A chemotaxonomic evaluation of the genus Beauveria

Mycol. Res. 92

(2): 199-209 (1989)

199

Printed in Great Britain

A chemotaxonomic evaluation of the genus Beauveria

LAURA MUGNAI Istituto di Patologia e Zoologia Fores/ale e Agraria, Piazzale delle Cascine, 28, 50144 Firenze, Italy

PAUL D. BRIDGE CAB International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, u.K.

HARR Y C. EVANS CAB International Institute of Biological Control, Silwood Park, Ascot, Berks. SL5 7PY, UK A chemotaxonomic evaluation of the genus Beauveria. Mycological Research 92 (2): 199-209 (1989). Intra- and interspecific variation of thirty-two isolates assignable to the genus Beauveria was evaluated using 64 morphological and biochemical characters. Two isolates of Tolypocladium cylindrosporum were included to test generic concepts. Seventeen cluster groups were obtained following a numerical taxonomic analysis, each group being separated by at least one character. Cultural characters were highly variable and could not be used reliably for species determination. Spore form was the most useful criterion to distinguish between species. Biochemical data generally supported species concepts based purely on morphology, with the exception of B. bassiana which comprised a heterogeneous assemblage of strains. There is evidence from API ZYM and esterase patterns that this variability is determined by host (substrate) and geographical origins. B. alba, although morphologically close to B. bassiana, could be separated readily on biochemical characters using principal component analysis. The following species of Beauveria are recognized: B. alba, B. amorpha, B. bassiana, B. brongniartii, B. velata, B. vermiconia and an undescribed taxon close to B. amorpha but morphologically and biochemically distinct. Both isolates of T. cylindrosporum cluster in the same group and, on the basis of present evidence, particularly conidiogenesis, the synonymy of this genus with Beauveria is questioned. Key words: Beauveria, Tolypocladium, Intraspecies variation, Interspecies variation, Numerical taxonomic analysis, API ZYM patterns, Esterase patterns.

The genus Beauveria has been the subject of considerable taxonomic investigation since its characterization by Vuillemin (1912). Petch (1926) undertook a cultural comparison of a range of Beauveria isolates, tentatively assigned to eight species groups, in order to determine the value of this method as a means of supplementing micromorphological data. Based on spore shape, he recognized only two species complexes and expressed reservation in using cultural characters for species typification. MacLeod (1954) reached similar conclusions after critically examining sixteen described species of Beauveria. De Hoog (1972) further endorsed these views and reduced the genus to three species. Several new taxa have been described since (de Hoog &: Rao, 1975; Samson &: Evans, 1982), and more recently Arx (1986) re-classified all Tolypocladium species within the genus Beauveria. The latter paper has broadened the concept of the genus but instead of establishing a firm basis for future taxonomic work, it has only served to confuse. Because of the actual and potential economic importance of the genus, both as a source of biological control agents of insect pests as well as novel metabolites (Dunn &: Mechalas, 1963; Ferron, 1978, 1981), its

taxonomy is of practical interest. Collections of Beauveriainfected insects, from tropical ecosystems, have revealed considerable variation between isolates which has made species identification difficult. The present study tests species concepts within the genus Beauveria by employing the multidisciplinary approach adopted by Bridge et al. (1985), comparing morphological and biochemical characters. MATERIALS AND METHODS Isolates examined

Isolates were obtained from culture collections held at the CAB International Mycological Institute (IMI), the CAB International Institute of Biological Control (CIBC) and the Centraalbureau voor Schimmelcultures (CBS). These are listed in Table 1 under their deSignated catalogue names and accession codes. Morphology

For macromorphological studies, isolates were grown in the dark at 20°C on yeast extract-sucrose agar (Scott et al., 1970),

