Phenotypic degeneration occurs during sector ... - Wiley Online Library

Journal of Applied Microbiology 2002, 93, 163–168

Phenotypic degeneration occurs during sector formation in Metarhizium anisopliae M.J. Ryan1*, P.D. Bridge2, D. Smith2 and P. Jeffries1 1

Research School of Biosciences, University of Kent, Canterbury and 2CABI Bioscience, UK Centre (Egham), Egham, Surrey, UK

345/10/01: received 31 October 2001, revised 19 March 2002 and accepted 3 April 2002

M . J . R Y A N , P . D . B R I D G E , D . S M I T H A N D P . J E F F R I E S . 2002.

Aims: The formation of sectors was observed during subculturing of an isolate of the entomopathogenic fungus Metarhizium anisopliae, a fungus used for biological control of insect pests. The aim of the investigation was to establish whether sector formation was accompanied by changes in physiological characters. Methods and Results: Four degenerative morphological states, with reduced sporulation capacity, were characterized. Subcultures were taken from each sector and four new culture lines established. The new lines were further subcultured every 21 d. A physiological assessment of each line was undertaken after 42 d using TLC of secondary metabolites and fluorogenic enzyme tests. Full sporulation capacity was not regained on subculture, although some cultures recovered partially. Changes in secondary metabolite profiles and the loss in detection of activity of specific enzymes were observed. Conclusions: Sector formation was frequently accompanied by changes in the ability to produce secondary metabolites and enzymes. Significance and Impact of Study: The results illustrate the importance of maintaining the stability of important cultures during routine subculture. The consequences could have significant implications if degenerate cultures are used as inocula for liquid fermentation cultures or industrial scale production.

INTRODUCTION Entomopathogenic species of Metarhizium are used commercially as biological control agents of insect pests such as the desert locust Schistocerca gregaria (Prior 1992). Successful application in the field requires accurate formulation to ensure viability and stability of the fungal product during inoculum storage (Moore et al. 1993). It is also necessary to maintain stock cultures of the appropriate fungal strains for inoculum production. Maintenance of parent cultures must be tightly controlled and monitored to ensure the fungus

Correspondence to: CABI Bioscience, UK Centre (Egham), Bakeham Lane, Egham, Surrey, UK, TW20 9TY (e-mail: [email protected]). * Present address: CABI Bioscience, UK Centre (Egham), Bakeham Lane, Egham, Surrey, TW20 9TY, UK. Present address: School of Biological and Chemical Sciences, Birkbeck College, University of London, Malet Street, London, WC1E 7HX, UK. ª 2002 The Society for Applied Microbiology

retains its pathogenicity, together with other characters important for biocontrol performance. Strains of Metarhizium are commonly maintained on Sabouraud Dextrose Agar, a nutrient agar that promotes excellent vegetative growth and sporulation. However, sectors are commonly formed on agar culture, that differ in cultural morphology from that exhibited by the parent culture; for example, some sectors do not sporulate. Kirk et al. (2001) describe the formation of sectors as ‘mutation’ or selection in plate cultures resulting in one or more sectors of the culture having a changed form of growth. Factors that reduce genetic stability in fungi range from spontaneous mutations during DNA replication to induced changes to genome structure and content (Smith 1999). Prosser (1993) concluded that sector formation could result from atypical growth, although the mechanism for this could not be explained. Kim (1997) observed attenuation in cultures of Fusarium oxysporum f.sp. niveum such that after 18 successive subcultures, sectors were detected that exhibited variation in colonial morphology and pigmentation.

