Coproduction of chitinolytic enzymes and biomass for biological control by Trichoderma harzianum on media containing chitin

BIOLOGICAL

CONTROL

2, 272-277

(1992)

Coproduction of Chitinolytic Enzymes and Biomass for Biological Control by Trichoderma harzianum on Media Containing Chitin ARNE Department

of Horticultural

Sciences,

New

York

TRONSMO~ State

AND

Agricultural

ReceivedMay 1,1992;

Experiment

harzianum; control; biomass; conidia; chitobiosidase; chitinase;

Gliocladium N-acetyl-/3chitinolytic

INTRODUCTION

Chitinolytic enzymes are potentially useful for eral purposes. The exoskeleton of certain shellfish, as shrimp and crabs, are composed largely of chitin, disposal of such materials as waste poses ecological

sevsuch and dif-

’ Present address:Department of Biotechnological Sciences,Agricultural University of Norway, P.O. Box 5040, N-1432 As, Norway.

1049.9644/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

212 Inc. reserved.

HARMAN Station,

Cornell

University,

Geneva,

New

York

14456

1992

ficulties. Enzymatic conversion of chitinous wastes to simple carbohydrates may provide a partial solution to such problems and provide simple sugars useful in other processes (Cosio et al., 1982). In addition, chitinolytic enzymes may control herbivorous insects and plant pathogenic fungi. Chitinolytic enzymes degrade polymeric chitin, and therefore they should weaken the exoskeleton and/or peritrophic membrane of insects and inhibit growth of fungi that contain chitin in their cell walls (Roberts and Selitrennikoff, 1988). Therefore, these enzymes are likely to be commercially useful as pure proteins. Species of Trichoderma and Gliocladium are known for their ability to provide biological control of plant pathogenic fungi; many authors have implicated chitinolytic enzymes in this biocontrol ability (Chet, 1987). These fungi not only produce large amounts of chitindegrading enzymes, but also a range of enzyme types (Harman et al., 1993; Tronsmo and Harman, 1993). However, in spite of the large number of published papers on chitinolytic enzymes produced by these fungi, there are very few reports on either optimization of production of these enzymes or on their purification and characterization. Many papers, because of their methodology, consider only N-acetyl-P-glucosaminidase. Trichoderma and Gliocladium could be important sources of chitin-degrading enzymes, they grow readily in culture, and are useful in biological control. Recently, a strain of G. uirens and one of T. harzianum have been registered by the U.S. Environmental Protection Agency, for use as microbial pesticides. Methods for inexpensive production of biomass usable in biocontrol will facilitate the use of these fungi in commercial agriculture. The coproduction of enzymes and biomass should provide material useful for a variety of commercial purposes, and the biomass will be valuable as a biocontrol product. Coproduction should reduce the cost of production since both the supernatant solution and the particulates will provide useful products. Biomass to be used for biological control must be composed of appropriate propagules; for this purpose conidia may function well (Harman et al., 1991). The objective of the research

Press, Inc.

KEY WORDS: Trichoderma

E.

acceptedDecember 2,

Production of N-acetyl-&glucosaminidase (EC 3.2. 1.30), chitobiosidase, and biomass by Trichoderma harzianum is dependent on media composition but not on inoculum concentration. Growth of T. harzianum in simple mineral salt solutions plus single carbon sources, including chitin, gave low enzyme activity in the culture filtrate and few conidia. Addition of any of several complex materials, such as VS juice, yeast extract, or proteose peptone, substantially increased enzyme and spore yield. If sucrose (0.5%) was added together with chitin, enzyme yields were increased after 6, but not after 3 days of culture, relative to chitin alone. The source of chitin was also important; higher yields of enzymes or conidia were obtained with a purified colloidal chitin than with an unpurified chitin from crab shells. Addition of insoluble polyvinylpyrrolidone to the culture increased yields of enzymes. All activity was present as extracellular enzymes; grinding fungal mycelium did not increase the amounts of enzymes detected. An economical medium consisting of a mineral salts solution, colloidal chitin, and sucrose that supported high yields of both conidia and chitinolytic enzymes was identified. Such coproduction may aid commercialization of T. hanianum by reducing the costs of two separate and valuable products. Q 1992 Academic

virens; biological glucosaminidase; enzymes.

