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 relat- ively 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 = 6- Benzylamino purine. Benomyl is methyl-(butylcarbamoyl) -2-benzimidazole carbamate.

MBC is Carbendazim (methyl benzimidazole -2-yl-carb- amate).

TBZ is Thiabendazole; 2-(Thiazol-4-y-1) benzimidazole. FBZ is Fenbendazole [5-(Phenylthio)-lH-benzimidazole-

2-yl] carbamic acid methyl ester.

Introduction Plant tissue cultures are sometimes found to be contamin- ated by microorganisms which were either present in the original explant or arise as laboratory contaminants. Gener- ally 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 sub- cultured 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 -20"C. Appropriate volumes of the concentrated solutions of fungicides were added to molten agar medium and plates poured immed- iately. 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 contain- ing 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 Czapek- Dox 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.

Fungic ides Benomyl and Carbendazim (MBC) from Dupont (U.K.) Ltd. Thiabendazole (TBZ) from Merk, Sharp and Dohme. Fenbendazole (FBZ) from Hoechs t Nystatin, Fungizone, Clotrimazole and Griseofulvin were

34

purchased from Sigma. Imazalil and miconazole were a gift from Janssen Pharm- aceutica, Beerse Belgium.

T e s t s o f f u n g i c i d e s o n p l a n t t i s s u e s

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.

2) Root cultures Root cultures in shaken liquid media (MS medium contain- ing 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 (tripli- cate 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.

4) Germination Tobacco seeds were germinated in water containing fungi- cides and germination assessed. Control germinated seeds were placed onto agar-solidified MS medium containing fungicide and progress of germinated seedlings assessed.

R e s u l t s

B e n z i m i d a z o l e f u n g i c i d e s

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 comp- ounds are fungicides, FBZ is used primarily as an anthel- minthic. 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 stimul- atory to callus and inhibitory to roots, at higher doses TBZ is

T a b l e 1

Fungus Type

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

A Paecilomyces B Penicillum (monoverticillate) m ~ m C Mycelia Sterilia

(similar to Rhizoctonia) D Paecilomyces m 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 ~

m

m

m

w

m

m

a

m

m

m

m

m

w

i

m

m

m m

! u

m

m

NT NT NT NT NT NT

Fungi were grown on solidifcd MS 3% sucrose media containing fungi- cides 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

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, IMZ = imazalik MIC = mieonazole, GRI = Grlseofulvin.

PROTOPLASTS

r~

Z~100 U E 8o ~s~

o 60 Z

~ 40 M ~ 20 >

< M

100

100., 80

60

40

20

. 5 10 20 50 100

80

60

CALLUS

~80 160 la0 120 100 80 6o - - - _ 4o 20 0

-20 -40

i~ i i i i ! _ 70 30 40 50

~ 80 ~ 60

• ~ 40 ¢~ 20 O 0

~ -20 -40

< - 6 o ~ - 8 o

" 100

0.5 1.0 5.0 10.0 20.0

10 20

40

20

0.5 1~.0 5'.0

,vO 6O 4O 2O 0

2O 4~) hl ~ SO

Ill 20 31) 40

÷

10 20 30 40 50

DOSE/~g/ml

inhibitory to both but benomyl only to callus. The reason for these discrepancies is unclear, tests with other fungicides shown in fig. 1 show approximate concordance with the effects of fungicides on protoplast derived cells, callus and roots, so it is unlikely that the assay system itself is at fault A more likely explanation is that these compounds are exerting both phytotoxic and hormone like effects. Benomyl has been shwon both to promote and inhibit root and shoot growth depending on its concentration and the plant under test. On the whole TBZ is inhibitory (Schreiber & Hock 1975). In germination tests tobacco seeds germinated normally in the presence of all the benzimidazoles, however subsequent seedling growth was strongly inhibited by TBZ, the seedlings became chlorotic and died (data not shown). The lack of inhibition of germination by benzimidazoles but subsequent inhibition of seedling growth has been noted by others (Schreiber & Hock 1975).

The benzimidazoles which have the least effect on growth at fungicidal concentrations are MBC and FBZ, of these FBZ is the least phytotoxic although it is not quite as effective a fungicide as MBC (table 1). We have used MBC successfully to rid tobacco callus cultures of penicillum contamination.

T h e N - s u b s t i t u t e d i m i d a z o l e s This is an important group of fungicides in clinical and agricultural use. Clotrimazole and miconazole are widely used clinically (Garrod et al 1981), econazole (a close relation to miconazole) has also been used successfully with animal cells in culture (Kunze & Todd 1983). Imazilil is one

ROOTS

1.21 •

1.0 - - ~ -

0.8 I

0.6

0.4

0.2

1.0 :~ ~ 0.8 © ~ 0.6

~ 0.-4 < M 0.2 ,~

1.0

0.8

0.6

04

O2

,; ~'o ;0 4'0 ;o

10 20 30 ~0 50

1'0 ;0 ;0 4

35

Fig. 1. The effects of various fungicides on the growth of plant tissues. Left hand column, effect on plating efficiencies of protoplast derived cells ofN. plumbaginifolia, middle column effect on growth on N. tabacum callus, fight hand column effect on growth of roots of N. taba- cum (transformed with Agrobacterium rhizo- genes A4). Plating efficiency is measured relative to con- trol (control plating efficiencies were greater than 50%). Growth of callus is measured by the increase/decrease in wet weight (meas- ured in mg .) after one week. The lower dotted line represents the initial weight, the upper line the weight increment attained by control call- us (i.e. in absence of fungicide). Relative growth of roots is measured by expressing the ratio of final root weight (after one week) to initial root weight in the presence of the fungicide and dividing this by the corres- ponding ratio for roots without fungicide. The upper dotted line is the level reached by control tissue, the lower represents no growth, below the lower dotted line is therfore weight lost. The first row are benzimidazole fungicides Abenomyl; • MBC; OTBZ; • FBZ. Second row are N substituted imidazole fung- icides • imizalil; m clotrimazole; • miconazole. Third row are fungicides in clinical use []nystatin; @fungizone, a n d + griseofulvin.

of several systemic agricultural fungicides in widespread use (Worthing 1979). The imidazoles are thought to interfere with sterol synthesis required for fungal cell walls (Van den Bossche et al 1980).