164 M . J . R Y A N ET AL.

However, it was concluded that sector characteristics remained stable, even after further subculture. Stock cultures of wood-decay basidiomycetes are also known to be subject to progressive senescence, and show an increased tendency to sector in culture (Gramss 1991). Any changes in the characteristics important for the efficacy of a commercially important fungus maintained in plate culture could be problematical, as for example in the loss of pathogenicity in a strain used for biological control programmes. Personnel who routinely subculture stock cultures usually recognize abnormal growth and avoid subculturing from sectored regions. However, inexperienced staff or covert sectoral changes may result in subcultures being initiated from sectored mycelium. In some cases, such subcultures revert to type, as in the red variant of Aspergillus nidulans, although the frequency of reversion may decrease on successive subculture (Grindle 1964). In this investigation, using Metarhizium, subcultures were deliberately taken from sectors, which were then maintained as independent lines. The changes in culture morphology and physiological activity of replicates of each line subcultured from the sectors was compared using TLC of secondary metabolites and extracellular enzyme tests. The objectives were to establish whether changes in the culture morphology of Metarhizium, as a result of sector formation, were accompanied with changes in physiology. M A T E R I A LS A N D M E T H O D S Culture maintenance and selection During a study of the effects of preservation on culture viability, a series of five replicate cultures were established at 25 C on Sabouraud Dextrose Agar (SDA; Oxoid, UK) from each of two different sources of the same strain of Metarhizium anisopliae IMI 382472. The sources of the parent cultures comprised either (i) cultures grown at 25 C on 2% (w/v) Malt Extract Agar (MEA; Oxoid, UK) maintained by frequent (every two months) subculture or (ii) cultures obtained from cryopreserved specimens. The strain had originally been isolated from Cochliotus melolonthoides in 1997 and preserved by continual subculture. After 28 d growth, sectoring was observed in one of the replicates of the fresh cultures from the continuously subcultured sample, and in three of the replicate cultures from the cryopreserved samples. The culture morphology of each sector was noted and then a further three replicate subcultures were established from these four sectored cultures by taking inoculum from sectored areas of the mycelium from the different sources (Fig. 1). The first (‘line A’) was subcultured from the limited sporulating sector of the culture that had been maintained by continual

Source* A

B

C

D

Parent cultures 1st subculture (Primary cultures) 2nd subculture† (Secondary cultures) Secondary cultures (grown for 21 d) 1 2 3 4 – – 5 6 – – 7 8 9 10 – Secondary cultures used for analysis

Fig. 1 Culture histories of lines investigated. (d, culture exhibiting sector; s, culture not exhibiting sector). *Culture line A was established from the limited sporulating sector of the culture that had been maintained by continual subculture on MEA. Culture lines B and C were established from cultures that had originally been cryopreserved from sectors showing limited sporulation. Culture line D was established from cultures that had originally been cryopreserved from a sector that was nonsporulating. Cultures 7 and 9 were taken from nonsectored regions whilst cultures 8 and 10 were taken from sectors exhibiting recovery of the ability to sporulate in the same primary subcultures

subculture on MEA. The three remaining subcultures came from sectors on the three cultures that had originally been cryopreserved: two (‘lines B and C’) from sectors showing limited sporulation, and the third (‘line D’) from another sector that was nonsporulating. Subcultures were taken as mycelial blocks (approx. 10 mm2) from each sector and maintained on SDA at 25 C for 21 d. At this stage each primary subculture was examined for cultural morphology and sporulation prior to a further set of three replicate secondary subcultures being made from each primary subculture. If the primary subcultures exhibited sectoring at this stage then two sets of secondary subcultures were made (Fig. 1), one from the nonsporulating sector and one from the sector showing recovery of sporulation. These were then incubated for a further 21 d. This resulted in a total of 45 cultures at 42 d after initial subculturing. From these, 10 cultures were selected as representing the different subcultural histories and each was subjected to physiological assessment using extracellular enzyme tests and TLC of secondary metabolites. Fluorogenic extracellular enzyme tests Ten 4-methylumbelliferyl (4MU; Sigma, UK) derivatives (Table 1) were selected for testing. The substrates were chosen because the enzymes assayed for had been shown to be present in the fungi considered. These included enzymes that had been considered as differential characters between fungal populations (Wasfy et al. 1987; Bridge et al. 1993) and enzymes where variability had previously been evident

ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 93, 163–168

SECTORIZATION IN METARHIZIUM

Table 1 Methylumbelliferyl (4MU) substrates used to detect a panel of 10 enzyme activities No.