GARY

COPRODLJCTION

OF

CHITINOLYTIC

ENZYMES

described here was to determine methods to optimize production of conidia and N-acetyl-fi-D-glucosaminidase (EC 3.2.1.30) and chitobiosidase from T. harzianum strain Pl. MATERIALS

AND

METHODS

Fungi and Media T. harzianum Pl (ATCC 74058) (Tronsmo, 1989a) was originally isolated from wood chips, while T. harzianum strain 1295-22 (ATCC 20847) was prepared using protoplast fusion (Stasz et al., 1988). G. virens strain 31 (ATCC 20903) was isolated from soil (Smith et al., 1990). All three are effective in controlling plant pathogenic fungi (Smith et al,, 1990; Harman and Stasz, 1991; Tronsmo, 1989b, 1991). Two basic mineral salts media were employed: a synthetic basal medium (SM) (Nelson et al., 1988) that contained 0.68 g KH,PO,, 0.87 g K,HPO,, 0.2 g KCl, 1.0 g NH,NO,, 0.2 g CaCl,, 0.2 g MgSO,. 7H,O, 2 mg FeSO,, 2 mg MnSO,, 2 mg ZnSO,, and 1000 ml H,O at pH 6.7 and a modified Richard (MR) medium that contained 10 g KNO,, 5 g KH,PO,, 2.5 g MgSO, .7H,O, 2 mg FeCl,, and 1000 ml H,O at pH 6.0. To these basic media were added, in various combinations and amounts, crab shell chitin flakes (Sigma Chemical Co., St. Louis, MO), purified colloidal chitin prepared as described by Vessey and Pegg (1973), glucose, sucrose, V8 juice (Campbell Soup Co., Camden, NJ), Proteose Peptone No. 3 (Difco Laboratories, Detroit, MI), and polyvinylpyrrolidone (PVP) (Polyclar AT, GAF Corp., Germany). Growth Conditions

and Enzyme Preparation

The fungi were grown in 100 ml of the respective media in 250-ml Erlenmeyer flasks. The flasks were inoculated with conidia from fungi grown on PDA plates (at the rate of 5 X lo6 conidia/ml final concentration in the inoculated media unless otherwise noted) and placed on an orbital shaker at 150 rpm and 23-25°C. At the termination of culture, biomass was separated from the extracellular liquid by centrifugation at 8000 g for 10 min, and this liquid was designated as the culture filtrate. In some experiments the biomass was ground before separation from the culture liquid using a Tekmar homogenizer (Thomas Scientific, Philadelphia, PA) at maximum speed for 2 min prior to separation of the biomass from the culture filtrate. Determination of N-Acetyl-P-D-glucosaminidase Chitobiosidase Activity in Culture Filtrates

and

Tests were developed specifically to quantify two different exochitinases. Exochitinases which release Nacetyl-P-glucosamine from chitin will be considered as N-acetyl-&glucosaminidase (hereafter described as

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273

glucosaminidase), whereas the enzymes that release dimerit units from chitin will be considered as 1,4-Pchitobiosidase (hereafter described as chitobiosidase) (Tronsmo and Harman, 1993). These activities were determined using a procedure similar to that of Roberts and Selitrennikoff (1988). Test samples (10 or 30 ~1) or 1:2 serial dilutions thereof were added to wells in a flatbottom-well microtest plate (Corning, Corning, NY). Fifty microliters of a substrate solution containing 300 yglml of either p-nitrophenyl-N-acetyl-P-D-glucosaminide or p-nitrophenyl-p-D-N,N’-diacetylchitobiose (both from Sigma) dissolved in 50 mM phosphate buffer, pH 6.7, were added using a multichannel pipette. The plates were incubated at 50°C by floating on styrofoam beads in a water bath. Reactions were terminated by the addition (with a multichannel pipette) of 50 ~10.4 M Na,CO, to each well, which also served to enhance the color of p-nitrophenol formed by the enzymatic cleavage of the substrate. Absorbance at 405 nm was measured using a Dynatech MR 700 microplate reader (Dynatech Laboratories, Guernsy, UK), and optical density values in the range 0.050 to 1.000 were used. One unit of enzyme activity was defined as the amount of enzyme that released 1 pmol of p-nitrophenol per milliliter enzyme per minute. Color released from p-nitrophenyl-N-acetyl-fl-D-glucosaminide or p-nitrophenylP-D-N,N’-diacetylchitobiose was considered as indicative of N-acetylglucosaminidase and chitobiosidase activity, respectively (Ohtakara et al., 1981; Roberts and Selitrennikoff, 1988; Tronsmo and Harman, 1993). The activities of these enzymes are associated with separate proteins, at least in T. harzianum Pl (Harman et al., unpublished information). For experiments in which the conidia were enumerated, the biomass was ground in a Tekmar homogenizer for 30 s. Samples so obtained were diluted lo-fold and counted in a Petroff-Hauser counting chamber (Thomas Scientific, Philadelphia, PA). Experimental

