Plant Cell Reports (1984) 3:33-36
Plant Cell Reports © Springer-Verlag 1984
Use of fungicides in plant tissue culture Robert Shields, Susan J. Robinson, and Pat A. Anslow Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Received October 17, 1983 / Revised version received January 25, 1984- Communicated by I. Potrykus
Abstract We have examined a number of antifungal agents which might prove useful in plant tissue culture. We find that carbendazim, fenbendazole and imazali! can be used relatively safely and also have a broad spectrum of antifungal activity. Fungicides in clinical use do not prove to be as effective.
Abbreviations YEPD = yeast potato dextrose, MS medium is Murashige and Skoog medium purchased from Flow Laboratories. 2,4 D = 2,4 dichlorophenoxyacetic acid. BAP = 6Benzylamino purine. Benomyl is methyl-(butylcarbamoyl) -2-benzimidazole carbamate. MBC is Carbendazim (methyl benzimidazole -2-yl-carbamate). TBZ is Thiabendazole; 2-(Thiazol-4-y-1) benzimidazole. FBZ is Fenbendazole [5-(Phenylthio)-lH-benzimidazole2-yl] carbamic acid methyl ester.
Introduction Plant tissue cultures are sometimes found to be contaminated by microorganisms which were either present in the original explant or arise as laboratory contaminants. Generally contaminated tissue is discarded but in the case of valuable cells it would be useful to use antimicrobial agents to eliminate infections. In a previous report (Pollock et al 1983) we showed that certain medically useful antibiotics have been used successfully to rid cultures of bacterial contamination. Contamination can also be caused by yeast and fungi; in this report we examine various fungicides to see if they can be used against these infections. To be effective, the ideal antifungal agent should be fungicidal in plant tissue culture medium, non toxic to plant cells and have a broad spectrum of fungicidal activity. In medical practice bacterial infection is more common than fungal but with plants the reverse is true. As a result a large number of fungicides are available which are active against plant fungal pathogens. However to our knowledge only a few of these have been tested for effects in plant tissue cultures (Brown et al 1982). Accordingly we have examined a number of fungicides used in animal cell culture as well as some in agricultural and clinical use to see which might be
suitable for use in plant cell culture. We tested the effects of a number of fungicides on (i) Fungi arising as laboratory contaminants of plant tissue culture (ii) Plating efficiency of protoplast- derived cells of haploid N.plumbaginifolia (iii) callus cultures of N. tabacum (iv) Root cultures of N. tabacurn (v) Germination of tobacco seeds (vi) Tobacco seedling growth.
Materials and Methods Growth of fungi Isolates of various fungi contaminating cultures in this laboratory were collected and maintained on solidified YEPD medium. They were subcultured either by taking a loop of spores or a piece of myceliumagitating in lml of Muller Hintons broth, dropping 0.1 ml of this suspension onto fresh agar plates and spreading the solution with a glass spreader. Fungi were cultured at 26 "C in the dark and subcultured every two to three weeks.
Tests of fungicides on funsal growth Fungicides were dissolved (after heating if necessary) in DMSO at 10-20mg/ml and stored a t - 2 0 " C . Appropriate volumes of the concentrated solutions of fungicides were added to molten agar medium and plates poured immediately. Fungi were spread on these plates as described above. Fungicides were tested both on YEPD agar and on solidified plant tissue culture medium (MS medium containing 3% sucrose 0.9% agar). Growth of fungi was assessed every two days for up to four weeks.
Identification of Fungi Fungal strains were placed on malt extract agar (Oxoid) pH 5.4, potato dextrose agar(Oxoid) pH 5.6 modified CzapekDox agar pH 6.8 and plate count agar pH 7.0. At regular intervals during incubation at 25"C for up to two months the morphological structures of each fungus were examined microscopically.
Fungicides Benomyl and Carbendazim (MBC) from Dupont (U.K.) Ltd. Thiabendazole (TBZ) from Merk, Sharp and Dohme. Fenbendazole (FBZ) from H o e c h s t Nystatin, Fungizone, Clotrimazole and Griseofulvin were
34
4) Germination
purchased from Sigma. Imazalil and miconazole were a gift from Janssen Pharmaceutica, Beerse Belgium. Tests of fungicides
Tobacco seeds were germinated in water containing fungicides and germination assessed. Control germinated seeds were placed onto agar-solidified MS medium containing fungicide and progress of germinated seedlings assessed.
on plant tissues
1) Protoplasts The effects of various doses of fungicides on the plating efficiency of haploid N. plumbaginifolia protoplasts was assessed as previously described (Pollock et al 1983). The original N. plumbaginifolia plants were a gift of J.P. Bourgin~INRA, Versailles, France.