Substrate

Enzyme detected

1 2 3 4 5 6 7 8 9 10

4MU 4MU 4MU 4MU 4MU 4MU 4MU 4MU 4MU 4MU

b-Glucosamidase a-Arabinofuranosidase b-Chitobiosidase a-Fucosidase b-Galactosidase b-Glucosidase b-Glucuronidase a-Mannosidase b-Xylosidase Esterase

N-acetyl-b-D-glucosamide a-L-arabinofuranoside b-D-N-N´-diacetylchitobioside a-L-fucoside b-D-galactoside b-D-glucoside b-D-glucuronide a-D-mannopyranoside b-D-xyloside butyrate

Table 2 Classification of cultural morphology Type

Culture morphology

Sporulation

1 2 3 4

Typical Typical Typical Atypical

Normal Reduced Non-sporulating Non-sporulating

(Bridge et al. 1986). The selected enzymes are produced at different stages of growth and so give an overall picture of the physiological status of the test organism (Barth and Bridge 1989). Substrates were prepared according to the method of Barth and Bridge (1989). In each case, 50 ll of culture fluid, taken from cultures grown in glucose yeast medium (GYM; Mugnai et al. 1989) for 7 d at 30 C was mixed with 50 ll of substrate solution in a well of a 96-well microtitre plate (U well, BDH, UK). Each test was duplicated. Controls of 50 ll substrate solution, 50 ll)1 water, and 50 ll sterile culture fluid 50 ll)1 water were included on each plate. Plates were incubated for 4 h at 37 C. Fifty microlitres of saturated sodium bicarbonate (BDH, UK) solution was then added to each reaction and the plates examined on a UV transilluminator (UVP, Cambridge, UK). Enzyme activity produced a blue fluorescence, whereas the absence of enzyme activity resulted in no fluorescence. For a reference control, similar enzyme Table 3 Summary of culture morphology exhibited by lines of Metarhizium anisopliae isolate IMI 382472, originally subcultured from sectors after 3 subcultures

165

assays were carried out on parent cultures that showed a typical colonial appearance of M. anisopliae and had no apparent sectors. TLC of extracellular secondary metabolites TLC was carried out using the agar plug method described by Paterson (1986). Isolates were grown in the dark on yeast extract-sucrose (YES; Scott et al. 1970) agar for 28 d at 25 C. Agar plugs were cut with a 5-mm diam cork borer and applied for 10 s to a Silica gel 60 TLC plate (BDHMerck, UK). Griseofulvin (1 lg ml)1; Sigma) in a two : one (v/v) mixture of chloroform/methanol was also applied to the plate as a reference compound of known RF. The experiment was run in duplicate. Once dry, plates were placed in an equilibrated TLC tank containing 100 ml of TEF (Toluene/Ethylene/Formic acid; 80 : 10 : 10 v/v/v) solvent. Plates were examined under white light, long-wave UV (365 nm), short-wave UV (254 nm) and a combination of short-wave followed by long-wave UV again and spot characters recorded. To allow the detection of additional secondary metabolites the plates were then sprayed with 0Æ5% (v/v) p-anisaldehyde (Sigma) in ethanol/acetic acid/ concentrated sulphuric acid (17 : 2 : 1 v/v/v) and heated for 8 min at 105 C before being examined again under the lighting regimes described above. To ensure correct classification of metabolites, profiles were compared after the mean and standard error RF values were calculated for individual spots showing similar properties (colour and RF value). Metabolite profiles from original parent cultures were again used as reference controls. RESULTS Culture characteristics The sectors formed in cultures of Metarhizium isolate IMI 382472 were classified into four types based on criteria derived from analysis of culture morphology and the extent of sporulation (Table 2). The typical culture morphology exhibited by the original isolate consisted of mycelium producing aerial hyphae and conidiophores. However, some

Replicate

Mycelium morphology

Sporulation

Type*

A1,2,3 B4 C5 C6 D7,9 D8,10

Typical Atypical Typical Typical Typical Typical

Limited sporulation Non-sporulating V. limited sporulation Limited sporulation Non-sporulating Limited sporulation

2 4 3 2 3 2

(2) (4) (2) (2) (3) (3)

* Culture morphology type (Table 2), brackets refer to morphology of original sector from which line was subcultured. ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 93, 163–168

X X X X X X X X X X X X X X X X X X X X X X X • X X X X • • X • • • • • • • • • • • • • • • • X • • • • • • • X • • • X X • • • • •

•, enzyme activity detected; X, enzyme activity not detected.

