Mycopathologia 118: 29-36, 1992. 9 1992 KluwerAcademic Publishers. Printedin the Netherlands.

Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi* G.N. E1-Sayed, 1 C.M. Ignoffo, 1 T.D. Leathers 2 & S.C. Gupta 2

1Biological Control of Insects Research Laboratory, USDA/ARS, P.O. Box 7629, Columbia, MO 65205 USA; 2Microbial Properties Research Center, USDA/ARS, I815N. University Street, Peoria, IL 61604 USA Received 4 February 1991; acceptedin revised form 25 July 1991

Key words: Beauveria, biochemistry, enzyme profiles, entomopathogenic fungi, insects, Metarhizium, Nomuraea, apiZYM system

Abstract

An apiZYM| with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae. Similar enzyme profiles were obtained for two of the N. rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates. Centroid-clustering analysis revealed three distinct enzyme profiles.

Introduction

teria [2-6] or fungi [7-9]. More recently, the apiZYM | system also was used to characterize whole culture filtrates of entomopathogenic fungi, i.e., Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii [10]. Our objectives were to determine if the apiZYM | system could be used to detect enzymes expressed during germination of conidia of entomopathogenic fungi and, also if the expressed enzymatic profiles could be used to differentiate between species or isolates of Nomuraea rileyi.

It would be highly desirable to have a rapid, specific, standardized micromethod to detect enzymes expressed by entomopathogenic fungi and to be able to use this system to differentiate between genera, species and/or isolates of entomopathogenic fungi. A system similar to that described by Buissi6re et al. [1], is currently commercially available under the trade name apiZYM @ (API Laboratory Products Ltd., Philpot House, Ryleigh, Essex, England). The apiZYM system, or similar systems, have been used to detect specific enzymes, and also to identify bac-

Materials and methods

* Mention of a proprietary product in this paper does not constitute a recommendationfor use by USDA.

Fungal isolates. The five isolates of Nomuraea rileyi used included one from Ecuador (Ec), one

30 from Mississippi (Ms), virulent, parent strain with green conidia (GV) and two mutant isolates of GV (a yellow avirulent isolate designated YAV and a yellow virulent isolate designated YV) [1113]. The isolate of Metarhizium anisopliae ( C M - 23) was obtained (in 1981) from Dr. A. Villacosta (Londrina, Brazil) and Beauveria bassiana (Bb-196), isolated in 1983 from Heliothis armigera on cotton in Cordoba, Spain by Dr. T. Poprowski.

Preparation of fungal supernatants for enzyme analysis. Conidia of each isolate were harvested from sporulating cultures grown on Sabouraudmaltose-agar fortified with 1% yeast extract [12]. The harvested conidia were stored at -20 ~ until needed for inoculation of flasks. The techniques for preparing cuticle and the supernatants for enzyme analysis were as previously described [14]. To summarize, ground cuticle (from 5th instar larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) was sterilized with ethylene oxide and then added (1% W/V) to 50 ml of sterilized distilled water (SDW) in 125ml Erlenmeyer flasks. Conidia were suspended in SDW and purified by filtration and centrifugation (8000 XG, 10 min at 4 ~ The resultant pellet was then resuspended in SDW containing 0.002% Tween 80 (v/v) and a combination of streptomycin/penicillin/gentamycin (0.01 ml/ml culture fluid). This preparation was used to inoculate each culture flask with 7.5 x 10 6 spores. After inoculation with conidia, the cultures were incubated at 25 ~ on a reciprocal shaker (150-180 rev/min). Cuticle in SDW without conidia was used as controls. In addition, conidia were also cultured on Sabouraud-maltose-yeast broth (SMYB). Aliquots (1 ml) of each culture were removed daily (for 3 days) for microscopy to determine that germination had occurred. After 24, 48 or 72 h incubation, each flask was placed in ice-water and the culture was sonicated (MSE MK 2, 150 Watts; amplitude 22 m) for 5 min to disrupt the eonidia. Sonication was used as the standard procedure since preliminary studies showed that the enzymatic profiles of sonicated or non-sonicated con-

