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Differential sensitivity of fungi to lithium chloride in culture media
mycological research 112 (2008) 717–724
journal homepage: www.elsevier.com/locate/mycres
Differential sensitivity of fungi to lithium chloride in culture media Dana L. RICHTERa,*, Sara C. ROBINSONa, Maria P. BEARDSLEEb, Maureen L. HABARTHb a
School of Forest Resources and Environmental Science, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA b Department of Civil and Environmental Engineering, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA
article info
abstract
Article history:
Forty species of fungi, representing a range of ecological and taxonomic groups, were tested
Received 23 January 2007
for their ability to grow on agar media amended with lithium chloride (LiCl) at 1.5, 3 and 6 g l1.
Received in revised form
Species of Trichoderma varied considerably in their sensitivity to LiCl; at one week on 6 g l1
7 January 2008
LiCl medium, the growth of seven species of Trichoderma was considerably inhibited; how-
Accepted 24 January 2008
ever, by three weeks at this level, four of the species tested were able to attain 30 % of control
Corresponding Editor: Karl Ritz
growth. Of the seven species tested, an isolate of T. viride was the most sensitive to LiCl in agar. Eleven other imperfect fungi also showed a range of ability to grow on agar amended with
Keywords:
LiCl, from total inhibition to complete lack of inhibition. Six ascomycete fungi were greatly
Fungus culture
inhibited by LiCl at all levels; however, an isolate of Chaetomium globosum was highly tolerant
Fungus isolation
of LiCl. Seven basidiomycete wood-decay fungi were quite sensitive to LiCl in agar, showing
Lithium chloride
total to nearly total inhibition even at the lowest level; however, after three weeks, an isolate
Media amendments
of Postia placenta was nearly uninhibited except at 6 g l1. Five ectomycorrhizal basidiomycete
Selective media
fungi were totally inhibited by all levels of LiCl; however, one ectomycorrhizal imperfect fun-
Trichoderma
gus (Cenococcum graniforme) was able to grow at 3 g l1 and was uninhibited at 1.5 g l1. Four
Wood-decay fungi
zygomycete fungus isolates were nearly unaffected in their growth by all levels of LiCl. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Lithium chloride (LiCl) is a non-toxic, alkali metal salt that is soluble in water; in its solid state, it is a white, odourless substance that resembles table salt. It is commonly used as a desiccant, as well as in metallurgy and pyrotechnics (Budavari et al. 1989). Lithium chloride was reported by Kado & Heskett (1970) for use in culture media to increase the efficiency of isolating plant pathogenic bacteria from soil (e.g. Agrobacterium, Corynebacterium, and Erwinia spp.). Its selective nature is due to the salt differentially affecting the permeability of bacterial membranes. During the course of their work, LiCl at 5–7 g l1
was also noted to inhibit fungal colonies that otherwise would obscure the observation of bacterial colonies (Kado & Heskett 1970). Trichoderma spp. can be aggressive colonizers of agar plates when attempting to isolate specific fungi from various substrates. Species of Trichoderma are ubiquitous in soil and rotten wood, and are also parasites of other fungi (Samuels 1996); therefore, they are naturally present wherever fungi exist. In the authors’ experience, Trichoderma spp. are common contaminants in agar plates when attempting to isolate wooddecay fungi from rotten wood and ectomycorrhizal fungi from root tips and mycelial strands.
* Corresponding author. Tel.: þ1 906 487 2149. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.01.013
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Following Kado & Heskett’s (1970) work, Wildman (1991) showed that LiCl at 6 g l1 could be used to inhibit the spore germination and growth of Trichoderma spp., increasing the efficiency of isolating other fungi from soil dilution plates; however, the sensitivity of Trichoderma to LiCl was not uniform among species in the genus. Based on the latter paper, LiCl has been used in malt agar plates to prevent overgrowth by Trichoderma spp. when isolating wood-inhabiting stain and decay fungi. However, at 6 g l1 it has been observed that LiCl has the ability to significantly inhibit wood decay and other fungi as well; therefore, 1.5–3 g l1 was adopted as a moderate level of LiCl to inhibit Trichoderma spp. when attempting to isolate stain and decay fungi from very decayed wood. Benomyl (Du Pont, Wilmington, Delaware) is also a common media amendment used in wood-decay and ectomycorrhizal fungus isolation laboratories, but along with preventing the growth of fast-growing imperfect fungi, such as Trichoderma spp. (Johnson 1995; Worrall 1991), Benomyl also
Materials and methods The base medium used for all tests was 2 % malt agar (2 M), prepared by suspending 20 g malt (Bacto, Difco, Lawrence,
Week 1 A
Key to Fungi
A A
90
T.h. = Trichoderma harzianum T.k. = Trichoderma koningii T.l. = Trichoderma longibrachiatum T.po. = Trichoderma polysporum T.ps. = Trichoderma pseudokoningii T.r. = Trichoderma reesei T.v. = Trichoderma viride
A 70 60 50
A
A A
40
T.v.
T.po.
T.h.
T.r.
6.0
A
A
B
A B
50
B B A
C
1.5
3.0
T.h.
B T.v.
T.ps. T.r.
T.h. T.k. T.l. T.po.
T.r. T.v.
T.l. T.po. T.ps.
T.h. T.k.
20
C B
B C
C C
T.v.
30
T.r.
A
40
T.po. T.ps.
Growth as % of Control
3.0
60
0
B C C B
A
70
10
B B
Week 2 A A
A
90 80
T.r. T.v.
1.5
B
C
T.k. T.l.
100
T.ps.
T.h.
T.k. T.l. T.po.
0
T.ps.
A
20 10
B
T.ps. T.r.
B
T.k.
A
A
T.l.
30
T.h.
B
T.k. T.l. T.po.
Growth as % of Control
80
T.v.
100
prevents the isolation of ascomycete fungi, which also cause stain and decay in wood. Rose Bengal is another media amendment used to inhibit fast-growing fungi, while allowing slower-growing fungi and bacteria to be isolated (Jarvis 1973; Mycological Society of America, Mycology Guidebook Committee 1974). Aside from the two papers cited in the previous paragraph, there appears to be little additional information on the use of LiCl in culture media to inhibit Trichoderma spp. or other fast-growing fungi. The purpose of this study was to expose a range of ecological and taxonomic groups of fungi to various levels of LiCl in agar to assess its use as an amendment to selectively inhibit Trichoderma spp. and possibly other fast-growing, contaminating fungi.
