Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

0038-0717/84 Copyright

PARASITISM ROLFSII

OF SCLEROTIA BY TRICHODERMA Y.

ELAD,

t’

IY84

$3.00 + 0.00

Pergamon

Press Ltd

OF SCLEROTIUM HARZIANUM I. CHET

RINA BARAK and

Dcpartmcnt of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew Jerusalem, Rchovot 76100, Israel

University

of

Summary-The

ability of Trichotkrtrrtr hurziunutn isolate 203 to attack the soil-borne plant pathogen Sclerotiunl rol/,kii is apparently connected with the production by the isolates of chitinase and /I-( l.3)-glucanase inside the attacked sclerotia during parasitism. SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytopiasmic contents. It is assumed that T. hurrirmunz utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.

MATERIALS AND METHODS

INTRODUCTION

The soil-borne plant-pathogenic fungus Sckrotium rolfsii Sacc. forms brown sclerotia which are very well organized compact structures, built of three layers: the rind, which is composed of empty melanized cells; the cortex cells. which are filled with vesicles, and the medulla (Chet. 1975). Chet et 01. (1967), finding that the melanin-like pigment of the sclerotia is absent from hyphal walls, concluded that this pigment is important in providing resistance to biological and chemical degradation. Willets (1971) suggested that the melanin acts as an inhibitor of chitinases and glucanases, which are associated with biological degradation. Several workers have reported the involvement of mycoparasites in decline of sclerotial fungi, e.g. Sporidesmium sclerotiorum parasitic on Sclerotinio minor, Sclerotiniu sclerotiorum and Sclerotium cepivorum (Adams and Ayers, 1981), Coniothyrium minituns on S. sclerotiorum (Trutmann et al., 1980) and Laterisporu breviramu on S. minor (Uecker et (II., 1982). Antagonism between Trichodermu spp and S. sclerotiorum, Rhixctoniu tulipurum and S. minor have also been reported (Huang, 1980; Davet, 1979; Gladders and Coley Smith, 1980). Shigemitsu et ul. (1981) published microscopic observations of Corricium ro&ii sclerotia infected by Asper~illus tcrreus. Parasitism of T. hurziunum on S. ro&kii sclerotia was used in successful biological control field-trials conducted by Wells et ul. (1972) and Elad et ul. (1982b). T. hur~iunum, isolated from the soil, excreted /j-( I ,3)-glucanase and chitinase when grown on mycelium and cell walls of either S. rolfsii or Rhizoctoniu soluni. Mycelial cell wall degradation, also observed under scanning and transmission electron microscopes (Elad et ul., 1983a, b), was followed by penetration of the antagonistic fungus into mycelium of the tested plant pathogens. We report the sequence of events and mode of interaction between T. harziunum and S. ro!fsii sclerotia.

Struins

and growth

conditions

Trichodermu hurzianum Rifai isolates 203, 250 and I IO were used. All three were capable of parasitizing Rhizoctonia solani Kiihn, but only 203 could attack sclerotia of Sclerotium rolfsii Sacc. (Elad el al., 1982b). S. rolfsii (type A, ATCC 26325) (Chet and Henis, 1969) and T. hurzianum isolates were grown

and maintained et ul.,

on a synthetic

1973) at

collected Enzymutic

28 f

1°C.

from 4-week-old

medium

S. rolfsii

(SM) (Okon sclerotia were

SM plates.