Chemotaxonomic evaluation of Beauveria

200

Table 1. List of fungal isolates evaluated Accession code

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

IMI 269037 CBS 604.80 CBS 605.80 CBS 606.80 IMI 160567 IMI 173 201 IMI 187643 CIBC 6 CIBC 40 CIBC 292 CIBC 294 CIBC 309 CIBC 310 CIBC 311 CIBC 313 C!BC 314 CIBC 315 C!BC 318 CIBC 320 CIBC 322 IMI 314128 IMI 228343 CIBC 291 IMI 12 942 IMI 12 944 CBS 607.80 CBS 608.80 C!BC 250 CIBC 257 CBS 645.74 CBS 849.73

Designated species

Host or substrate

Country of origin

Accession date

Beauveria alba (Limber) Saccas B. amorpha (Hahnel) Samson & Evans B. amorpha

Cystoseira sp. (alga) Coleoptera adult

Australia

1982

Brazil

1979

Coleoptera adult

Brazil

1978

B. amorpha

Coleoptera adult

Brazil

1977

B. bassiana (Bals.) Vuill. B. bassiana

Eucalyptus sp.

Australia

1971

Horse

U.K.

1973

B. bassiana

Seawater

Adriatic

1974

B. bassiana B. bassiana B. bassiana

Lepidoptera pupa Lepidoptera moth Coleoptera adult (Hypothenemus hampei) Mirid (Heteroptera)

Philippines Indonesia Jamaica

1985 1985 1987

Papua New Guinea

1986

Ecuador

1987

B. bassiana

Coleoptera adult (H. hampei) Lepidoptera pupa

Ecuador

1974

B. bassiana

Ant (Hymenoptera)

Ecuador

1974

B. bassiana

Derrnaptera

Ecuador

1974

B. bassiana

Lepidoptera larva

Ecuador

1974

B. bassiana

Pentatomid (Heteroptera)

Ecuador

1974

B. bassiana

Lepidoptera larva

Ecuador

1975

B. bassiana

Ant (Hymenoptera)

Ecuador

1975

B. bassiana

Lepidoptera pupa

Ecuador

1974

B. bassiana

Homoptera

India

1987

B. brongniartii (Sacc.) Petch B. brongniartii

Coleoptera adult

Malawi

1978

Lepidoptera larva

Mexico

1986

B. densa (Link) Picard B. stephanoderis (Bally) Petch B. ve/ata Samson & Evans B. velata

Diptera

Sri Lanka

1926

Coleoptera adult (H. hampei) Lepidoptera larva (Arctiidae) Lepidoptera larva

Sri Lanka

1926

Ecuador

1975

Ecuador

1974

Lepidoptera pupa (7Megalopygidae) Lepidoptera pupa

Ecuador

1975

Ecuador

1976

Volcanic ash

Chile

1974

Volcanic ash

Chile

1973

B. bassiana B. bassiana

1

1

B. ve/ata B. velata B. vermiconia de Hoog & Rao B. vermiconia

Laura Mugnai, P. D. Bridge and H. C. Evans

201

Table I, (Cont.)

Accession code

Designated species

Host or substrate

Country of origin

Accession date

32

CIBC

Beauveria sp.

Orthoptera

Kenya

1986

33

170 1M1

Tolypocladium cylindrosporum Cams Tolypocladium cylindrosporum

Diptera

U.S.A.

1979

Diptera

U.K.

1987

240363 34

1M1 313 449

1

2

= B. bassiana (fide de Hoog, 1972).

2

= B. cylindrospora (fide Arx, 1986).

potato dextrose agar and oatmeal agar. The centre of the plates was inoculated with a 5 mm mycelial plug and radial growth assessed after 3 and 6 weeks, together with cultural characteristics. For micromorphological studies, isolates were grown using the slide culture technique (Booth, 1971; Hawksworth, 1974) and examined directly under a light microscope.

tris, 0'525% citric acid, pH 9) using a 2'8% stacking gel and a 9'7% separating gel. Gels were loaded with 45 III of sample and 5 III of 0'1 % bromophenol blue in 2 M sucrose, run in trisglycine buffer (pH 8'3) at 20 rnA (constant current) until the bromophenol blue had travelled 5 em, and then stained for acetyl esterase bands (Lawrence et al., 1960). Duplicate samples were run on at least two occasions.