6.0
LiCl Concentration (g L-1) Fig 1 – Growth as percent of control of seven species of Trichoderma after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
Differential sensitivity of fungi to lithium chloride
719
Kansas) and 15 g agar (Difco, Lawrence, Kansas) in 1 l distilled water. LiCl (Fisher Scientific, Pittsburgh, PA) was added at 1.5, 3, or 6 g l1 before autoclaving. Approximately 20 ml were poured into 100 mm plates and stored 3–7 d before use. All fungi were grown at these levels of LiCl and compared with growth on control 2 M agar plates (0 g l1 LiCl). All procedures were conducted under sterile conditions in a laminar-flow hood. Six groups of fungi represented by 40 species were tested for growth on LiCl media. These fungi represented the major classes of fungi (ascomycetes, basidiomycetes, imperfect fungi, and zygomycetes) and functional groups of fungi
commonly isolated in forest pathology laboratories (saprotrophs, mould, wood decay, wood stain, ectomycorrhizal). Seven species of Trichoderma were also tested. All fungi are maintained at the School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI. Inoculum for test plates was prepared by growing test fungi on 2 M plates at 22 C to achieve colonies ca 2–4 cm diam (usually 1–2 weeks). Plugs of inoculum 5 mm diam and the thickness of the agar were cut with a sterile cork borer from the perimeter of an actively growing fungus colony. Inoculum
Week 1 100 90
A
A
A
A
A
A
A
A
A
A
70
AB
B
60 50 A
B
A.a. A.c. A.n. A.p. B.c. C.c. C.g.
A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c.
A.a.
10 0
A
A
1.5
C
C
B A
3.0
6.0
Week 2 100 90
A
A
A
A
A
A
A
Key to Fungi
A
A
A
A.a. = Alternaria alternata A.c. = Arthrographis cuboidea A.n. = Aspergillus niger A.p. = Aureobasidium pullulans B.c. = Botrytis cinerea C.c. = Cladosporium cladosporioides C.g. = Cenococcum graniforme P.p. = Penicillium purpurogenum P.v. = Paecilomyces variotii S.c. = Stachybotrys chartarum S.l. = Scytalidium lignicola
A
B
70
B B
50
AB
C
A A
A
40 30
A
B
20 A
1.5
A
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
0
B
A
10
C
A
3.0
C
A C B
B
A
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
Growth as % of Control
80
60
A
A C B
C
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
A
20
P.p. P.v. S.c. S.l.
30
A
B
A
A
B
B
B
40
S.l.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 2 – Growth as percent of control of 11 species of imperfect fungi after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
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D. L. Richter et al.
plugs were firmly placed mycelium side down in the centre of a test plate. Three replicate plates were prepared for each LiCl level (1.5, 3, and 6 g l1) for each fungus. Test plates were either wrapped in Parafilm (to contain overgrowth by some fungi) or closed in plastic bags and incubated in the upright position in the dark at 22 C. Growth of fungi was measured weekly for three weeks following inoculation; due to their slower growth mycorrhizal fungi were also measured at six weeks. Growth was measured as the radius of mycelial extension from the edge of the inoculum plug. If the growth of the colony was uniformly circular, only one measurement was taken. If the growth of the colony was irregular, three points of greatest radial extent were measured and an average of these was used to represent total growth. If the growth of a colony reached the edge of the test plate, this was considered ‘maximum growth’. Maximum growth was equal to the radius of the test plate (42.5 mm) minus the radius of the inoculum plug (2.5 mm), or 40 mm. Mean growth of the three replicates for each fungus on each LiCl concentration was determined.
As the fungi tested grow at widely different rates, fungus growth rates on LiCl-amended media were converted to growth as a percent of fungus growth in the absence of LiCl (growth as percent of control), in order to compare growth among fungi within a group (Trichoderma spp., ascomycetes, basidiomycetes, imperfect fungi, and zygomycetes). Following arc sine square root transformation, growth as percent of control data for each fungus response to LiCl level by week were subjected to repeated measures analysis of variance (ANOVA) followed by Tukey’s HSD (P 0.05). Analysis was conducted using SAS software, Version 9.1 of the SAS System for Windows Version 5.1 (SAS Institute 2002–2003).
Results LiCl exerted a wide range of effects on fungus growth depending on the fungus species and the amount of LiCl added to the media. Generally, species of zygomycetes, ascomycetes, and imperfect fungi were more able to tolerate LiCl in media
Week 1 100
Key to Fungi C.a. = Ceratocystis adiposa C.c. = Ceratocystis coerulescens C.f. = Ceratocystis fagacearum C.pic. = Ceratocystis piceae C.pil. = Ceratocystis pilifera C.g. = Chaetomium globosum
A
90
70 60 50
A
A
A
A
3.0
A C.g.
C.pic.
C.a.
B C.c.
C.g.
B
B
C.f.
A C.pil.
C.f.
C.a. C.c.
1.5
C.pic.
B
B C.g.
C.pic.
A
C.pil.
0
C.f.
A C.a.
10
C
A
C.pil.
30 20
6.0
Week 2
100
A
A
90
A
A
80 B
A
70 60 A
B
40
C
30
A
3.0
C
A C.pil. C.g.
C.f.
C
C.pic.
B
C.a. C.c.
A C.g.
C.c.
C.g.
C.pil.
C.f. C.pic. 1.5
B C.a.
0
C.c.
10
B
A
C.f. C.pic.
20
C.pil.
50
C.a.
Growth as % of Control
A
B
40
C.c.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 3 – Growth as percent of control of six species of ascomycete fungi after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
Differential sensitivity of fungi to lithium chloride
721
than basidiomycetes; ectomycorrhizal basidiomycetes were the most sensitive of all groups tested. Figs 1–5 show the growth of the fungi tested as a percent of control fungus growth, by fungus group tested, measured after one, two, or three weeks incubation depending on the general growth rate of the group. Different letters above error bars indicate significant differences between LiCl levels by fungus species within week. In some cases, a fungus grew to 100 % of control growth by week one or two; thereafter, no additional growth was recorded due to the limit of the Petri plates. For some species, sensitivity to LiCl decreased over the course of three weeks, as mycelial growth increased from week 1 to week 2 and/or from week 2 to week 3. This pattern of growth was especially evident for Trichoderma koningii, T. polysporum, Ceratocystis adiposa, Chaetomium globosum, Trametes versicolor, and Irpex lacteus, and may represent
adaptation to LiCl in agar media. In the majority of cases, maximum growth by a fungus isolate as percent of control growth was observed during the first two weeks of incubation. Among species of Trichoderma (Fig 1), at one week most isolates were considerably inhibited by 6 g l1 LiCl, being able to attain only 0–8.6 % of control growth, except for T. longibrachiatum, which attained 18.5 % of control growth. At one week 1.5 g l1 LiCl did little to inhibit the species of Trichoderma; most grew from 45–100 % of controls, except for T. koningii and T. viride, which grew to 23 and 21 % of controls, respectively. At week 1, T. harzianum, T. polysporum, and T. reesei grew significantly more than other fungi on 1.5 g l1 LiCl. At two weeks, most species of Trichoderma could grow from 14–23 % of controls on 6 g l1, except T. pseudokoningii and
Week 2 100
Key to Fungi G.t. = Gloeophyllum trabeum I.l. = Irpex lacteus N.l. = Neolentinus lepideus P.g. = Phlebia gigantea P.o. = Pleurotus ostreatus P.p. = Postia placenta T.v. = Trametes versicolor
A
90 B
70 60 A 50
A
40 30
1.5
3.0
B
T.v.