Fig. 2. The effect of selected fungicides on root cultures. Acontrol, B 10/xg/m| imazalil; C 10/xg/ml clotrimazole. The experiment is described in the legend to fig. 1.

36

While miconazole and clotrimazole kill protoplast derived cells at levels which are fungicidal (fig 1), imazalil has relatively little toxic effect. Clotrimazole has an extremely detrimental effect on root and callus growth (fig. 1 and 2) as well as on seed germination (not shown). Clotrimazole has however been used successfully in this laboratory to eliminate yeast contaminating coconut embryos without harming the tissue. Imazalil slows germination, has little effect on callus or root growth (fig. 1) but did induce a deformed thickening of lateral roots (fig. 2). Miconazole was toxic to protoplast derived cells (the only tissue tested).

Of the imidazole fungicides tested only imazalil could be recommended for use with plant tissues, and then caution should be exercised. It does however have a fairly broad antifungal spectrum (table 1). The Polyenes This clinically important group of fungicides has also been used with plant and animal cell cultures (Laskey 1970, Watts and King 1973, Garrod et al 1981). These agents appear to function by increasing fungal cell wall permeabil- ity (Medoff and Kobayashi 1980).

We find amphotericin B (Fungizone) is more toxic than nystatin to protoplast derived cells (fig. 1); both drugs inhibit root and callus cultures to various degrees. Other workers have noted that nystatin is less toxic than fungizone to tobacco protoplasts and callus (Watts and King 1973, Chin et al 1979. Rimocidin, another polyene has been shown to be relatively non toxic in short term protoplast cultures (Bancroft et al 1975). The fungicidal activity of these agents in our hands is rather

poor compared with the benzimidazole and N substituted imidazole fungicides (table 1), however we have success- fully used short incubations with nystatin at 10/zg/ml to rid plant cultures of an unidentified yeast contamination (data not shown). In our tests we find nystatin and fungizone will often slow fungal growth considerably without abolishing it. It may be possible to get better fungicidal cover with nystatin if it is frequently replaced in the medium or combined with rifampicin (Beggs et al 1976). Griesofulvin This fungicide is useful clinically (Garrod et al 1981). It works by interfering with cytokinesis but in a different manner than the benzimidazoles. We find that it is relatively non toxic to plant tissues at the concentrations tested (fig. 1). It is also a poor fungicide in our test system (table 1). Griesofulvin at high concentrations causes non disjunction in plant cells (Lo Schiavo et al 1980). Discussion We have tested the effects of a number of agricultural and medically useful fungicides for their effects on numerous fungi as well as on plant cells and tissues. The aim of the work was to see if fungicides could be safely used in plant tissue culture to rid cultures of fungal contamination. The ideal fungicide would kill all species of contaminating fungi without harming plant tissue. While no single fungicide combined both these properties some came close to this ideal. The fungicides which appear most useful in our hand are the benzimidazoles MBC and FBZ (at 30/xg/ml) or imazalil(10--20/xg/ml) amongst the imidazoles. Imazalil does however slow seed germination and affect root growth but has a broad spectrum of antifungal activity.

While we have tested only a small proportion of the large

number of agricultural fungicides available, we have tried most of the fungicides that are in clinical use. On the whole the agricultural fungicides appear to be more useful. If fungi arise in plant cell cultures which cannot be dealt with by the fungicides recommended above, other benzimidazole fungi- cides might be tried. Another large group of systemic fungicides we have not tested are the triazoles which have a similar mode of action to the N-substituted imidazoles. These too may prove useful. Another possibility is to combine non toxic levels of amphotericin or nystatin with rifampicin (which is not toxic at 20/xg/ml (Pollock et al 1983))to provide broad spectrum antifungal activity (Beggs et al 1976) Other possible fungicides that may prove useful are discussed in Brown et al 1982.

We have tested the phytotoxicity of the fungicides on a limited number of tissues from two species of NieotiancL It is possible that the toxicities vary from tissue to tissue and species to species (Schreiber and Hock 1975,Brown et al 1982). In our tests (with the exception of the benzimidaz- oles) there is good concordance between phytotoxicities measured with protoplast derived cells, callus and roots. So far no compound shown to be non toxic in plating efficiency tests has shown to be toxic with other plant tissues (fig. 1, Pollock et al 1983, unpublished observations). The conven- ience and sensitivity of the plating efficiency assay makes it the method of choice for revealing phytotoxicity.

The question also arises as to whether these compounds have deleterious effects not revealed in our tests. Long term effects of the benzimidazoles on plants should be revealed by their widespread agricultural use. The benzimidazoles have been shown to be weakly mutagenic in bacteria (Seller 1975) but not in yeast(Wood 1982). In fungi and plants (at high doses) these compounds can cause chromosomal non disjunction.The N substituted imidazoles do not appear to be mutagenic (Voogd and Van der Stel 1983). In a previous publication we showed that certain antibiotics

can be used safely with plant cells (Pollock 1983),here we show that the principle of using antimicrobial agents in plant cell culture can be extended to fungicides.

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