Biochemistry

Data analysis

The following tests were carried out with each isolate using two or more replicates. Carbon sources. The ability to utilize lactose or citrate as sole carbon sources was tested at 1% (w Iv) concentration in medium B (Lynch et aI., 1981), using the methodology of Bridge (1985) with 0'005 % bromocresol purple as pH indicator. Enzymes. The hydrolysis of casein, gelatin and Tween (20 and 80) was investigated as described previously (Bridge, 1985). Urease production was analysed in a two-medium system: the basal medium being medium B supplemented with 1% (w Iv) glucose, adjusted to pH 4'5 and solidified with 1'2 % (w Iv) agar (Oxoid No.3). Control and test media contained 0'005 % (w Iv) bromocresol purple whilst the test medium had an additional 0'2% (wi v) urea. A rise in pH after growth on the test medium and not in the control was considered a positive result. API ZYM (API-bio Merieux Ltd.) and acetyl esterase tests (Bridge & Hawksworth, 1984, 1985; Bridge etal., 1985). Cultures were grown for 9 days in glucose-yeast medium (GYM) consisting of liquid medium B supplemented with 1% (w Iv) glucose, 0'5 % (w Iv) yeast extract, 0'005 and 0'001 % (w/v) CuS0 4 :5H2 0 and ZnS0 4 :7H2 0, respectively. The supernatant fraction was used as inoculum for the API ZYM strips, which were incubated at 37° for 4 h and read according to the manufacturer's instructions. The mycelial pellet fraction from the GYM cultures was disrupted by freezing at - 20 0, and then grinding with carborundum and 1-2 ml tris-glycine buffer (0'3% tris, 1'44% glycine at pH 8'3) at 4°. The resultant slurry was clarified by centrifugation and the supernatant further clarified by filtration with a 0'45 Ilm cellulose nitrate filter. The protein content of the extracts was calculated using the method of Lowry et ai. (1951). Extracts were concentrated by freeze-drying to give a final concentration of 5 mg ml- 1 . Electrophoresis was carried out in polyacrylamide tube gels in a discontinuous system with a tris-citrate gel buffer (1'37%

Biochemical test results were coded using 0 for negative, 2 for positive and I for weak or missing results. Similarities were calculated using Gower's coefficient, discounting matching negative results, and were clustered using the average linkage UPGMA algorithm (Sneath & SokaL 1973). All computations were performed with the MINIPAC numerical taxonomy package (Bridge & White, unpubl.) on an Amstrad PCW 8512 microcomputer. Under data handling, the results of the chemotaxonomic evaluation were coded to give 64 characters. Macromorphological characters were recorded as all present, for example a white colony with a yellow tint was coded as both white and yellow. Spore shapes were coded as 2 for predominant shape and I for alternative forms.

RESULTS AND DISCUSSION The clusters obtained by analysing all the morphological and biochemical data in dendrogram form (Fig. I) are shown as groups in Table 2. Only 58 of the 64 characters are considered to be relevant, the others being excluded because of difficulties in interpretation. The similarities expressed are lower than those usually reported in numerical taxonomy, partly because in this study all matching negative results have been discounted and partly due to the diversity existing between isolates.

Morphology Macromorphological characters were generally found to be highly variable, making them unreliable for species determination. In certain instances, however, colony colour can aid separation of closely related taxa. For example, Beauveria sp. (isolate 32) can be distinguished from B. amorpha by its consistent orange colour. Synnematal formation was considered initially to be a good diagnostic feature but species such as B. amorpha which produce well-organized structures

Chemotaxonomic evaluation of Beauveria

202

Fig. 1. UPGMA dendrogram based on all test results. Similarities derived from Gower's coefficient discounting matching negative results.

20 I

30 I

40 I

Percentage similarity 60 SO

70 I

I

!