P.g. P.o.
I.l.
N.l.
C C B
G.t.
T.v.
P.p.
P.o.
B
A B
P.p.
B A B P.g.
N.l.
B I.l.
T.v.
P.g.
B P.p.
A
G.t.
0
A
P.o.
A I.l.
10
B
A
N.l.
20
G.t.
Growth as % of Control
80
6.0
Week 3
100
A
90
A A
A
70 60 50 40
A
B
30
1.5
B
3.0
B T.v.
B
P.p.
A
P.o
N.l.
I.l.
B C B G.t.
B T.v.
B
P.o. P.p.
A P.g.
I.l.
G.t.
T.v.
B
N.l.
B
P.g.
A P.p.
I.l.
A P.g.
0
G.t.
10
A
P.o.
20
N.l.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 4 – Growth as percent of control of seven species of basidiomycete wood decay fungi after 2 and 3 weeks on 1.5, 3.0, and 6.0 g LL1 LiCl-amended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
722
D. L. Richter et al.
Key to Fungi M.sp.1 = Mucor sp. 1 M.sp.2 = Mucor sp. 2 Mo.sp. = Mortierella sp. Z.sp. = Zygorhynchus sp.
Week 1
100
A
A
A
A
90
A
A
B A
B
80
Growth as % of Control
A
70 60 B
50 40 30
C
0
3.0
Z. sp.
Mo. sp.
M. sp. 2
M. sp.1
Z. sp.
Mo. sp.
M. sp.1
1.5
M. sp .2
Z. sp.
Mo. sp.
M. sp. 2
10
M. sp.1
20
6.0
LiCl Concentration (g L-1) Fig 5 – Growth as percent of control of four isolates of zygomycete fungi after 1 week growth on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
T. viride, which were still totally or nearly totally inhibited. T. longibrachiatum grew significantly less than all other fungi on 1.5 g l1 LiCl, whereas T. harzianum, T. polysporum, T. pseudokoningii, and T. reesei were uninhibited and grew as much as controls. Growth of T. viride was significantly reduced by 3 g l1 LiCl compared with growth on 1.5 g l1. Considering all levels of LiCl together, at two weeks, the mycelial growth sensitivity of seven Trichoderma isolates could be ranked generally in the order (from most sensitive to most tolerant): T. viride > T. longibrachiatum > T. koningii > T. pseudokoningii > T. harzianum > T. reesei > T. polysporum. At 1.5 g l1 LiCl, four species of Trichoderma were uninhibited in growth, whereas three species were able to attain growth of 50–80 % of control. Additionally, at this level, T. longibrachiatum was more sensitive to LiCl in media than T. viride. After three weeks at 6 g l1 LiCl, T. pseudokoningii and T. viride were still totally inhibited, whereas mycelial growth of all other species of Trichoderma ranged from 20–50 % of control. The mycelial growth sensitivity of the 11 imperfect fungi tested was highly variable depending on LiCl concentration (Fig 2). Among the fungus isolates, Alternaria alternata and Aspergillus niger were the most tolerant, demonstrating complete lack of inhibition with even the highest level of LiCl in the media to week 3. Penicillium purpurogenum and Scytalidium lignicola were consistently the most sensitive to LiCl at week 2; P. purpurogenum was able to attain only 10 % of control on 1.5 g l1 LiCl, and S. lignicola was completely inhibited by all
levels of LiCl. Generally, at the highest levels, LiCl slowed the growth of most imperfect fungi considerably; however, lower levels were only moderately inhibitory. Cenococcum graniforme was the only isolate of all fungi tested that exhibited growth stimulation by LiCl, as by week 3, colonies on 1.5 g l1 LiClamended media grew slightly more than control colonies. For the imperfect fungi at week 1, there were no significant differences in growth between levels of LiCl for Alternaria alternata, Aureobasidium pullulans, Cladosporium cladosporioides, P. purpurogenum, and S. lignicola. The aforementioned fungi, along with Aspergillus niger, also had no significant differences in growth between levels of LiCl at week 2. Sensitivity of six species of ascomycete fungi to LiCl after two weeks (Fig 3) could generally be ranked in the order (most sensitive to most tolerant): Ceratocystis pilifera > C. fagacearum > C. adiposa > C. piceae > C. coerulescens > C. globosum, with the exception that C. coerulescens was more sensitive than C. piceae at 1.5 g l1 LiCl. Whereas the isolate of C. pilifera was completely inhibited at all levels of LiCl, the isolate of C. globosum was highly tolerant of LiCl at all levels and grew nearly as much as controls, even by two weeks. At week 1, C. adiposa, C. fagacearum and C. pilifera were considerably inhibited in their growth by all levels of LiCl. C. coerulescens grew in the presence of LiCl, but had significantly different growth between all levels of LiCl. C. globosum was moderately affected by LiCl in media; however, there were no significant differences in growth between levels. At
Differential sensitivity of fungi to lithium chloride
week 2, C. globosum grew nearly as much as controls and again there were no significant differences in growth between levels of LiCl for this isolate. The sensitivity of seven species of basidiomycete wooddecay fungi to LiCl after two and three weeks growth (Fig 4) could be ranked in the order (most sensitive to most tolerant): Phlebia gigantea > Neolentinus lepideus > Pleurotus ostreatus > G. trabeum > Trametes versicolor > Irpex lacteus > Postia placenta. P. gigantea did not grow on any level of LiCl in the media at up to 3 weeks, whereas P. placenta exhibited growth at all levels of LiCl. P. placenta and I. lacteus were the only two fungi to exhibit mycelial growth at 3 g l1, and P. placenta was the only fungus to show any growth at 6 g l1 LiCl. Five species of basidiomycete ectomycorrhizal fungi tested (figure not shown) were completely inhibited in growth by all levels of LiCl, even after six weeks of incubation. However, one imperfect ectomycorrhizal fungus, Cenococcum graniforme, was able to grow considerably at 3 g l1 LiCl and was uninhibited by 1.5 g l1 LiCl (Fig 2). Due to their rapid growth rate, it was most useful to examine differences in LiCl sensitivity of zygomycete fungi after only one week (Fig 5). Although a limited number of species were tested, the isolates of zygomycetes appear to be relatively less affected by LiCl in culture media than other groups of fungi. Although one isolate of Mucor sp. showed sensitivity to LiCl, the other isolate of Mucor sp. was completely uninhibited at all levels of LiCl. Among the zygomycetes, the isolate of Zygorhynchus sp. demonstrated the most sensitivity to LiCl in culture media.