Design

Each experiment contained three replicates of each treatment, and each experiment was repeated twice. RESULTS

The first experiment compared the ability of the two test fungi to produce N-acetyl-P-D-glucosaminidase and chitobiosidase in SM or MR in the presence of chitin, colloidal chitin, and V8 juice. In most media, T. harzianum Pl produced more enzyme activity, as measured by micromoles nitrophenol per milliliter per minute, than G. virens 31. In general, more enzyme activity was produced in MR than in SM media with G. virens 31, while the converse was true with T. harzianum Pl. V8 juice substantially enhanced enzyme production by

274

TRONSMO

AND

n Pl Nagase q 31 Nagase

n Pl Biase W 31Biase

FIG. 1. Effect of media on the production of N-acetyl-fl-glucosaminidase (Nagase, A) and chitobiosidase (Biase; B) by 2’. harzianum Pl (Pl) or G. virens 31 (31) after 5 days on the mineral medium MR or SM with 1% chitin (Cs), 0.5% colloidal chitin (Cp), 15% V8 juice (V8), and 0.5% sucrose (S). There were three replicates of each treatment and the standard deviations are marked on the bars.

both fungi. Colloidal chitin induced more enzyme production than nonpurified chitin, especially with T. harzianum Pl (Fig. 1). Grinding cultures prior to separating biomass from culture filtrate had no effect on enzyme activity (data not shown). Preliminary experiments showed that the addition of insoluble PVP to the growth media increased the Nacetyl-p-D-glucosaminidase and chitobiosidase activity in the culture filtrate; however, the addition of 0.2 to 5% (w/v) PVP was equally effective (data not shown). Similarly, inoculum concentrations of 1 X lo5 to 1 X lo7 conidia/ml had no significant effect on either N-acetyl-fiD-glucosaminidase or chitobiosidase activity (data not shown). The influence of pH on sporulation was also determined. The maximum levels of sporulation with both G.

HARMAN

virens 31 and T. harzianum Pl were obtained in media initially adjusted to pH 6.7 and for T. harzianum 129522 at pH 5.0 (Fig. 2). The influence of PVP on enzyme activity was examined over time with both fungi. The addition of PVP resulted in a substantial increase in activity with G. uirens 31; less effect was seen with T. harzianum Pl. In both cases, a peak of enzyme activity was noted at 6 or 5 days, respectively, followed by a decrease and then a stabilization or gradual increase (Figs. 3A-3C). Media containing colloidal chitin gave rise to 2 to 3 times more of both enzymes from T. harzianum Pl than did crab shell chitin. The influence of a number of combinations of ingredients on N-acetyl-p-glucosaminidase and chitobiosidase production was determined with T. harzianum Pl in the SM basal medium (Figs. 4). Small amounts of enzyme were produced on glucose, sucrose, and chitin, all with and without 15% V8 juice, or sucrose plus yeast extract. Substantially larger amounts of both enzymes were produced when the fungi were grown in media containing colloidal chitin with V8 juice, sucrose, yeast extract, proteose peptone, or sucrose + proteose peptone. The amount of enzyme increased from 3 to 6 days in media containing colloidal chitin with glucose and glucose + V8 juice. With media containing colloidal chitin, PVP had variable effects. In some media combinations, the presence of PVP appeared to enhance enzyme activity, while in others it decreased or had no effect upon activity. After 3 days of incubation, the highest level of activity of both enzymes was produced on colloidal chitin with V8 juice or yeast extract, and after 6 days on colloidal chitin with sucrose and PVP. The level of production in these media was 50-80 times higher than in SM plus glucose (Fig. 4). 10 1295 9 2 .g ‘2 8