80 I

90 I

100

Cluster

I

InIU

34 ] 33

IU I

26 ] 27 21 28 ] 29 24 13 ] 16 20 18 25 14 19 32

J

n

5 30 ] 3\

12 ] 23 22

Taxa

A

Beauveria. bassiana

B C

B. bassiana Toiypoc/adium cylindrosporum

D

B. bassiana

E F G

H I

B. bassiana B. veiata B. bassiana B. velata B. densa

J

B. bassiana

K L M

B. stephanoderis B. bassiana Beauveria sp.

N 0

P

B. amorpha B. bassiana B. vermiconia

Q

B. brongniartii

Fig. 2. UPGMA dendrogram based on physiological data only. Similarities derived from Gower's coefficient discounting matching negative results.

20

30

40

I

I

Percentage similarity SO 60 70 I

I

80

90

100

I

I

In I; ]

24 5 22

n

17

'-----I

I[~.--------c:====

Lf-L--.c=============== on the host rarely develop them in culture, conversely several isolates of B. bassiana readily form synnemata of varying complexity in vitro. There is a similar unpredictability about the aggregation of the conidiogenous cells of B. bassiana which typically occur in dense clusters or heads in vivo. Certain isolates in culture, however, never show this clustering and conidiogenous cells are sparse to solitary, particularly in slide culture. The distinction between B. bassiana and those species with globose conidia becomes difficult. Nevertheless, the most useful micromorphological character for rapid species

I~ ] 16 18

Cluster A,Di I,O,Qi B

Dii Ji G

U

N

19 20

26 27 ] 28 29 14 34 ] 33 25

I

32 21 30 ] 31 23

Li

Iii

F,H

Lii C K E M

G P

Qii

identification is spore form. Thus, B. velata, B. vermiconia, B. amorpha and Beauveria sp. separate readily on conidial shapes (Figs 4-9). Spore size can be subject to considerable in vitro variation and should be employed, therefore, only in conjunction with other differential characters. In other species, spore morphology in vitro can be misleading since in slide culture a number of isolates produce ellipsoidal to cylindrical secondary spores (MacLeod, 1954) termed blastospores by Thomas et al. (1987). These differ considerably in size and shape from conidia on the host. For example, several of the

203

Laura Mugnai, P. D. Bridge and H. C. Evans Fig. 3. Principal components analysis of strains of B. bassiana and closely related taxa. Group A: strains from Coleoptera; group B: strains from Hymenoptera from Ecuador; group C: strains from Lepidoptera from Ecuador.

4--.-------------------------------, _6

17 •

I.

• 24

.11

0-

-23 _ 21

.25 /'

--/.'20 C',

!) 13

I8::!,,!}_~

-4-+----------------,-1--------------1 -6

0

6

First principal component

Table 2. Cluster groups and morphological and biochemical data evaluated Cluster groups

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Characters

A

B

Growth on lactose Base from citrate Gelatin hydrolysis Casein hydrolysis Urease Tween 20 hydrolysis Tween 80 hydrolysis RNAase activity API-ZYM 5 API·ZYM 6 API-ZYM 7 API-ZYM 8 API-ZYM 9 API-ZYM 10 API-ZYM 13 API-ZYM 16 API-ZYM 17 API-ZYM 20 Colony pulverulent Colony fluffy Colony felty Colony cream Colony white Colony apricot Colony citron yellow Colony grey Colony light brown Colony pink Colony orange Reverse ochraceous Reverse cinnamon

+

+

V

+ +

+ + + + + + +

C

+ + + + + + V

0

E

+

+ +

V V

F

G

H

K

+ + +

V V

V

+

+

+

V V V

V

V V

+ V V V

+ + + +

+ + +

+ + + +

+ + +

+ + + +

+ + +

L

+ + +

V V

+ + +

+ + +

+ + +

+ +

0 0 0 0 0 0 0 0 0 0

+

V

V V

V

+ + +

+ + +

V

+ +

+

+

V

+ + +

+ +

+

+

+

+ + + +

+ +

V V

0

+

V

+

+

+

+

+

V

+ +

+

V V V

V V

V V V V

V

+ +

+ +

+ +

+

+

+

+

+

+ + +

+ V

V

+ + V

+ + +

V

+

+ +

Q

+ + + +

+ V

P

V

V V V V

N

+

+ V V

M

+

+ V V V

+ V V V

V

+

V V

V

+

V

+ V

V V

V

V

V

+ V

V

+

+

V V

+

V V

+ V

+

V V

+

Chemotaxonomic evaluation of Beauveria

204

Table 2. (Cont.)