Discussion This research demonstrated that there is a remarkable range of response to LiCl among fungi, even within a taxonomic group. However, as single isolates of species were used in the testing, it is not known how the effect of LiCl may vary within a species due to variation among fungus strains. In addition, LiCl may also alter hyphal density of a fungus colony, and therefore, radial growth may not represent an actual reduction in fungal biomass. However, hyphal extension is a highly important characteristic when isolating fungi on agar plates from a substrate containing a number of different fungi. Knowing which species of fungi are able to grow or which are inhibited by LiCl in culture media is important in the use of this amendment when selecting for specific fungi during isolations from wood, soil or root tips. Using the hyphal growth data at a mid-concentration of LiCl (3 g l1) after two weeks of growth, the fungus isolates tested can be sorted into generalized tolerance groups (Table 1), from a group of fungi intolerant of LiCl (no growth on LiCl-amended media) to a group highly resistant to LiCl (75–100 % of control growth). Fourteen of the 40 species tested could not grow on a moderate level of LiCl, whereas ten of the species tested were highly resistant to LiCl in culture media. Although the only species of Penicillium tested (P. purpurogenum) was the most sensitive of the imperfect fungi to LiCl, Wildman (1991), using media amended with 6 g l1 LiCl and soil dilution plates, showed that there was considerable variation among species of Penicillium with respect to their
723
Table 1 – Generalized ranking of fungi according to response to lithium chloride (LiCl) in culture media; data after two weeks growth on 3 g lL1 LiCl-amended media (N [ 3) Tolerance group Intolerant fungi ¼ no growth Ceratocystis adiposa Ceratocystis pilifera Gloeophyllum trabeum Hebeloma arenosa Laccaria bicolour Laccaria laccata Neolentinus lepideus Penicillium purpurogenum Phlebia gigantea Pleurotus ostreatus Rhizopogon rubescens Scytalidium lignicola Suillus granulatus Trametes versicolor
Isolate numbera DR 163-1 ISK ATCC 60758 ATCC 11539 DR 195 DR 378 DR 137 ATCC 12653 ATCC 52427 BURD 5192 MAD 542 DR 401 Highley F-64-F DR 383 MAD 697
Sensitive fungi ¼ growth to 25 % of control Arthrographis cuboidea DR 451 Ceratocystis fagacearum DR 420 Cladosporium cladosporioides ATCC 6271 Irpex lacteus ATCC 11245 Trichoderma viride ATCC 32630 Moderately sensitive fungi ¼ growth between 25–50 % of control Aureobasidium pullulans Forintek 132E Ceratocystis piceae Forintek 387A Trichoderma koningii Forintek 230D Trichoderma longibrachiatum Forintek 259A Trichoderma pseudokoningii Forintek 228B Moderately tolerant fungi ¼ growth between 50–75 % of control Cenococcum graniforme DR G-5-2 Ceratocystis coerulescens DR-ISK-100 Paecilomyces variotii DR 455 Postia placenta MAD 698 Stachybotrys chartarum ATCC 16026 Trichoderma harzianum Highley T-25 J Resistant fungi ¼ growth between 75–100 % of control Alternaria alternata DR 406 Aspergillus niger ATCC 6275 Botrytis cinerea ATCC 28466 Chaetomium globosum ATCC 6205 Mortierella sp. DR 432 Mucor sp. 1 DR 434 Mucor sp. 2 DR 435 Trichoderma polysporum Forintek 260E Trichoderma reesei ATCC 46480 Zygorhynchus sp. DR 433 a Highley, Terry L. Highley, Forest Products Lab, Madison, WI; Forintek, Forintek Mycology Labs, Ottawa, ON; ATCC, American Type Culture Collection, Beltsville, MD; DR, Dana L. Richter; MAD, Forest Products Laboratory, Madison, WI; BURD, Harold H. Burdsall, Forest Products Lab, Madison, WI.
tolerance or sensitivity to LiCl. Wildman (1991) also demonstrated that it was possible to isolate other imperfect fungi from soil on media amended with 6 g l1 LiCl, including Aspergillus spp. and Paecilomyces sp. The present study confirms this, as the isolates of these fungi tested were also able to grow on LiCl-amended media.
724
Wildman (1991) focused on Trichoderma spp. response to LiCl, finding that the sensitivity to LiCl is not uniform throughout the genus and that LiCl resistant strains exist. This study verifies these results. However, alhough Wildman (1991) found that T. longibrachiatum was the most sensitive to 6 g l1 LiCl, the current study found that T. pseudokoningii and T. viride were the most sensitive, and that T. longibrachiatum was only moderately inhibited by 6 g l1 LiCl. Conversely, at 6 g l1 LiCl, Wildman (1991) found that T. koningii and T. harzianum were the most tolerant of the species of Trichoderma tested, whereas this study found these species to be only moderately tolerant at this level of LiCl, and found that T. polysporum was the most tolerant of the species of Trichoderma tested. This apparent variation in response to LiCl by the same species of Trichoderma may be due to intraspecific variation within the species tested. Also, Wildman (1991) used a malt agar with 30 g malt þ 5 g peptone l1, whereas the media in this study contained simply 20 g malt l1. Whereas Benomyl is useful to prevent growth of ascomycete and imperfect fungi when isolating basidiomycetes from soil or wood, LiCl may be used to isolate certain ascomycete and imperfect fungi, especially those fungi tolerant to LiCl. LiCl-amended media may be useful in inhibiting certain Trichoderma species when attempting to isolate fungi from wood or soil. However, as some species of Trichoderma and other fast-growing fungi will be inhibited, some target fungi may also be inhibited, even on the lowest level of LiCl. However, this would not work for the isolation of basidiomycetes (except possibly Postia placenta), which generally are slower growing fungi and highly inhibited by LiCl in media. It is striking that among the basidiomycetes, several species in the wood-decay group were able to tolerate LiCl in the media, whereas all basidiomycete ectomycorrhizal fungi tested were totally inhibited.
D. L. Richter et al.
Acknowledgements Peter E. Laks, MTU Wood Protection Group, provided partial funding and laboratory space, and offered valuable discussions regarding testing of fungi. The authors express appreciation to three anonymous reviewers who provided helpful comments to improve the paper.
references
Budavari S, O’Neil MJ, Smith A, Heckelman PE (eds), 1989. The Merck Index: an Encyclopedia of Chemicals, Drugs and Biologicals, 11th edn. Merck, Rahway, NJ. Jarvis B, 1973. Comparison of an improved rose–bengal–chlortetracycline agar with other media for the selective isolation and enumeration of molds and yeasts in food. The Journal of Applied Bacteriology 36: 723–727. Johnson GC, 1995. A comparison of the efficacy of six media for isolating wood-decaying Hymenomycetes. Canadian Journal of Microbiology 41: 104–107. Kado CI, Heskett G, 1970. Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas, and Xanthomonas. Phytopathology 60: 969–976. Mycological Society of America, Mycology Guidebook Committee, 1974. Handling fungi. In: Mycology Guidebook. University of Washington Press, Seattle, pp. 3–27. Samuels GJ, 1996. Trichoderma: a review of biology and systematics of the genus. Mycological Research 100: 923–935. SAS Institute, 2002–2003. Version 9.1 of the SAS System for Windows Version 5.1. SAS Institute, Cary, NC, USA. Wildman HG, 1991. LiCl as a selective inhibitor of Trichoderma species on soil isolation plates. Mycological Research 95: 1364–1368. Worrall JJ, 1991. Media for selective isolation of hymenomycetes. Mycologia 83: 296–302.