Results Benzimidazole
2) Root cultures Root cultures in shaken liquid media (MS medium containing 3% sucrose ) were initiated from roots of tobacco plants regenerated fromAgrobacterium rhizogenes (Strain A4) transformed tobacco tissue (gift of D. Tepfer, I.N.1LA., Versailles, France). Root cultures proliferated rapidly and after a week in culture a dense root mat was formed (fig. 2). The roots were blotted dry and cut into sections weighing from 8 0 - 1 0 0 m g . These sections were weighed, placed in suspension culture with various doses of fungicides (triplicate flasks for each dose) and cultured for a week. The roots were then blotted and reweighed. Growth is expressed as the ratio of final to initial root weight, and relative growth is the ratio of this fraction to that of control tissue (i.e. incubated without fungicides). Similar results were obtained if the roots were dried in a vacuum oven, weighed and growth expressed as a ratio of weight of dried roots to that of control roots.
3) Callus cultures Growth of callus was assessed by plating 0.5 ml of a rapidly growing suspension culture of N. tabacum onto 7 cm filter papers (Whatman no.2) placed on solidified culture medium (MS 3% sucrose, 0.5mg/12,4 D; 0.5mg/1BAP, 0.9% agar) containing fungicides in 10cm petri dishes. After a week the filter papers and callus were weighed and growth measured. Growth is expressed as the increment/decrement in weight of callus after one week. Table
fungicides
These compounds are in widespread use as systemic agricultural fungicides and appear to act by interfering with fungal microtubules and hence fungal cytokinesis (Roy and Fantes 1982 ). We tested Benomyl, Carbendazim (MBC) Thiabendazole (TBZ) and Fenbendazole (FBZ). The first three compounds are fungicides, FBZ is used primarily as an anthelminthic. Benomyl is broken down in use to MBC and butyl isocyanate, MBC is considered to be the active fungicide (Hammerschlag & Sisler 1973). The benzimidazoles had good antifungal activity against the fungi tested (table 1). Fungi that are resistant to the benzimidazoles are resistant up to 100/xg/ml of the drugs. As expected the antifungal activities of benomyl and MBC are very similar, the exception may be due to the activity of the volatile butyl isocyanate breakdown product of benomyl which has been observed to be fungicidal by others (Hammerschlag & Sisler 1973). The plating efficiencies of protoplast derived cells of N. plumbaginifolia in the presence of these fungicides is shown in fig. 1. Benomyl was completely toxic at all doses, since MBC is far less toxic we assume that the volatile breakdown product butylisocyanate is killing the cells in the tight lidded petri dishes used in these experiments. The least toxic compounds of this group in this assay is FBZ. FBZ is also non toxic to callus and root cultures and may even be stimulatory to callus at low concentration (fig 1). MBC also has little detrimental effect on callus or roots, however TBZ and Benomyl at low doses appear stimulatory to callus and inhibitory to roots, at higher doses TBZ is
1
Fungus Type A Paecilomyces B Penicillum (monoverticillate) C Mycelia Sterilia (similar to Rhizoctonia) D Paecilomyces E Actinomycete F Dematiaceous hyphomycete G Mycelia Sterilia H Mycelia Sterilia I Yeast (Rhodoturula) J Unidentified sporulating fungi K NT L Ascomycete M NT N Paecilomyces
FUNGICIDE MBC FBZ TBZ BEN NYS FUNG CLO IMZ MIC GRI 30~g/ml 30/xg/ml 50/.tg/ml 50/zg/ml 10/xg/ml 10/.tg/ml 10/xg/ml 20/xg/ml 20#g/ml 20~g/ml m
~
m
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m
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NT NT
NT NT
NT NT
m
Fungi were grown on solidifcd MS 3% sucrose media containing fungicides as described in materials and methods. If no fungai growth was observed after 3 weeks of growth the fungicides were deemed effective. T h e - s y m b o l indicates no growth. Not all the fungi could be classified as
m
w
they failed to sporulate. Fungi J, K and M were lost before they could be examined, NT means not tested, BEN = Benomyl, NYS = nystatin, FLING = Fungizone, CLO = elotrimazole, I M Z = imazalik MIC = mieonazole, GRI = Grlseofulvin.
100.,
35
60 40 20 .