Cluster groups Characters 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

47 48 49 50 51 52 53 54 55 56 57 58

Reverse honey Reverse amber Growth rate 2 Growth rate 3 Esterase band 1 (rf 0'07) Esterase band 2 (rf 0'15-0·16) Esterase band 3 (rf 0'21-0·26) Esterase band 4 (rf 0·31) Esterase band 5 (rf 0·38) Esterase band 6 (rf 0·405) Esterase band 7 (rf 0'44) Esterase band 8 (rf 0'49--Q'50) Esterase band 9 (rf 0'53-0'57) Esterase band 10 (rf 0'58-0'64) Esterase band 11 (rf 0'71-0'74) Esterase band 12 (rf 0'77) Esterase band 13 (rf 0'945) Esterase band 14 (rf 0'82-0'84) Raised stroma Spores globose Spores ellipsoidal Spores reniform Spores cylindrical Spores falcate Mucoidal sheath Slimy heads Verticillate heads

A

+

B

+

C

v

D

v v v

E

F

+

+

v v

+

+

+

G

H

K

+

L

M

+

v v

N

0

v

Q

v

v

V

+

+ V

+

P

+

v

+

v v

v v

+

v

v

v v

v

v v

v

v

+

v v

+

v

+

+

+

+ +

+

+

v

+

+ +

v

v

v

v

v

v

v

v

+

v v

+ +

+ +

v

+ v

+ +

+ +

v

+ +

+ +

+

+ +

+ v

+

+

v v

v

+ +

+

+ + +

+ + +

+ +

v

+ v

+

+

+ v

+, Positive result; -, negative result; v, variable within the group; 0, tested but strips consistently masked by an opaque orange reaction. API:lYM codes: 5, lipase (C14); 6, leucine arylamidase; 7, valine arylamidase; 8, cystine arylamidase; 9, trypsin; 10, chymotrypsin; 13, galactosidase; 16, glucosidase; 17,13 glucosidase; 20, fucosidase.

South American isolates (15, 16, 18, 19) of B. bassiana were originally identified as B. brongniartii due to the preponderance of ellipsoidal spores in culture. Isolate 25, probably a paratype of B. stephanoderis (Petch, 1926), forms only ellipsoidal to reniform spores (Figs 11, 12), and consistently separates out from the B. bassiana complex with which it is now considered synonymous (de Hoog, 1972). It is possible that continuous subculturing of this old isolate has resulted in complete loss of the globose conidia (Fig. 10). Ornamentation of the spore

surface is also subject to cultural variation, and in B. velata becomes more pronounced in old cultures. Indeed isolates 28 and 29 were not included originally in the description of this taxon (Evans & Samson, 1982) as the mucoid spore covering was not immediately evident from the in vivo samples. The relationship only became clear after examination of old cultures and a more critical evaluation of host specimens. B. alba and B. bassiana possess conidia which overlap in size and shape (Figs 10, 13), but the former species can be delimited by

Laura Mugnai, P. D. Bridge and H. C. Evans

205

Figs 4-9. Spore morphology in slide cultures of Beauveria spp. Figs 4-5. Isolate 31 (CBC 849.73), B. vermiconia (x 1600). Fig. 6. Isolate 3 (CBS 605.80), B. amorpha with small reniform conidia and large cylindrical blastospores (x 1000). Fig. 7. Isolate 32 (CIBC 170), Beauveria sp. with significantly larger, curved or reniform conidia compared with B. amorpha (x 1000). Figs 8-9. Above isolate, showing typical reniform conidia and atypical ellipsoidal spores (x 1600).

its erect conidiophores and verticillate conidiogenous cells. The presence of cylindrical conidia produced from phialides in slime heads are highly significant morphological characters to separate T. cylindrosporum from all other isolates evaluated here, questioning the validity of the recent placement of Tolypocladium W. Gams (Gams, 1971) in synonymy with Beauveria (Arx, 1986).