journal homepage: www.elsevier.com/locate/mycres
Differential sensitivity of fungi to lithium chloride in culture media Dana L. RICHTERa,*, Sara C. ROBINSONa, Maria P. BEARDSLEEb, Maureen L. HABARTHb a
School of Forest Resources and Environmental Science, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA b Department of Civil and Environmental Engineering, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA
article info
abstract
Article history:
Forty species of fungi, representing a range of ecological and taxonomic groups, were tested
Received 23 January 2007
for their ability to grow on agar media amended with lithium chloride (LiCl) at 1.5, 3 and 6 g l1.
Received in revised form
Species of Trichoderma varied considerably in their sensitivity to LiCl; at one week on 6 g l1
7 January 2008
LiCl medium, the growth of seven species of Trichoderma was considerably inhibited; how-
Accepted 24 January 2008
ever, by three weeks at this level, four of the species tested were able to attain 30 % of control
Corresponding Editor: Karl Ritz
growth. Of the seven species tested, an isolate of T. viride was the most sensitive to LiCl in agar. Eleven other imperfect fungi also showed a range of ability to grow on agar amended with
Keywords:
LiCl, from total inhibition to complete lack of inhibition. Six ascomycete fungi were greatly
Fungus culture
inhibited by LiCl at all levels; however, an isolate of Chaetomium globosum was highly tolerant
Fungus isolation
of LiCl. Seven basidiomycete wood-decay fungi were quite sensitive to LiCl in agar, showing
Lithium chloride
total to nearly total inhibition even at the lowest level; however, after three weeks, an isolate
Media amendments
of Postia placenta was nearly uninhibited except at 6 g l1. Five ectomycorrhizal basidiomycete
Selective media
fungi were totally inhibited by all levels of LiCl; however, one ectomycorrhizal imperfect fun-
Trichoderma
gus (Cenococcum graniforme) was able to grow at 3 g l1 and was uninhibited at 1.5 g l1. Four
Wood-decay fungi
zygomycete fungus isolates were nearly unaffected in their growth by all levels of LiCl. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Lithium chloride (LiCl) is a non-toxic, alkali metal salt that is soluble in water; in its solid state, it is a white, odourless substance that resembles table salt. It is commonly used as a desiccant, as well as in metallurgy and pyrotechnics (Budavari et al. 1989). Lithium chloride was reported by Kado & Heskett (1970) for use in culture media to increase the efficiency of isolating plant pathogenic bacteria from soil (e.g. Agrobacterium, Corynebacterium, and Erwinia spp.). Its selective nature is due to the salt differentially affecting the permeability of bacterial membranes. During the course of their work, LiCl at 5–7 g l1
was also noted to inhibit fungal colonies that otherwise would obscure the observation of bacterial colonies (Kado & Heskett 1970). Trichoderma spp. can be aggressive colonizers of agar plates when attempting to isolate specific fungi from various substrates. Species of Trichoderma are ubiquitous in soil and rotten wood, and are also parasites of other fungi (Samuels 1996); therefore, they are naturally present wherever fungi exist. In the authors’ experience, Trichoderma spp. are common contaminants in agar plates when attempting to isolate wooddecay fungi from rotten wood and ectomycorrhizal fungi from root tips and mycelial strands.
* Corresponding author. Tel.: þ1 906 487 2149. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.01.013
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D. L. Richter et al.
Following Kado & Heskett’s (1970) work, Wildman (1991) showed that LiCl at 6 g l1 could be used to inhibit the spore germination and growth of Trichoderma spp., increasing the efficiency of isolating other fungi from soil dilution plates; however, the sensitivity of Trichoderma to LiCl was not uniform among species in the genus. Based on the latter paper, LiCl has been used in malt agar plates to prevent overgrowth by Trichoderma spp. when isolating wood-inhabiting stain and decay fungi. However, at 6 g l1 it has been observed that LiCl has the ability to significantly inhibit wood decay and other fungi as well; therefore, 1.5–3 g l1 was adopted as a moderate level of LiCl to inhibit Trichoderma spp. when attempting to isolate stain and decay fungi from very decayed wood. Benomyl (Du Pont, Wilmington, Delaware) is also a common media amendment used in wood-decay and ectomycorrhizal fungus isolation laboratories, but along with preventing the growth of fast-growing imperfect fungi, such as Trichoderma spp. (Johnson 1995; Worrall 1991), Benomyl also
Materials and methods The base medium used for all tests was 2 % malt agar (2 M), prepared by suspending 20 g malt (Bacto, Difco, Lawrence,
Week 1 A
Key to Fungi
A A
90
T.h. = Trichoderma harzianum T.k. = Trichoderma koningii T.l. = Trichoderma longibrachiatum T.po. = Trichoderma polysporum T.ps. = Trichoderma pseudokoningii T.r. = Trichoderma reesei T.v. = Trichoderma viride
A 70 60 50
A
A A
40
T.v.
T.po.
T.h.
T.r.
6.0
A
A
B
A B
50
B B A
C
1.5
3.0
T.h.
B T.v.
T.ps. T.r.
T.h. T.k. T.l. T.po.
T.r. T.v.
T.l. T.po. T.ps.
T.h. T.k.
20
C B
B C
C C
T.v.
30
T.r.
A
40
T.po. T.ps.
Growth as % of Control
3.0
60
0
B C C B
A
70
10
B B
Week 2 A A
A
90 80
T.r. T.v.
1.5
B
C
T.k. T.l.
100
T.ps.
T.h.
T.k. T.l. T.po.
0
T.ps.
A
20 10
B
T.ps. T.r.
B
T.k.
A
A
T.l.
30
T.h.
B
T.k. T.l. T.po.
Growth as % of Control
80
T.v.
100
prevents the isolation of ascomycete fungi, which also cause stain and decay in wood. Rose Bengal is another media amendment used to inhibit fast-growing fungi, while allowing slower-growing fungi and bacteria to be isolated (Jarvis 1973; Mycological Society of America, Mycology Guidebook Committee 1974). Aside from the two papers cited in the previous paragraph, there appears to be little additional information on the use of LiCl in culture media to inhibit Trichoderma spp. or other fast-growing fungi. The purpose of this study was to expose a range of ecological and taxonomic groups of fungi to various levels of LiCl in agar to assess its use as an amendment to selectively inhibit Trichoderma spp. and possibly other fast-growing, contaminating fungi.