0 B

-I --t-

Pl

-

31

8

7

2

3

4

5

6

tl

I PH

FIG. 2. Effect of initial pH of the medium on the production of conidia after 6 days of growth of 2’. harzianum Pl (Pl), T. harzianum 129522 (1295), and G. virens 31 (31) on SM medium, with 0.05 A4 phosphate buffer adjusted with HCl or NaOH if necessary and 0.5% colloidal chitin.

COPRODUCTION

1.4

A

18

=E

0.8

8 -% &

0,6

‘2 2

0.4

zi

0,2

CHITINOLYTIC

-

31CsEiase

-

31 Cs PVP Biase

-----o-

31 Cs PVP Nagase

B -

Pl Cp PVP Nagase

-----P-

Pl Cs PVP Nagase

-

Pl Cs Nagase

10

.22 2 f -, e 8 B 3 T; E 3.

03’

AND

.c ;3 E 2 c

BIOMASS

BY

7’. harzianum

275

4

W

Nagase3D

3

Pl C$ Nagase

n

1

ENZYMES

31 Cs Nagase

1.2 .eE :

OF

C

1,2

2

1.0

W

Biase3D

20 s

nP

.;

Pl Cp Biase

-

Pl Cp PVP Eiase

-

Pl Cs Biase

--o-

Pl Cs PVP Biase

02’

61

FIG. 3. Time course of N-acetyl$-glucosaminidase (Nagase) and chitobiosidase (Biase) production by G. k-ens 31 (31) on MR medium with 15% V8 juice and 1% chitin (Cs) with or without 1% PVP (A), or 2’. harzianum Pl (Pl) on SM medium with 15% V8 juice and 1% chitin, or 0.5% colloidal chitin (Cp) with or without 1% PVP (B, C!). The averages of four replicates of each treatment are given.

Media that supported production of high levels of chitinolytic enzymes also supported production of large numbers of conidia. MR or SM basal media were equally effective in supporting conidial production of T. harzianum Pl when colloidal chitin was used as a carbon source (Fig. 5) whereas SM media were more effective with chitin as the carbon source. SM basal medium

FIG. 4. Effect of glucose (Glu), sucrose (S), chitin (Cs), colloidal chitin (Cp), V8 juice (V8), yeast extract (Y), proteose peptone (Pep), and polyvinylpyrrolidone (PVP) on the production of N-acetyl-@glucosaminidase (Nagase) or chitobiosidase (Biase) by 2’. harzianum Pl on SM medium after 3 (3D) and 6 (6D) days. The number before the substances refers to the concentration in percentage. There were three replicates of each treatment and the standard deviations are marked on the bars.

276

TRONSMO

FIG. 5. Effect of mineral solution (SM or MR) and colloidal tin (Cp), chitin (Cs), and 15% V8 juice (15V8) on the production conidia by !I’. harzianum Pl, 5 days after inoculation. There three replicates of each treatment and the standard deviations marked on the bars.

AND

chiof were are

HARMAN

Probably the sugar enhances the growth of the fungus early in the culture cycle, but after 6 days the sucrose is fully utilized and no longer functions as a catabolic inhibitor. Enzyme production reached its peak after 5 days for T. harzianum Pl and 6 days for G. virens 31. Thereafter the activity in the culture filtrate dropped to about l/3 of the maximal activity, perhaps because the protein in the culture filtrate was used as a carbon source when other nutrients were depleted. Especially for G. uirens 31, activity increased again later (Fig. 3). This latter increase was perhaps associated with autolysis of older hyphae. Since Trichoderma and Gliocladium cell walls contain high proportions of chitin, chitinolytic enzymes are no doubt involved in growth and differentiation of these fungi. On media that supported high enzyme production, polyvinylpyrrolidone sometimes increased the enzyme activity in the culture filtrate. This is probably not caused by stimulation of the production, but by mini-