Biochemistry Data analysis involving only biochemical characters is presented in Figs 2,3. Generally, there is a good correlation with accepted species concepts within the genus Beauveria. Isolates of B. amorpha and B. vermiconia retain the same duster groups as in Fig. 1 whilst the four isolates of B. ve/ata are also dose, although differing in some esterase bands. This biological

Chemotaxonomic evaluation of Beauveria

206

Figs 10-14. Conidiogenesis and conidial morphology in slide cultures of Beauveria spp. Fig. 10. Isolate 24 (IMI 12942), Beauveria 'densa' with predominantly globose, B. bassiana-type conidia (x 1000). Figs 11-12. Isolate 25 (lMI 12944), B. 'stephanoderis' with atypical ellipsoidal conidia (x 1000, x 1600). Figs 13-14. Isolate 1 (IMI 269037), B. alba with both globose and ellipsoidal conidia on long, often solitary conidiogeneous cells (x 1600).

variation may be due to host specialization within the Lepidoptera (group F on Ardiidae larvae, group H on pupae of 7Megalopygidae) rather than geographical origins, since they were collected in the same locality. Similarly, the two B. brongniartii isolates, although clustering in Fig. 1, drift

significantly in Figs 2 and 3, which could be due to both host and geographical variability. In the principal component analysis, isolate 22 from a wood-boring coleopteran host occurs with two B. bassiana isolates from a similar host source. This pattern is also repeated with B. bassiana isolates from

207

Laura Mugnai, P. D. Bridge and H. C. Evans Table 3. Most useful differential characters

Beauveria

Characters/ isolates 2 4 9 11 20 22 27 29 44 48 51 52 53 54 55 56 58

Base from citrate Casein hydrolysis API ZYM 5 API ZYM 7 Colony fluffy Colony cream Colony light brown Colony orange Esterase band 9 Esterase band 13 Spores globose Spores ellipsoidal Spores reniform Spores cylindrical Spores vermiform Mucoidal sheath Slimy heads

bassiana

densa

stephanoderis

+ +

v v v

+

v v -

+ +

alba

amorpha

w

+

+ + +

+

brongniartii

velata

Beauveria

sp.

v v

+

+ +

vermiconia

0 0

T. cylindrospomm

+ + +

+ +

v

+ v

+ v

+ +

+ + + +

+ + + +

v

+

v

+ + +

+

+ +

+ + +

+

+

+ + +

+, Positive result; -, negative result; v, variable reaction; w, weak reaction; 0, masked reaction. API ZYM 5, lipase. API ZYM 7, valine arylamidase.

lepidopteran hosts, and to a lesser extent with hymenopteran hosts, from Ecuador (Fig. 3). Conversely, isolates of B. bassiana on other hosts from the same collecting locality differ significantly in their biochemical properties. B. bassiana isolates from non-insect sources also tend to separate on this basis, varying from the 'normal' isolates principally in their API ZYM and esterase patterns. B. alba shows considerable biochemical separation from B. bassiana, and can be distinguished from all other taxa by its enzyme spectrum. Beauveria sp. splits from B. amorpha in its inability to react with citrate which is additional evidence for species delimitation. Once again, the T. cylindrosporum isolates, although geographically remote, cluster together and are biochemically distinct from the Beauveria isolates.