6.0
LiCl Concentration (g L-1) Fig 1 – Growth as percent of control of seven species of Trichoderma after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
Differential sensitivity of fungi to lithium chloride
719
Kansas) and 15 g agar (Difco, Lawrence, Kansas) in 1 l distilled water. LiCl (Fisher Scientific, Pittsburgh, PA) was added at 1.5, 3, or 6 g l1 before autoclaving. Approximately 20 ml were poured into 100 mm plates and stored 3–7 d before use. All fungi were grown at these levels of LiCl and compared with growth on control 2 M agar plates (0 g l1 LiCl). All procedures were conducted under sterile conditions in a laminar-flow hood. Six groups of fungi represented by 40 species were tested for growth on LiCl media. These fungi represented the major classes of fungi (ascomycetes, basidiomycetes, imperfect fungi, and zygomycetes) and functional groups of fungi
commonly isolated in forest pathology laboratories (saprotrophs, mould, wood decay, wood stain, ectomycorrhizal). Seven species of Trichoderma were also tested. All fungi are maintained at the School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI. Inoculum for test plates was prepared by growing test fungi on 2 M plates at 22 C to achieve colonies ca 2–4 cm diam (usually 1–2 weeks). Plugs of inoculum 5 mm diam and the thickness of the agar were cut with a sterile cork borer from the perimeter of an actively growing fungus colony. Inoculum
Week 1 100 90
A
A
A
A
A
A
A
A
A
A
70
AB
B
60 50 A
B
A.a. A.c. A.n. A.p. B.c. C.c. C.g.
A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c.
A.a.
10 0
A
A
1.5
C
C
B A
3.0
6.0
Week 2 100 90
A
A
A
A
A
A
A
Key to Fungi
A
A
A
A.a. = Alternaria alternata A.c. = Arthrographis cuboidea A.n. = Aspergillus niger A.p. = Aureobasidium pullulans B.c. = Botrytis cinerea C.c. = Cladosporium cladosporioides C.g. = Cenococcum graniforme P.p. = Penicillium purpurogenum P.v. = Paecilomyces variotii S.c. = Stachybotrys chartarum S.l. = Scytalidium lignicola
A
B
70
B B
50
AB
C
A A
A
40 30
A
B
20 A
1.5
A
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
0
B
A
10
C
A
3.0
C
A C B
B
A
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
Growth as % of Control
80
60
A
A C B
C
A.a. A.c. A.n. A.p. B.c. C.c. C.g. P.p. P.v. S.c. S.l.
A
20
P.p. P.v. S.c. S.l.
30
A
B
A
A
B
B
B
40
S.l.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 2 – Growth as percent of control of 11 species of imperfect fungi after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
720
D. L. Richter et al.
plugs were firmly placed mycelium side down in the centre of a test plate. Three replicate plates were prepared for each LiCl level (1.5, 3, and 6 g l1) for each fungus. Test plates were either wrapped in Parafilm (to contain overgrowth by some fungi) or closed in plastic bags and incubated in the upright position in the dark at 22 C. Growth of fungi was measured weekly for three weeks following inoculation; due to their slower growth mycorrhizal fungi were also measured at six weeks. Growth was measured as the radius of mycelial extension from the edge of the inoculum plug. If the growth of the colony was uniformly circular, only one measurement was taken. If the growth of the colony was irregular, three points of greatest radial extent were measured and an average of these was used to represent total growth. If the growth of a colony reached the edge of the test plate, this was considered ‘maximum growth’. Maximum growth was equal to the radius of the test plate (42.5 mm) minus the radius of the inoculum plug (2.5 mm), or 40 mm. Mean growth of the three replicates for each fungus on each LiCl concentration was determined.
As the fungi tested grow at widely different rates, fungus growth rates on LiCl-amended media were converted to growth as a percent of fungus growth in the absence of LiCl (growth as percent of control), in order to compare growth among fungi within a group (Trichoderma spp., ascomycetes, basidiomycetes, imperfect fungi, and zygomycetes). Following arc sine square root transformation, growth as percent of control data for each fungus response to LiCl level by week were subjected to repeated measures analysis of variance (ANOVA) followed by Tukey’s HSD (P 0.05). Analysis was conducted using SAS software, Version 9.1 of the SAS System for Windows Version 5.1 (SAS Institute 2002–2003).
Results LiCl exerted a wide range of effects on fungus growth depending on the fungus species and the amount of LiCl added to the media. Generally, species of zygomycetes, ascomycetes, and imperfect fungi were more able to tolerate LiCl in media
Week 1 100
Key to Fungi C.a. = Ceratocystis adiposa C.c. = Ceratocystis coerulescens C.f. = Ceratocystis fagacearum C.pic. = Ceratocystis piceae C.pil. = Ceratocystis pilifera C.g. = Chaetomium globosum
A
90
70 60 50
A
A
A
A
3.0
A C.g.
C.pic.
C.a.
B C.c.
C.g.
B
B
C.f.
A C.pil.
C.f.
C.a. C.c.
1.5
C.pic.
B
B C.g.
C.pic.
A
C.pil.
0
C.f.
A C.a.
10
C
A
C.pil.
30 20
6.0
Week 2
100
A
A
90
A
A
80 B
A
70 60 A
B
40
C
30
A
3.0
C
A C.pil. C.g.
C.f.
C
C.pic.
B
C.a. C.c.
A C.g.
C.c.
C.g.
C.pil.
C.f. C.pic. 1.5
B C.a.
0
C.c.
10
B
A
C.f. C.pic.
20
C.pil.
50
C.a.
Growth as % of Control
A
B
40
C.c.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 3 – Growth as percent of control of six species of ascomycete fungi after 1 and 2 weeks on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
Differential sensitivity of fungi to lithium chloride
721
than basidiomycetes; ectomycorrhizal basidiomycetes were the most sensitive of all groups tested. Figs 1–5 show the growth of the fungi tested as a percent of control fungus growth, by fungus group tested, measured after one, two, or three weeks incubation depending on the general growth rate of the group. Different letters above error bars indicate significant differences between LiCl levels by fungus species within week. In some cases, a fungus grew to 100 % of control growth by week one or two; thereafter, no additional growth was recorded due to the limit of the Petri plates. For some species, sensitivity to LiCl decreased over the course of three weeks, as mycelial growth increased from week 1 to week 2 and/or from week 2 to week 3. This pattern of growth was especially evident for Trichoderma koningii, T. polysporum, Ceratocystis adiposa, Chaetomium globosum, Trametes versicolor, and Irpex lacteus, and may represent
adaptation to LiCl in agar media. In the majority of cases, maximum growth by a fungus isolate as percent of control growth was observed during the first two weeks of incubation. Among species of Trichoderma (Fig 1), at one week most isolates were considerably inhibited by 6 g l1 LiCl, being able to attain only 0–8.6 % of control growth, except for T. longibrachiatum, which attained 18.5 % of control growth. At one week 1.5 g l1 LiCl did little to inhibit the species of Trichoderma; most grew from 45–100 % of controls, except for T. koningii and T. viride, which grew to 23 and 21 % of controls, respectively. At week 1, T. harzianum, T. polysporum, and T. reesei grew significantly more than other fungi on 1.5 g l1 LiCl. At two weeks, most species of Trichoderma could grow from 14–23 % of controls on 6 g l1, except T. pseudokoningii and
Week 2 100
Key to Fungi G.t. = Gloeophyllum trabeum I.l. = Irpex lacteus N.l. = Neolentinus lepideus P.g. = Phlebia gigantea P.o. = Pleurotus ostreatus P.p. = Postia placenta T.v. = Trametes versicolor
A
90 B
70 60 A 50
A
40 30
1.5
3.0
B
T.v.