idia (after 48 h incubation) were essentially identical. Only 3/171 possible comparisons (19 enzyme x 9 fungal isolates) were positive for a non-sonicated sample when the sonicated sample was rated negative. In these instances, reactions were weakly positive (N. atypicola, trypsin; N. rileyi GV, a-galactosidase; B. bassiana, a-glucosidase). Conidial suspensions after sonication were clarified by filtration (Whatman 2 filter paper) and centrifugation (15,000 XG for 20 min at 4 ~ The resultant pellet was discarded, the supernatant of each culture, adjusted to pH = 6.0 (with NaOH) to minimize protein breakdown, was adjusted to give 1 mg of protein/ml [15].

Enzymataic analysis. The apiZYM | system consists of microcupules containing dehydrated chromogenic enzyme substrates and buffer. These substrates were capable of detecting the following 19 enzymes: alkaline and acid phosphatases; butyrate esterase (C4); caprylate esterase lipase (Cs); myristate lipase (C14); leucine, valine, and cystine aminopeptidases; trypsin; chymotrypsin; phosphoamidase; a-galactosidase; ~-galactosidase; /3-glucuronidase; a-glucosidase; /3-glucosidase; N-acetyl-/3-glucosaminidase; a-mannosidase; and a-fucosidase. To detect an enzyme, 0.05ml of the fungal supernatant was added to each microcupule of the apiZYM | gallery. After incubation in the dark at 37 ~ for 4 h (in a humidification chamber containing 5 ml SDW), 0.025 ml of reagents A and B were added to each microcupule. The gallery was kept in the dark for 5 min, then illuminated 20s using a 1000 W to optimize the colour reaction. Colour reactions, after comparison to the apiZYM| our-reaction chart, were rated from 0 (no colour above background) to 5 (darkest colour); the reaction rating scale we used was: 0, no reaction; 1, weakly positive; 2, 3 or 4, moderately positive; 5, strongly positive.

Enzyme profiles. A centroid clustering method, described by Pielou [16], was used to analyze the enzyme profiles of all species and isolates. The analysis was based on the numerical ratings (0, 1,

31 2, 3, 4 or 5) of each of the 19 enzymes for conidia that were incubated on T. ni cuticle for 48 h.

Results and discussion

All fungal isolates tested exhibited some level of enzyme activity for alkaline and acid phosphatases, phosphoamidase, butyrate esterase, caprylate, esterase-lipase, leucine aminopeptidase, valine aminopeptidase, N-acetyl-/3-glucosaminidase, ~-galactosidase and /3-glucosidase (Table 1). In contrast, o~-glucuronidase and a-fucosidase could not be detected in any of the nine fungal isolates. Also, all fungal isolates produced significantly more acid than alkaline-phosphatase. The following lists other distinct differences (Table 2) that were detected in enzyme profiles of germinating conidia: N. rileyi isolates generally were negative or weakly positive for myristate lipase, cystine aminopeptidase, trypsin, chymotrypsin, ~-galactosidase and o~-glucosidase; Isolate YAV or M. anisopliae was negative for leucine and valine aminopeptidase or N-acetyl-/3glucosaminidase, respectively; leucine, valine and cystine aminopeptidases and/3-galactosidase were not detected in isolate YAV; N. atypicola was moderately to strongly positive for aminopeptidases and chymotrypsin; N. anemonoides was moderately to strongly positive for leucine aminopeptidase, a- and/3-galactosidase, a- and fi-glucosidase and N-acetyl-/3-glucosaminidase; and B. bassiana was moderately to strongly positive for leucine and valine aminopeptidases, c~- and /3galactosidase, and/3-glucosidase. The summarized results (Table 1) generally demonstrated that time of incubation did not alter a decision whether the enzyme was present or absent for any particular fungal isolate. In all instances (except two) when the enzyme was definitively present (2 to 5 rating) it was initially detected after 24 h of incubation. The two exceptions were leucine aminopeptidase and/3-galactosidase for the Ec isolate of N. rileyi and N. atypicola, respectively. These enzymes were initially