X X X X X X X X X X X • • • • X • • • • • •

• • • • • • • • • • •

b-Xylosidase a-Mannosidase b-Glucuronidase b-Galactosidase b-Glucosidase a-Fucosidase b-Chitobiosidase

1 2 3 4 5 6 7 8 9 10 Control (from original culture)

The formation of sectors in cultures of Metarhizium anisopliae IMI 382472 is a degenerative process. Sectors differed from the parent culture in morphology, secondary metabolite and enzyme production. As each parameter was

a-Arabinofuranosidase

DISCUSSION

b-Glucosamidase

The original culture produced a profile comprised of eight extracellular secondary metabolites. These profiles changed dramatically on sectoring, and only replicate six exhibited the extracellular secondary metabolite profile that was characteristic of the original isolate (Table 5). All of the other replicates lost or gained additional extracellular secondary metabolites. For example, replicates four, seven and nine gained two extra metabolites [RF (· 100) 59Æ3, 61Æ3] and lost one [RF (· 100) 55Æ7]. The remaining replicates lost either one or two metabolites from the original extracellular secondary metabolite profile.

Culture

TLC of secondary metabolites

Enzyme assayed

The enzyme activities of replicates from each line are presented in Table 4. Four subcultures (two, seven, eight and 10) retained the enzyme profile that was characteristic of the original isolate, shown as the control. However, the others did not. Of these, six of the cultures failed to produce one or more enzymes as compared to the control (Table 4). Replicate four did not produce b-glucosidase. a-Fucosidase activity was only retained by replicates two, three, four, seven, eight, nine and 10. b-Glucosamidase activity was retained by all replicates except five.

Table 4 Enzyme activities of lines and replicates derived from sectors of Metarhizium anisopliae isolate IMI 382472

Fluorogenic enzyme assays

Esterase

sectors exhibited atypical culture morphology, which consisted of resupinate mycelium. None of the sectors formed in this investigation produced conidia to the extent exhibited by the mycelium from which they arose and some sectors ceased to sporulate altogether. Subcultures of the lines derived from sectors rarely recovered the cultural morphology exhibited by the original isolate (Table 3). However, on primary subculture, two replicates subcultures from line D and one replicate culture from line C, originally subcultured from a nonsporulating sector exhibiting typical culture morphology (type three), formed further sectors which recovered the ability to sporulate. However, sporulation was limited and no cultures recovered levels of sporulation typical of the original isolate. Further sectorization was observed in many of the subcultures, but they either retained a cultural morphology similar to that exhibited by the mycelium from which they arose or they degenerated further. Subcultures from atypical sectors of line B (type four) did not show any evidence of recovery.

• • • • • • • • • • •

166 M . J . R Y A N ET AL.


SECTORIZATION IN METARHIZIUM

167

Table 5 Extracellular secondary metabolite profiles of the lines of Metarhizium anisopliae isolate IMI 382472 derived from sectors Mean RF (· 100) ± S.E.

RF 1 7Æ6 ± 0Æ1

RF 2 11Æ0 ± 0Æ1

RF 3 16Æ2 ± 0Æ2

RF 4 30Æ3 ± 0Æ3

RF 5 39Æ5 ± 0Æ3

RF 6 49Æ8 ± 0Æ3

RF 7 55Æ7 ± 0Æ5

RF A 59Æ3 ± 0Æ1

RF B 61Æ3 ± 0Æ1

RF 8 96Æ5 ± 1Æ6

Line/rep 1 2 3 4 5 6 7 8 9 10 Control

• • • • • • • • • • •

• • • • • • • • • • •

X • • • • • • • • • •

• • • • X • • X • • •

X X X • X • • • • • •

• • • • • • • • • • •

• • • X • • X X X X •

X X X • X X • X • X X

X X X • X X • X • X X

• • • • • • • • • • •

RF’s 1–8 refer to the original extracellular secondary metabolite profile of Metarhizium spp. isolate IMI 382472; RF A and RF B are additional secondary metabolites to the original profile; •, metabolite detected; X, metabolite not detected; control, from nonsectoring replicate of isolate IMI 382472.