P.g. P.o.
I.l.
N.l.
C C B
G.t.
T.v.
P.p.
P.o.
B
A B
P.p.
B A B P.g.
N.l.
B I.l.
T.v.
P.g.
B P.p.
A
G.t.
0
A
P.o.
A I.l.
10
B
A
N.l.
20
G.t.
Growth as % of Control
80
6.0
Week 3
100
A
90
A A
A
70 60 50 40
A
B
30
1.5
B
3.0
B T.v.
B
P.p.
A
P.o
N.l.
I.l.
B C B G.t.
B T.v.
B
P.o. P.p.
A P.g.
I.l.
G.t.
T.v.
B
N.l.
B
P.g.
A P.p.
I.l.
A P.g.
0
G.t.
10
A
P.o.
20
N.l.
Growth as % of Control
80
6.0
LiCl Concentration (g L-1) Fig 4 – Growth as percent of control of seven species of basidiomycete wood decay fungi after 2 and 3 weeks on 1.5, 3.0, and 6.0 g LL1 LiCl-amended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
722
D. L. Richter et al.
Key to Fungi M.sp.1 = Mucor sp. 1 M.sp.2 = Mucor sp. 2 Mo.sp. = Mortierella sp. Z.sp. = Zygorhynchus sp.
Week 1
100
A
A
A
A
90
A
A
B A
B
80
Growth as % of Control
A
70 60 B
50 40 30
C
0
3.0
Z. sp.
Mo. sp.
M. sp. 2
M. sp.1
Z. sp.
Mo. sp.
M. sp.1
1.5
M. sp .2
Z. sp.
Mo. sp.
M. sp. 2
10
M. sp.1
20
6.0
LiCl Concentration (g L-1) Fig 5 – Growth as percent of control of four isolates of zygomycete fungi after 1 week growth on 1.5, 3.0, and 6.0 g LL1 LiClamended media. Mean growth of 3 plates. Error bars represent one standard deviation. Letters above columns compare individual fungi by LiCl level within week ( p 0.05).
T. viride, which were still totally or nearly totally inhibited. T. longibrachiatum grew significantly less than all other fungi on 1.5 g l1 LiCl, whereas T. harzianum, T. polysporum, T. pseudokoningii, and T. reesei were uninhibited and grew as much as controls. Growth of T. viride was significantly reduced by 3 g l1 LiCl compared with growth on 1.5 g l1. Considering all levels of LiCl together, at two weeks, the mycelial growth sensitivity of seven Trichoderma isolates could be ranked generally in the order (from most sensitive to most tolerant): T. viride > T. longibrachiatum > T. koningii > T. pseudokoningii > T. harzianum > T. reesei > T. polysporum. At 1.5 g l1 LiCl, four species of Trichoderma were uninhibited in growth, whereas three species were able to attain growth of 50–80 % of control. Additionally, at this level, T. longibrachiatum was more sensitive to LiCl in media than T. viride. After three weeks at 6 g l1 LiCl, T. pseudokoningii and T. viride were still totally inhibited, whereas mycelial growth of all other species of Trichoderma ranged from 20–50 % of control. The mycelial growth sensitivity of the 11 imperfect fungi tested was highly variable depending on LiCl concentration (Fig 2). Among the fungus isolates, Alternaria alternata and Aspergillus niger were the most tolerant, demonstrating complete lack of inhibition with even the highest level of LiCl in the media to week 3. Penicillium purpurogenum and Scytalidium lignicola were consistently the most sensitive to LiCl at week 2; P. purpurogenum was able to attain only 10 % of control on 1.5 g l1 LiCl, and S. lignicola was completely inhibited by all
levels of LiCl. Generally, at the highest levels, LiCl slowed the growth of most imperfect fungi considerably; however, lower levels were only moderately inhibitory. Cenococcum graniforme was the only isolate of all fungi tested that exhibited growth stimulation by LiCl, as by week 3, colonies on 1.5 g l1 LiClamended media grew slightly more than control colonies. For the imperfect fungi at week 1, there were no significant differences in growth between levels of LiCl for Alternaria alternata, Aureobasidium pullulans, Cladosporium cladosporioides, P. purpurogenum, and S. lignicola. The aforementioned fungi, along with Aspergillus niger, also had no significant differences in growth between levels of LiCl at week 2. Sensitivity of six species of ascomycete fungi to LiCl after two weeks (Fig 3) could generally be ranked in the order (most sensitive to most tolerant): Ceratocystis pilifera > C. fagacearum > C. adiposa > C. piceae > C. coerulescens > C. globosum, with the exception that C. coerulescens was more sensitive than C. piceae at 1.5 g l1 LiCl. Whereas the isolate of C. pilifera was completely inhibited at all levels of LiCl, the isolate of C. globosum was highly tolerant of LiCl at all levels and grew nearly as much as controls, even by two weeks. At week 1, C. adiposa, C. fagacearum and C. pilifera were considerably inhibited in their growth by all levels of LiCl. C. coerulescens grew in the presence of LiCl, but had significantly different growth between all levels of LiCl. C. globosum was moderately affected by LiCl in media; however, there were no significant differences in growth between levels. At
Differential sensitivity of fungi to lithium chloride
week 2, C. globosum grew nearly as much as controls and again there were no significant differences in growth between levels of LiCl for this isolate. The sensitivity of seven species of basidiomycete wooddecay fungi to LiCl after two and three weeks growth (Fig 4) could be ranked in the order (most sensitive to most tolerant): Phlebia gigantea > Neolentinus lepideus > Pleurotus ostreatus > G. trabeum > Trametes versicolor > Irpex lacteus > Postia placenta. P. gigantea did not grow on any level of LiCl in the media at up to 3 weeks, whereas P. placenta exhibited growth at all levels of LiCl. P. placenta and I. lacteus were the only two fungi to exhibit mycelial growth at 3 g l1, and P. placenta was the only fungus to show any growth at 6 g l1 LiCl. Five species of basidiomycete ectomycorrhizal fungi tested (figure not shown) were completely inhibited in growth by all levels of LiCl, even after six weeks of incubation. However, one imperfect ectomycorrhizal fungus, Cenococcum graniforme, was able to grow considerably at 3 g l1 LiCl and was uninhibited by 1.5 g l1 LiCl (Fig 2). Due to their rapid growth rate, it was most useful to examine differences in LiCl sensitivity of zygomycete fungi after only one week (Fig 5). Although a limited number of species were tested, the isolates of zygomycetes appear to be relatively less affected by LiCl in culture media than other groups of fungi. Although one isolate of Mucor sp. showed sensitivity to LiCl, the other isolate of Mucor sp. was completely uninhibited at all levels of LiCl. Among the zygomycetes, the isolate of Zygorhynchus sp. demonstrated the most sensitivity to LiCl in culture media.