5
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50 100
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Plant Cell Reports © Springer-Verlag 1984
Use of fungicides in plant tissue culture Robert Shields, Susan J. Robinson, and Pat A. Anslow Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Received October 17, 1983 / Revised version received January 25, 1984- Communicated by I. Potrykus
Abstract We have examined a number of antifungal agents which might prove useful in plant tissue culture. We find that carbendazim, fenbendazole and imazali! can be used relatively safely and also have a broad spectrum of antifungal activity. Fungicides in clinical use do not prove to be as effective.
Abbreviations YEPD = yeast potato dextrose, MS medium is Murashige and Skoog medium purchased from Flow Laboratories. 2,4 D = 2,4 dichlorophenoxyacetic acid. BAP = 6Benzylamino purine. Benomyl is methyl-(butylcarbamoyl) -2-benzimidazole carbamate. MBC is Carbendazim (methyl benzimidazole -2-yl-carbamate). TBZ is Thiabendazole; 2-(Thiazol-4-y-1) benzimidazole. FBZ is Fenbendazole [5-(Phenylthio)-lH-benzimidazole2-yl] carbamic acid methyl ester.
Introduction Plant tissue cultures are sometimes found to be contaminated by microorganisms which were either present in the original explant or arise as laboratory contaminants. Generally contaminated tissue is discarded but in the case of valuable cells it would be useful to use antimicrobial agents to eliminate infections. In a previous report (Pollock et al 1983) we showed that certain medically useful antibiotics have been used successfully to rid cultures of bacterial contamination. Contamination can also be caused by yeast and fungi; in this report we examine various fungicides to see if they can be used against these infections. To be effective, the ideal antifungal agent should be fungicidal in plant tissue culture medium, non toxic to plant cells and have a broad spectrum of fungicidal activity. In medical practice bacterial infection is more common than fungal but with plants the reverse is true. As a result a large number of fungicides are available which are active against plant fungal pathogens. However to our knowledge only a few of these have been tested for effects in plant tissue cultures (Brown et al 1982). Accordingly we have examined a number of fungicides used in animal cell culture as well as some in agricultural and clinical use to see which might be
suitable for use in plant cell culture. We tested the effects of a number of fungicides on (i) Fungi arising as laboratory contaminants of plant tissue culture (ii) Plating efficiency of protoplast- derived cells of haploid N.plumbaginifolia (iii) callus cultures of N. tabacum (iv) Root cultures of N. tabacurn (v) Germination of tobacco seeds (vi) Tobacco seedling growth.
Materials and Methods Growth of fungi Isolates of various fungi contaminating cultures in this laboratory were collected and maintained on solidified YEPD medium. They were subcultured either by taking a loop of spores or a piece of myceliumagitating in lml of Muller Hintons broth, dropping 0.1 ml of this suspension onto fresh agar plates and spreading the solution with a glass spreader. Fungi were cultured at 26 "C in the dark and subcultured every two to three weeks.
Tests of fungicides on funsal growth Fungicides were dissolved (after heating if necessary) in DMSO at 10-20mg/ml and stored a t - 2 0 " C . Appropriate volumes of the concentrated solutions of fungicides were added to molten agar medium and plates poured immediately. Fungi were spread on these plates as described above. Fungicides were tested both on YEPD agar and on solidified plant tissue culture medium (MS medium containing 3% sucrose 0.9% agar). Growth of fungi was assessed every two days for up to four weeks.
Identification of Fungi Fungal strains were placed on malt extract agar (Oxoid) pH 5.4, potato dextrose agar(Oxoid) pH 5.6 modified CzapekDox agar pH 6.8 and plate count agar pH 7.0. At regular intervals during incubation at 25"C for up to two months the morphological structures of each fungus were examined microscopically.
Fungicides Benomyl and Carbendazim (MBC) from Dupont (U.K.) Ltd. Thiabendazole (TBZ) from Merk, Sharp and Dohme. Fenbendazole (FBZ) from H o e c h s t Nystatin, Fungizone, Clotrimazole and Griseofulvin were
34
4) Germination
purchased from Sigma. Imazalil and miconazole were a gift from Janssen Pharmaceutica, Beerse Belgium. Tests of fungicides
Tobacco seeds were germinated in water containing fungicides and germination assessed. Control germinated seeds were placed onto agar-solidified MS medium containing fungicide and progress of germinated seedlings assessed.
on plant tissues
1) Protoplasts The effects of various doses of fungicides on the plating efficiency of haploid N. plumbaginifolia protoplasts was assessed as previously described (Pollock et al 1983). The original N. plumbaginifolia plants were a gift of J.P. Bourgin~INRA, Versailles, France.