detected after 48 h of incubation. Variations in levels of enzyme activity after 24, 48 or 72 h of incubation may reflect different phases of germination and germ tube growth [17]. Recently, we detected (unpublished data) a significant release of cuticle-degrading enzymes (within hours of contact with the host cuticle) by germinating conidia of N, rileyi. Enzyme activity for the chromogenic aryl amide peptides was the most variable (ratings of 0 to 5) with N. atypicola and B. bassiana producing the most activity. The enzyme profiles of the parent GV, YV and YAV isolates of N. rileyi were remarkably similar (Table 1) even though the YAV is an avirulent isolate while GV and YV are both virulent isolates. The only major difference in the enzyme profiles was that leucine and valine aminopeptidases, which were absent in the YAV isolate, were present in both the GV and YV isolate. The absence of aminopeptidases in the avirulent YAV isolate supports the contention that proteases (20) may be the most important enzymes in integumental penetration by germinating conidia. It does not, however, exclude the possibility that chitinase (4) as well as other hydrolytic enzymes, also may contribute to the integumental penetration process. We also were surprised to find that trypsin and ch~motrypsin could not be detected by apiZYM "~ in any isolate of N. rileyi since both enzymes were detected when a quantitative analysis of germinating conidia of both the Ms and Ec isolate of N. rileyi was conducted (unpublished). Three trypsin-like enzymes have been detected in culture filtrates of 5 entomopathogenic fungi, i.e., M. anisopliae, B. bassiana, V. lecanii, N. rileyi and Aschersonia aleyrodis [18, 19]. Possibly the trypsin-like enzymes of N. rileyi were too specific to be detected, or some other factor such as enzyme binding or blocking [19] prevented or obscured the color reaction of the apiZYM | system. The absence of N-acetyl-/3-glucosaminidase may be useful as a phenotype character for detecting M. anisopliae. Weak to moderate N-acetyl-/3-glucosaminidase activity was detected (apiZYM| in 5-day culture filtrates of most

5,5,5 3,5,5 3,5,5 5,5,2 5,5,3 1,1,4 2,2,3 4,5,5 3,5,5

Phosphoamidase

1,1,2 1,1,1 1,1,i 4,1,1, 2,4,1, 1,2,2, 1,1,1 1,1,1 1,1,1

Alkaline phosphatase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 2,3,3 4,4,5 3,3,3 3,4,5

agalactosidase

3,3,3 3,3,2 4,3,3 4,2,3 4,3,3 2,3,3 2,2,2 3,3,3 2,2,3

Butyrate esterase

4,3,3 2,3,3 2,3,4 3,4,4 3,3,3 0,2,2 3,3,4 3,5,5 1,2,2

/3galactosidase

2,2,3 3,2,2 4,3,2 2,2,3 3,2,2 2,4,4 2,2,3 3,3,3 1,1,2

Caprylate esteraselipase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0

aglucuronidase

0,0,0 1,1,1 0,0,0 0,0,0 0,0,0 1,1,1 1,1,1 1,1,1 1,1,1

Myristate lipase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 2,2,3 0,0,0 1,1,1

aglucosidase

1,1,2 0, i ,2 1,1,3 1,1,2 0,0,0 4,5,5 4,4,5 4,5,4 1,1,3

Leucine aminopeptidase

2,2,2 1,1,1 2,4,3 3,2,3 1,5,2 1,1,1 4,5,5 5,5,5 1,2,2

/3glucosidase

0,0,1 0,0,1 1,1,2 1,2,2 0,0,0 4,5,5 1,1,1 5,5,5 1,1,2

Valine aminopeptidase

5,4,4 2,4,3 4,5,5 4,5,5 5,5,3 1,2,2 4,4,5 5,5,5 0,0,0

N-acetyl/3-glucosaminidase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 2,2,2 1,1,1 1,1,1 1,1,2