assessed independently, no direct correlations were assumed between enzyme and secondary metabolite production. Culture morphology could be classified into one of four types based on cultural morphology and sporulation. Type four represented the most severe form of degeneration as no spores or aerial conidiophores were produced. Sporulation was reduced and completely absent in some lines. Once the ability to sporulate was lost it was rarely recovered. However, replicates of some lines that exhibited a complete loss of sporulation, formed further sectors, some of which recovered limited sporulation. Kim (1997) found that sector morphology in Fusarium remained stable during subsequent subcultures. In our investigation, although cultural morphology usually remained unchanged or degenerated further on subculture, the formation of further sectors could result in a limited recovery of cultural morphology. The loss of sporulation capacity of whole cultures of Fusarium following a period of successive subculture is well known and nonsporulating mycelial sectors may occur through similar degradative changes. Wing et al. (1995) showed that sporulative degeneration was not necessarily associated with a loss of ability to produce characteristic secondary metabolites, as also noted here for Metarhizium. Formation of sectors also resulted in changes in enzyme production. Only three replicates of line D and one replicate of line A exhibited enzyme profiles that were characteristic of that produced by the original isolate. All other replicates lost enzyme activities relative to the expected profile. One replicate (five) lost the activities of three enzymes relative to the original profile, which indicates more serious changes in physiological activity. Bridge et al. (1993) showed that isolates of M. anisopliae could be distinguished from those of M. flavoviride by the production of a-fucosidase. The loss of

this enzyme activity from three of the subcultures suggests that this character might not be stable. The results of the enzyme tests and TLC of secondary metabolites suggested that the formation of sectors was associated with physiological instability. Formation of sectors also resulted in frequent changes in secondary metabolite production, but in this case, gains as well as losses were recorded. Only one subculture from the lines originally cultured from a sector exhibited an extracellular secondary metabolite profile that was characteristic of the original isolate. Changes in the extracellular secondary metabolite profiles were associated with the degenerative states of cultural morphology. Three out of the four test replicates that exhibited type three or type four degenerative cultural morphology differed most from the metabolite profile exhibited by the original isolate. The mechanisms responsible for the formation of sectors and changes in physiological integrity have not yet been determined. For example, are changes due to gradual or spontaneous physiological or environmental adaptations or due to phenotypic or genetic factors such as movement of transposons or parasexual processes? Stability is important in fungi that are used in biocontrol programmes, such as Metarhizium. If an isolate has failed to sporulate, it may also fail to produce blastospores that are essential in inducing pathogenesis. Passaging experiments would need to be undertaken to establish whether an isolate that has undergone changes in cultural morphology as a result of the formation of sectors retained pathogenicity. The characteristics of a fungus may recover after passaging through the original host. For example, Prenerova (1994) reported improvement in the bioactivity of Paecilomyces farinosus isolates after passage through the Cephalcia abietis host.