Discussion This research demonstrated that there is a remarkable range of response to LiCl among fungi, even within a taxonomic group. However, as single isolates of species were used in the testing, it is not known how the effect of LiCl may vary within a species due to variation among fungus strains. In addition, LiCl may also alter hyphal density of a fungus colony, and therefore, radial growth may not represent an actual reduction in fungal biomass. However, hyphal extension is a highly important characteristic when isolating fungi on agar plates from a substrate containing a number of different fungi. Knowing which species of fungi are able to grow or which are inhibited by LiCl in culture media is important in the use of this amendment when selecting for specific fungi during isolations from wood, soil or root tips. Using the hyphal growth data at a mid-concentration of LiCl (3 g l1) after two weeks of growth, the fungus isolates tested can be sorted into generalized tolerance groups (Table 1), from a group of fungi intolerant of LiCl (no growth on LiCl-amended media) to a group highly resistant to LiCl (75–100 % of control growth). Fourteen of the 40 species tested could not grow on a moderate level of LiCl, whereas ten of the species tested were highly resistant to LiCl in culture media. Although the only species of Penicillium tested (P. purpurogenum) was the most sensitive of the imperfect fungi to LiCl, Wildman (1991), using media amended with 6 g l1 LiCl and soil dilution plates, showed that there was considerable variation among species of Penicillium with respect to their
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Table 1 – Generalized ranking of fungi according to response to lithium chloride (LiCl) in culture media; data after two weeks growth on 3 g lL1 LiCl-amended media (N [ 3) Tolerance group Intolerant fungi ¼ no growth Ceratocystis adiposa Ceratocystis pilifera Gloeophyllum trabeum Hebeloma arenosa Laccaria bicolour Laccaria laccata Neolentinus lepideus Penicillium purpurogenum Phlebia gigantea Pleurotus ostreatus Rhizopogon rubescens Scytalidium lignicola Suillus granulatus Trametes versicolor
Isolate numbera DR 163-1 ISK ATCC 60758 ATCC 11539 DR 195 DR 378 DR 137 ATCC 12653 ATCC 52427 BURD 5192 MAD 542 DR 401 Highley F-64-F DR 383 MAD 697
Sensitive fungi ¼ growth to 25 % of control Arthrographis cuboidea DR 451 Ceratocystis fagacearum DR 420 Cladosporium cladosporioides ATCC 6271 Irpex lacteus ATCC 11245 Trichoderma viride ATCC 32630 Moderately sensitive fungi ¼ growth between 25–50 % of control Aureobasidium pullulans Forintek 132E Ceratocystis piceae Forintek 387A Trichoderma koningii Forintek 230D Trichoderma longibrachiatum Forintek 259A Trichoderma pseudokoningii Forintek 228B Moderately tolerant fungi ¼ growth between 50–75 % of control Cenococcum graniforme DR G-5-2 Ceratocystis coerulescens DR-ISK-100 Paecilomyces variotii DR 455 Postia placenta MAD 698 Stachybotrys chartarum ATCC 16026 Trichoderma harzianum Highley T-25 J Resistant fungi ¼ growth between 75–100 % of control Alternaria alternata DR 406 Aspergillus niger ATCC 6275 Botrytis cinerea ATCC 28466 Chaetomium globosum ATCC 6205 Mortierella sp. DR 432 Mucor sp. 1 DR 434 Mucor sp. 2 DR 435 Trichoderma polysporum Forintek 260E Trichoderma reesei ATCC 46480 Zygorhynchus sp. DR 433 a Highley, Terry L. Highley, Forest Products Lab, Madison, WI; Forintek, Forintek Mycology Labs, Ottawa, ON; ATCC, American Type Culture Collection, Beltsville, MD; DR, Dana L. Richter; MAD, Forest Products Laboratory, Madison, WI; BURD, Harold H. Burdsall, Forest Products Lab, Madison, WI.
tolerance or sensitivity to LiCl. Wildman (1991) also demonstrated that it was possible to isolate other imperfect fungi from soil on media amended with 6 g l1 LiCl, including Aspergillus spp. and Paecilomyces sp. The present study confirms this, as the isolates of these fungi tested were also able to grow on LiCl-amended media.
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Wildman (1991) focused on Trichoderma spp. response to LiCl, finding that the sensitivity to LiCl is not uniform throughout the genus and that LiCl resistant strains exist. This study verifies these results. However, alhough Wildman (1991) found that T. longibrachiatum was the most sensitive to 6 g l1 LiCl, the current study found that T. pseudokoningii and T. viride were the most sensitive, and that T. longibrachiatum was only moderately inhibited by 6 g l1 LiCl. Conversely, at 6 g l1 LiCl, Wildman (1991) found that T. koningii and T. harzianum were the most tolerant of the species of Trichoderma tested, whereas this study found these species to be only moderately tolerant at this level of LiCl, and found that T. polysporum was the most tolerant of the species of Trichoderma tested. This apparent variation in response to LiCl by the same species of Trichoderma may be due to intraspecific variation within the species tested. Also, Wildman (1991) used a malt agar with 30 g malt þ 5 g peptone l1, whereas the media in this study contained simply 20 g malt l1. Whereas Benomyl is useful to prevent growth of ascomycete and imperfect fungi when isolating basidiomycetes from soil or wood, LiCl may be used to isolate certain ascomycete and imperfect fungi, especially those fungi tolerant to LiCl. LiCl-amended media may be useful in inhibiting certain Trichoderma species when attempting to isolate fungi from wood or soil. However, as some species of Trichoderma and other fast-growing fungi will be inhibited, some target fungi may also be inhibited, even on the lowest level of LiCl. However, this would not work for the isolation of basidiomycetes (except possibly Postia placenta), which generally are slower growing fungi and highly inhibited by LiCl in media. It is striking that among the basidiomycetes, several species in the wood-decay group were able to tolerate LiCl in the media, whereas all basidiomycete ectomycorrhizal fungi tested were totally inhibited.
D. L. Richter et al.
Acknowledgements Peter E. Laks, MTU Wood Protection Group, provided partial funding and laboratory space, and offered valuable discussions regarding testing of fungi. The authors express appreciation to three anonymous reviewers who provided helpful comments to improve the paper.
references
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