Cystine aminopeptidase

0,0,0 0,0,0 0,0,0 1,1,1 0,2,1 1,2,2 0,0,0 1,1,2 0,0,0

amannosidase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,1 0,0,0 0,1,1 0,0,1

Trypsin

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0

afucosidase

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 3,4,5 0,0,0 1,1,1 0,1,1

Chymotrypsin

5,5,5 4,5,5 5,4,5 5,5,5 5,5,3 1,2,5 4,5,5 4,5,5 4,5,5

Acid phosphatase

a Culture medium was 1% (w/v) cuticle + sterile distilled water (SDW). Each culture flask was inoculated with 7.5 • 106 conidia and incubated at 25 ~ Control flasks with only SDW + cuticle or conidia + SDW showed no enzyme color reaction. b Data in each enzyme column is the reaction rated after 24, 48 or 72 h incubation, respectively. The reaction ruling scale was: 0, no colour change; 1, weakly positive; 2, 3 or 4, moderately positive; 5, strongly positive.

Nomuraea rileyi Mississippi Ecuador GV YV YAV Nomuraea atypicola Nomuraea anemonoides Beauveria bassiana Metarhizium anisopliae

Species/isolate

Nomuraea rileyi Mississippi Ecuador GV YV YAV Nomuraea atypicola Nomuraea anemonoides Beauveria bassiana Metarhizium anisopliae

Species/isolate

apiZYM @ enzymesb

Table 1. Enzymatic profiles after 24, 48 and 72 h incubation of conidia of Nomuraea rileyi, Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium ansiopliae on cuticle of Trichoplusia ni a

to

33 Table

2.

Important

enzymes

that may be used to distinguish between

some isolates, species and genera

of entomopathogenic

fungi Enzymes a Species/isolate

Leucine

Valine

Cystine

Chymo-

N-acetyl-

a-galac-

amino-

amino-

amino-

trypsin

/3-glucos-

tosidase

peptidase

peptidase

peptidase

Mississippi

+

-

-

-

+

-

-

Ecuador

+

-

-

-

+

-

-

GV

+

+

-

-

+

-

YV

+

-

-

+

-

YAV

.

+

Nomuraea

c~-glucosidase

aminidase

rileyi

+

-

-

Nomuraea

atypicola

+

+

+

+

+

+

-

Nomuraea

anernonoides

+

-

-

-

+

+

+

+

+

-

-

+

+

-

-

+

+

-

-

+

-

Beauveria Metarhizium

bassiana anisopliae

.

.

.

a A c t i v i t y c l a s s i f i e d a s f o l l o w s : - , w e a k (1) o r n o a c t i v i t y ( 0 ) ; + p o s i t i v e a c t i v i t y (2 t o 5).

isolates of M. anisopliae, but not in isolate RS543 [10]. Also, when M. anisopliae was grown on locust cuticle, N-acetyl-/3-glucosaminidase appeared after the onset of proteolytic enzymes activity and was less inducible than endochitinase [18]. The high levels of glucosidases and galactosidases in N. anemonoides may reflect their importance in degrading plant debris by this soil-inhabiting fungus. The 1,4-glucosidases, as an example, complete cellulolysis by hydrolysis of cellobiose and cellodextrins to glucose [20]. Glucosidases and galactosidases may also intracellularly lower nucleotide sugar levels and turn off synthesis [24]. Also, the presence of moderate to strong levels of activity for acid phosphatase, phosphoamidase,/3-glucosidase and/3-galactosidase may reflect their role in the breakdown of phenolic compounds (i.e., glycosides, and galactosides). There was essentially no difference in the 48hr enzyme profiles of germinating conidia of the Ms isolate of N. rileyi whether SMY or cuticle of T. hi, H. zea or H. virescens cuticle was used as the substrate (Table 3). Two exceptions to this generalization, however, were obtained. In the first exception, moderate reactions with/3-glucosidase were detected for SMY (+3) or T. ni (+2) and H. zea (+3) cuticle, while a weakly positive