168 M . J . R Y A N ET AL.

From the results in this experiment, degeneration of cultural morphology was associated with changes in physiological stability. Cultures derived from sectors may affect the ability of a line to initiate and maintain pathogenesis in the target organism, as there were substantial changes in the enzyme and secondary metabolite profiles. Successful pathogenesis requires the synthesis of cuticle-degrading enzymes and secondary metabolites such as the destruxins. Therefore, disruption in the synthesis of these compounds may prevent successful pathogenesis. Because inexperienced workers could inadvertently transfer atypical sectors during subculture, research aimed at identifying and developing new biological control agents should focus on isolates that are found to be relatively stable in plate culture. The physiological, genetic and cultural stability of an isolate should be matched to an original standard. However, this would necessitate recording the characteristic morphology and physiology of a new isolate when it is first isolated from the environment so that future comparisons could be made. ACKNOWLEDGEMENTS The authors would like to thank Ms D.A. Evans (UKC) for technical support. The BBSRC and GlaxoSmithKline (GSK) are gratefully acknowledged for their support of M.J. Ryan through a BBSRC Industrial studentship. Dr D. Langley and Dr B.S. Lane (GSK) are thanked for their useful suggestions. REFERENCES Barth, M.G.M. and Bridge, P.D. (1989) 4-Methylumbelliferyl substituted compounds as fluorogenic substrates for extracellular enzymes. Letters in Applied Microbiology 9, 9–12. Bridge, P.D., Hawksworth, D.L., Kozakiewicz, Z., Onions, A.H. and Paterson, R.R.M. (1986) Morphological and biochemical variation in single isolates of Penicillium. Transactions of the British Mycological Society 87, 389–396. Bridge, P.D., Williams, M.A.J., Prior, C. and Paterson, R.R.M. (1993) Morphological, biochemical and molecular characteristics of Metarhizium anisopliae and M. flavoviride. Journal of General Microbiology 139, 1163–1169.

Gramss, G. (1991) Appearance of senescent sectors in the ageing vegetative thallus of several basidiomycetous fungi held in pure culture. Journal of Basic Microbiology 31, 113–120. Grindle, M. (1964) Nucleo–cytoplasmic interactions in the red cytoplasmic variant of Aspergillus nidulans. Heredity 19, 75–95. Kim, D.H. (1997) Induced change in DNA methylation of Fusarium oxysporum f.sp. niveum due to successive transfer. Journal of Biochemistry and Molecular Biology 30, 216–221. Kirk, P.M., Cannon, P.F., David, J.C. and Stalpers, J.A. (2001). Ainsworth and Bisby’s Dictionary of the Fungi, 9th edn. Wallingford, UK: CAB International. Moore, D., Bridge, P.D., Higgins, P.M., Bateman, R.P. and Prior, C. (1993) Ultra-violet radiation damage to Metarhizium flavoviride conidia and the protection given by vegetable and mineral oils and chemical sunscreens. Annals of Applied Biology 122, 605–616. Mugnai, L., Bridge, P.D. and Evans, H.C. (1989) A chemotaxonomic evaluation of the genus Beauveria. Mycological Research 92, 199–209. Paterson, R.R.M. (1986) Standardized one- and two-dimensional thin layer chromatographic methods for the identification of secondary metabolites in Penicillium and other fungi. Journal of Chromatography 368, 249–264. Prenerova, E. (1994) Pathogenicity of Paecilomyces farinosus toward Cephalcia abietis eonymphs (Insecta, Hymenoptera): enhancement of bioactivity by in vivo passaging. Journal of Invertebrate Pathology 64, 62–64. Prior, C. (1992) Discovery and characteristics of fungal pathogens for locust and grasshopper control. In Biocontrol of Locusts and Grasshoppers ed. Lomer, C.J. and Prior, C. pp. 159–180. Wallingford, UK: CAB International. Prosser, J.I. (1993) Growth kinetics of mycelial colonies and aggregations of ascomycetes. Mycological Research 97, 513–528. Scott, P.M., Lawrence, J.W. and van Walbeck, W. (1970) Detection of mycotoxins by thin-layer chromatography: application to screening of fungal extracts. Applied Microbiology 220, 280–283. Smith, M.L. (1999) Genetic stability in fungal mycelia. In The Fungal Colony ed. Gow, N.A.R., Robson, G.D. and Gadd, G.M. pp. 283– 301. Cambridge University Press, UK. Wasfy, E.H., Bridge, P.D. and Brayford, D. (1987) Preliminary studies on the use of biochemical and physiological tests for the characterisation of Fusarium isolates. Mycopathologia 99, 9–13. Wing, N., Burgess, L.W. and Bryden, W.L. (1995) Cultural degeneration in two Fusarium species and its effects on toxigenicity and cultural morphology. Mycological Research 99, 615–620.