Results Benzimidazole
2) Root cultures Root cultures in shaken liquid media (MS medium containing 3% sucrose ) were initiated from roots of tobacco plants regenerated fromAgrobacterium rhizogenes (Strain A4) transformed tobacco tissue (gift of D. Tepfer, I.N.1LA., Versailles, France). Root cultures proliferated rapidly and after a week in culture a dense root mat was formed (fig. 2). The roots were blotted dry and cut into sections weighing from 8 0 - 1 0 0 m g . These sections were weighed, placed in suspension culture with various doses of fungicides (triplicate flasks for each dose) and cultured for a week. The roots were then blotted and reweighed. Growth is expressed as the ratio of final to initial root weight, and relative growth is the ratio of this fraction to that of control tissue (i.e. incubated without fungicides). Similar results were obtained if the roots were dried in a vacuum oven, weighed and growth expressed as a ratio of weight of dried roots to that of control roots.
3) Callus cultures Growth of callus was assessed by plating 0.5 ml of a rapidly growing suspension culture of N. tabacum onto 7 cm filter papers (Whatman no.2) placed on solidified culture medium (MS 3% sucrose, 0.5mg/12,4 D; 0.5mg/1BAP, 0.9% agar) containing fungicides in 10cm petri dishes. After a week the filter papers and callus were weighed and growth measured. Growth is expressed as the increment/decrement in weight of callus after one week. Table
fungicides
These compounds are in widespread use as systemic agricultural fungicides and appear to act by interfering with fungal microtubules and hence fungal cytokinesis (Roy and Fantes 1982 ). We tested Benomyl, Carbendazim (MBC) Thiabendazole (TBZ) and Fenbendazole (FBZ). The first three compounds are fungicides, FBZ is used primarily as an anthelminthic. Benomyl is broken down in use to MBC and butyl isocyanate, MBC is considered to be the active fungicide (Hammerschlag & Sisler 1973). The benzimidazoles had good antifungal activity against the fungi tested (table 1). Fungi that are resistant to the benzimidazoles are resistant up to 100/xg/ml of the drugs. As expected the antifungal activities of benomyl and MBC are very similar, the exception may be due to the activity of the volatile butyl isocyanate breakdown product of benomyl which has been observed to be fungicidal by others (Hammerschlag & Sisler 1973). The plating efficiencies of protoplast derived cells of N. plumbaginifolia in the presence of these fungicides is shown in fig. 1. Benomyl was completely toxic at all doses, since MBC is far less toxic we assume that the volatile breakdown product butylisocyanate is killing the cells in the tight lidded petri dishes used in these experiments. The least toxic compounds of this group in this assay is FBZ. FBZ is also non toxic to callus and root cultures and may even be stimulatory to callus at low concentration (fig 1). MBC also has little detrimental effect on callus or roots, however TBZ and Benomyl at low doses appear stimulatory to callus and inhibitory to roots, at higher doses TBZ is
1
Fungus Type A Paecilomyces B Penicillum (monoverticillate) C Mycelia Sterilia (similar to Rhizoctonia) D Paecilomyces E Actinomycete F Dematiaceous hyphomycete G Mycelia Sterilia H Mycelia Sterilia I Yeast (Rhodoturula) J Unidentified sporulating fungi K NT L Ascomycete M NT N Paecilomyces
FUNGICIDE MBC FBZ TBZ BEN NYS FUNG CLO IMZ MIC GRI 30~g/ml 30/xg/ml 50/.tg/ml 50/zg/ml 10/xg/ml 10/.tg/ml 10/xg/ml 20/xg/ml 20#g/ml 20~g/ml m
~
m
m
i
m
m
m
a
m
m
~ ~ ~
m
m
m
~
m
!
w
m
u
m
m
~
~
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~
m
m
NT NT
NT NT
NT NT
m
Fungi were grown on solidifcd MS 3% sucrose media containing fungicides as described in materials and methods. If no fungai growth was observed after 3 weeks of growth the fungicides were deemed effective. T h e - s y m b o l indicates no growth. Not all the fungi could be classified as
m
w
they failed to sporulate. Fungi J, K and M were lost before they could be examined, NT means not tested, BEN = Benomyl, NYS = nystatin, FLING = Fungizone, CLO = elotrimazole, I M Z = imazalik MIC = mieonazole, GRI = Grlseofulvin.
100.,
35
60 40 20 .
5
10
20
50 100
E 8o ~s~ o 60 Z
_
M ~ 20 >