reaction was obtained for H. virescens cuticle. In the other exception, a strong reaction of /3galactosidase was recorded on SMY while moderate reactions were obtained using T. ni (+3), H. zea (+4) or H. virescens (+2) substrates. Whenever an enzyme was not detected or weakly positive for one substrate it also was absent or weakly positive for all other substrates. Enzyme profiles, similar to those at 48 hrs, were obtained when germinating conidia were assayed after 24 or 72 hrs of incubation. Results of enzyme profiles with the Ec isolate of N. rileyi were similar to enzyme profiles of the Ms isolate except for the following: a moderate (+2) caprylate esterase-lipase reaction at 48 hrs was detected on SMY, T. ni or H. zea cuticle and a weakly positive reaction on H. virescens cuticle; a strong reaction of/3-galactosidase was obtained on SMY while moderate reactions were detected on r. ni (+3), H. zea (+2) or H. virescens (+2) cuticle. As with the Ms isolate, whenever an enzyme was not detected for one substrate using Ec, it also was not detected in all other substrates (Table 3). To summarize, the enzyme profiles of germinating conidia after 24, 48 and 72 h of incubation were essentially the same for both the Ec and Ms isolates regardless of the culturing substrate. Although both isolates produced moderate to

34

Table 3. Effects of a culturing on Sabouraud-maltose-yeast (SMY) substrate or substrates of cuticle from larvae of Trichoplusia ni, Heliothis zea, and H. virescens on the enzyme profiles of germinating conidia of a Mississippian and Ecuadoran isolate of Nomuraea rileyi after 48 h incubation a ApiZYM@ enzymes

Submerged substrate b

T. ni cuticle Alkaline phosphatase Butyrate esterase Caprylate esterase-lipase Myrisate lipase Leucine amino-peptidase Valine amino-peptidase Cystine amino-peptidase Trypsin Chymotrypsin Acid phosphatase Phosphoamidase a-galactosidase /3-galactosidase a-glucuronidase a-glucosidase /3-glucosidase N-acetyl-/3-glucosaminidase a-mannosidase a-fucosidase

