminants were observed from the compost, even though mushroom compost harbors a substantial and diverse microbial community (10) .
We thank S . Muthumeenakshi for T . harzianum cultures, Helen Grogan for A . bisporus cultures and advice, and Paul Christy for assistance with statistical analyses .
J.W . was supported by a BBSRC CASE studentship .
* Corresponding author . Mailing address: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom . Phone: 44 0 1225 383051 . Fax: 44 0 1225 826779 . Email: bssrmc{at}bath.ac.uk .
Present address: Molecular Sensing plc, Melksham, Wiltshire SN12 8HL, United Kingdom .
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