with glucose, sucrose, or crab shell chitin gave low levels of conidial production; however, colloidal chitin increased conidial levels approximately loo-fold (Fig. 6). Addition of V8 juice enhanced spore production regardless of other amendments to the medium. Yeast extract was also effective in enhancing sporulation. Several media gave spore concentration near log/ml after 6 days, with the greatest concentration in media containing 0.5% colloidal chitin and 15% V8 juice, 0.5% glucose and 0.5% colloidal chitin, 0.5% sucrose and 0.5% colloidal chitin, and 0.1% yeast extract and 0.5% colloidal chitin (Fig. 6). Addition of PVP had no effect upon conidial production (data not shown). DISCUSSION

Low levels of both N-acetyl-P-D-glucosaminidase and chitobiosidase were produced even in the absence of chitin, but higher levels were produced in the presence of chitin, particularly colloidal chitin. These data indicate that these fungi have a low level of constitutive production of chitinolytic enzymes, but that these enzymes are induced in the presence of the appropriate substrate. Production of both N-acetyl-@-D-glucosaminidase and chitobiosidase appears to be coordinately regulated, since activities of both enzymes were affected similarly by similar alterations in the media employed. Glucose and sucrose generally inhibited production of these enzymes by T. harzianum and G. virens, indicating that these sugars function as catabolic inhibitors. However, the presence of low amounts (0.5%) of sucrose may enhance the activity measured after 6 days of growth.

6

FIG. 6. Effect of glucose (Glu), sucrose (S), chitin (Cs), colloidal chitin (Cp), V8 juice (V8), yeast extract (Y), and proteose peptone (Pep) on the conidial spore production of 2’. harzianum Pl in SM medium after 3 (A) or 6 (B) days after inoculation. The number before the substances refers to the concentration as a percentage. There were three replicates of each treatment and the standard deviations are marked on the bars.

COPRODUCTION

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ENZYMES

mizing inhibitory effects of phenolic substances (Loomis and Battaile, 1966). The experiments illustrate that by selecting appropriate media components, high levels of chitinolytic enzymes can be obtained, especially from T. harzianum Pl. This fungus therefore seems to be a useful source of chitin-degrading enzymes because it is easy to grow and produces high amounts of enzymes on simple defined media. Levels of enzyme produced are reasonably high and are, for example, higher than that which we used successfully for purification and characterization of enzymes (Harman et al., 1993). The chitinolytic enzymes from T. harzianum are likely to be useful as isolated proteins; the purified enzymes from this organism act synergistically to provide control of plant pathogenic fungi at protein concentrations substantially below that obtained with other reported chitinolytic enzymes (Lorito et al., 1993). The enzymes can be kept as dried proteins or in solution for several months without loss of activity (Harman and Tronsmo, unpublished information). Further, they act synergistically with a variety of fungicides used in medicine or agriculture and reduce the quantity of synthetic pesticide required for control. Consequently, strategies can be envisioned where a topical application of a low level of fungicide plus chitinolytic enzyme will provide the appropriate therapeutic or prophylactic effect but where human exposure to the synthetic fungicide can be reduced. For example, application of a low level of fungicide plus an enzyme to fruit to be protected from rots may provide control equivalent to that obtained with a higher quantity of the fungicide alone. Many of the same media that supported high levels of chitinolytic enzyme production also provided high levels of conidial production. Conidia are not usually produced in submerged culture; so, media providing high levels of conidial production are useful (Harman et al., 1991). The levels of conidial production noted in this study are comparable to or higher than those reported earlier (Harman et al., 1991). Some of the best media discovered in this study supported high levels of both chitinolytic enzymes and conidia production. The combination of SM synthetic salt solution and 0.5% colloidal chitin, 0.5% sucrose, and 1% or less of PVP seems promising for coproduction ofboth enzymes and biomass. Such coproduction may be commercially useful for producing biomass for biological control and chitinolytic enzymes. ACKNOWLEDGMENT

Financial support from the Agricultural Agricultural edged.

Research

Council

of Norway

University of Norway and are greatfully acknowl-

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7’. harziunum

REFERENCES

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and

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enzyme Br. Mycol.

of in