H. zea cuitcle

H. virescens cuticle

SMY Broth

24

48

72

24

48

72

24

48

72

24

48

72

1,1 3,3 2,3 0,1 1,0 0,0 0,0 0,0 0,0 5,4 5,3 0,0 4,2 0,0 0,0 2,1 5,2 0,0 0,0

1,1 3,3 2,2 0,1 1,i 0,0 0,0 0,0 0,0 5,5 5,5 0,0 3,3 0,0 0,0 2,2 4,4 0,0 0,0

2,1 3,2 3,2 0,1 2,2 1,1 0,0 0,0 0,0 5,5 5,5 0,0 3,3 0,0 0,0 2,2 4,3 0,0 0,0

1,1 3,4 1,1 0,1 1,0 0,0 0,0 0,0 0,0 4,5 3,4 0,0 4,3 0,0 0,0 3,2 4,3 0,0 0,0

2,1 3,4 2,2 0,1 1,1 1,0 0,0 0,0 0,0 5,4 4,4 0,0 4,2 0,0 0,0 3,2 4,4 0,0 0,0

2,1 3,3 2,2 0,i 1,1 1,0 0,0 0,0 0,0 5,5 4,3 0,0 5,2 0,0 0,0 3,2 5,5 0,0 0,0

1,1 2,2 1,i 0,1 0,0 0,0 0,0 0,0 0,0 4,4 3,4 0,0 1,2 0,0 0,0 1,1 3,2 0,0 0,0

1,1 2,2 1,1 0,1 0,0 0,0 0,0 0,0 0,0 5,5 4,3 0,0 2,2 0,0 0,0 1,1 5,2 0,0 0,0

1,1 3,3 2,1 0,1 0,0 0,0 0,0 0,0 0,0 5,5 4,3 0,0 2,2 0,0 0,0 1,1 5,2 0,0 0,0

1,1 2,2 2,2 0,0 0,0 0,0 0,0 0,0 1,0 5,4 4,3 1,0 5,5 0,0 1,0 2,2 2,3 0,1 0,0

1,1 3,2 2,2 0,0 0,0 0,0 0,0 0,0 1,0 5,5 5,4 1,0 5,5 0,0 1,0 3,2 4,3 0,1 0,0

1,1 4,3 3,2 0,0 0,0 0,0 0,0 0,0 1,0 5,5 5,3 1,0 5,4 0,0 1,0 3,2 5,3 0,1 0,0

aCulture medium of 1% (w/v) cuticle + sterile distilled water (SDW). Each culture was inoculated with 7.5 x 1 0 6 conidia and incubated at 25~ Control flasks with only cuticle + SDW or conidia + SDW showed no enzyme color reaction. bData in each column for each incubation period is the rating for the Mississippian and Ecudoran isolate, respectively. The reaction rating scale was: 0, no colour change; 1, weakly positive; 2, 3 or 4, moderately positive; 5, strongly positive.

strong reactions for release of acid-phosphatase, N-acetyl-fl-glucosaminidase and p h o s p h o a m i d ase, the individual ratings for the Ms isolate were generally higher than those of the Ec isolate. T h r e e e n z y m e profiles were schematically expressed (Figure 1). T h e first group contained zll 5 isolates of N. rileyi (Ec, Ms, YAV, Y V , G V ) . Within this group, isolate Ms and E c were m o r e closely clustered than isolates YAV, Y V and G V . T h e second group included B. bassiana, N. anemonoides and M. anisopliae. Within the second group, B. bassiana and N. anemonoides were closely clustered and distinct f r o m M. anisopliae. Nomuraea atypicola was the only r e p r e s e n t a t i v e of the third group. Cluster analysis of the e n z y m e profiles f r o m germinating conidia, could differ-

entiate b e t w e e n species of Nomuraea (rileyi vs. anemonoides vs. atypicola), but not isolates of N. | rileyi. T h e a p i Z Y M profile we o b t a i n e d f r o m o u r isolate of B. bassiana was only slightly different f r o m that r e p o r t e d by Mugnai et al. [21], w h o d e m o n s t r a t e d 'great h e t e r o g e n e i t y within the species c o m p l e x ' w h e n their evaluation of the genus was conducted using 64 m o r p h o l o g i c a l and biochemical characters. T h e clustering of e n z y m e profiles into three related but distinct groups m a y reflect the differences in host and ecological niches of these fungi [21-23]. N. rileyi primarily attacks larvae which feed on foliage; B. bassiana, M. anisopliae and N. anemonoides all are c o m m o n l y isolated f r o m soil and b o t h B. bassiana and M. anisopliae cus-

35

90

-

80

-

2.

70

3.

u

g

g

60

50

--

40

-

30

-

4.

5.

20

6. 10 I

',ll 5"

I ~

5"

7.

8.

9. Fig. I. Schematic depiction of the centroid-ctustering analysis of enzyme profiles of nine isolates of entomopathogenic fungi (Nomuraea rileyi; Mississippi, Ecuador, green virulent, yellow virulent, yellow avirulent; Nomuraea atypicola; Nomuraea anemonoides; Beauveria bassiana; Metarhizium anisopliae).

Acknowledgment We sincerely appreciate the indispensable technical advice and assistance of Clem Garcia and the statistical expertise of Jim Grasela who conducted the centroid-clustering analysis. We acknowledge also the significant constructive comments of the following reviewers: Drion G. Boucias, Raymond J. St. Leger, Wayne Gardner, Tariq M. Butt, Cynthia Lenz, Iran F. Salkin and Geoffrey A. Land.

10.

11.

12.

13.

14.

15.

16.

References 1. Buissi6re J, Fourcard A, Colobert L. Usage de substrates

17.

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18.

19.

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Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi.

An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola,...
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