GENERAL DISCUSSION AND CONCLUSIONS The most useful characters to separate species within the genus Beauveria are listed in Table 3. B. densa and B. stephanoderis which are conserved as distinct taxa in Herb. IMI are considered here to be aberrant strains of B. bassiana and these epithets are redundant (de Hoog, 1972). The description of B. stephanoderis by Petch (1926) makes no mention of the predominantly ellipsoidal spores and these may represent the secondary spores illustrated by MacLeod (1954) and Thomas et al. (1987). The latter workers reported that this spore type is predominantly produced in complex growth media. It is possible that physiologically stressed or old cultures (isolate 25 dates from 1926) lose the ability to sporulate normally. Indeed, the slide culture technique appears to induce the formation of abnormal secondary spores (blastospores) in 15

most of the isolates examined (Fig. 6). Those isolates of B. bassiana which have a tendency to form ellipsoidal conidia on standard culture media may be confused with B. brongniartii. However, in B. bassiana, a varying proportion of the spores are always globose whilst B. brongniartii has only ellipsoidal conidia. The isolation of B. bassiana from animal (horse nasal swabs) and marine substrates cannot be explained satisfactorily apart from airborne spore contamination. These isolates do, however, possess different biochemical characters when compared with those from insect hosts. Nevertheless, the great heterogeneity within this species complex permits few firm conclusions to be drawn. There are indications, however, of biochemical homogeneity amongst isolates from related hosts and to some extent from the same geographical areas. Such geographical groupings based on esterase patterns have been demonstrated recently in B. bassiana isolates from both mirid and weevil hosts (Riba et at 1986a; Poprawski et a/., 1988). The biochemical uniformity of the B. amorpha isolates from the same host and same locality further confirms the usefulness and validity of the methodology employed to delimit not only taxa, but also host - and/or geographically specific strains. The isolates of B. ve/ata show similarities in their biochemistry, especially in esterase patterns, although there are sufficient variables to separate two cluster groups, suggesting the presence of host-specific strains within the Lepidoptera. In fact, these two groups or strains can also be distinguished macromorphologically on their respective hosts (Evans & Samson, in prep.). There are now thought to be sufficient characters, both macro- and micromorphologicaL as well as biochemicaL to justify separation of Beauveria sp. MYC 92

Chemotaxonomic evaluation of Beauveria (isolate 32) from B. amorpha at the species level (Evans & Samson, in prep.). There is no evidence that either B. alba or B. vermiconia is entomogenous in habit, but all the isolates included in the present study have the ability to utilize chitin (Mugnai, unpub!.) and possibly exploit these substrates saprophytically in the soil (Evans, 1982). The former two species may represent older, more primitive or less specialized members of the genus before the entomogenous habit was adopted (Evans, 1988). Isozyme patterns have been evaluated and compared in several species of the genus Tolypocladium by Riba et al. (1986 b), who reported highly significant differences between T. cylindrosporum and a morphologically similar taxon, T. extinguens Samson & Soares. There was also considerable intraspecific variation in T. cylindrosporum, although isolates from the same host and geographical area were found to be genotypically homogeneous. The two dipteran isolates studied here were also biochemically uniform, despite wide geographical separation, indicating that in this case homogeneity is determined by host rather than geographical origin. The interpretation by Arx (1986) that Tolypocladium should be placed in synonymy with Beauveria requires verification. The latter genus is typified by dry blastic conidia formed on sympodially elongating conidiogenous cells whilst Tolypocladium has slime spores arising from non-percurrent phialides. Morphologically, the distinction would seem to be clear. On the basis of the biochemical data, however, the isolates show no unique patterns or properties and are not widely separated from the mass of Beauveria isolates. Perhaps this should not be surprising, since it is likely that both genera share a common teleomorph genus, almost certainly belonging to the Clavicipitales and possibly Cordyceps. Other entomogenous hyphomycete genera, such as HirsutelIa, Paecilomyces and Verticillium sensu stricto (Evans & Samson, 1986), are probably interrelated and, consequently, should possess similar isozymes to those reported here. Clarification of generic concepts and affinities within this group of economically important fungi is required and a chemotaxonomic approach may be rewarding. We wish to thank Mrs Devota Kavishe and Ms Georgina Godwin for technical assistance. The senior author received financial support during this study from the Italian Consiglio Nazionale delle Ricerche (C.N.R.).

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(Received for publication 29 March 1988)

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