uctivity

S. roI/Gi sclerotia (5 gg ‘) and a spore suspension of T. hurzknum ( IO4 g- ‘) were mixed in an autoclaved, washed, loamy-sand soil (Elad et ul., 1982a). A week

later the sclerotia were separated from the soil on a metal mesh (500 pm grid). Ten ml of 0. I N phosphate buffer (pH 5) were added to either log soil or I g sclerotia which were then shaken for 20 min (200 rev min-‘) and centrifuged at 5OOOg for IO min. The supernatant served as crude enzyme and was examined for its enzymatic activity and protein content after lyophilization as described by Elad et al. (1982b). /3-(l,3)-glucanase (EC 3.2.2.39) was assayed by following the release by culture lyophilizate of free glucose from laminarin, using the glucose oxidase reagent (Sigma Chemical Co., MO 63178, USA) which is based on enzymatic oxidation of glucose and the appearance of brown colour. The amount of glucose was determined calorimetrically at 450 nm. Specific activity (GLJ) was expressed as pmol glucose mg protein-’ h-‘. The reaction mixture, containing culture lyophilizate in 2.0 ml 0.1 M citrate buffer (pH 4.7) and 1.6 mg soluble laminarin, was incubated at 40°C for I h and was stopped by boiling. Chitinase (EC 3.2.1.14) was assayed by following the release of IV-acetyl-glucosamine according to the method of Reissig CI al. (1955). Specific activity (CU) 381

382

Y.

ELAII 6’1 (Ii.

was expressed as /~mol N-acetylglucosamine mg protein’ h ‘. The reaction mixture, containing culture lyophilizate in 2.0 ml 0.1 M phosphate buffer (pH 5.1) and 1.6 mg colloidal chitin, was incubated at 37°C for 2 h and was stopped by boiling. Protein content of the enzyme solutions was determined by the Folin phenol reagent (Lowry er al., 1951). The experiment was repeated three times. Mycoparusitism

The following system was used to observe parasitism: a cellophane membrane, well washed in hoiiing. distilled water, was placed on the surface ofwater agar. An agar disc (SM), covered with T. /rar;iunum, Isolate 203, mycelium, was placed on one end of the cellophane membrane and another disc covered with S. m&ii mycelium, was placed on the other. The mycoparasite and its host grew towards each other (at 28 + 1°C for 4 days) and the hyphae intermingled on the cellophane membrane. Ten days later parasitism of T. Aur:icltw~~ was observed macroscopically on sclerotia of the plant pathogen (Chet et ul., 1981). S. ro&ii sclerotia were removed and iixed for 12 h in 3”/Qgiutara~dehyde (Sigma) in 0.1 M phosphate buffer (pH 7.0) in 4’C. The specimens were then dehydrated in a graded acetone series and half of them were cut with a scalpel and critical-point dried (Polaron Equipment Ltd, Watford. England). Dried specimens were coated with gold palladium using a Polaron ES00 (Poiaron Equipment Ltd) and viewed in a scanning electron microscope (Jeol JSM 35C). TIM

rn&kii sclerotia was examined. Among three T. Irarziunum isolates, only 203 was capable of attacking S. ro@ii. All three were inoculated into autoclaved soil

containing sclerotia. Enzymatic activity was tested in extracts of both the attacked sclerotia and the soil. T. horziunum isolate 203, capable of attacking S. ro&ii, excreted more B-( I ,3)-giucanase and chitinase than isolates 250 and 1IO which are incapable of attacking this pathogen (Table I). With the SEM it was seen that S. w&ii sclerotia parasitized by T. /~ur~~unut?lhad lost their regular shape and that holes had appeared in them (Figs I and 2). The Trichodwnu mycelium grew over the sclerotia and appeared to penetrate in between rind cells (Fig. 3). Over the surface of the scierotium it is possible to observe aggregates of conidia of the mycoparasite (Fig. 3). Parasitized sclerotia were cut and observed under SEM, and hyphae of Trichodermcc were detected inside the cut sclerotium (Fig. 4) while many conidia of T. hurAmum were also found on the cut surface of a sclerotium (Fig. 5). The mode of parasitism by T. hurziunum was further investigated by examining parasitized sclerotia with TEM, T. hur5unum hyphae penetrated medullar sclerotial cells (Fig. 6) and this myceliai invasion is apparently associated with hydrolysis of the host cell walls. A progressive invasion resulted in loss of cytoplasm in host cells. In general, the cells of the scierotiai host which are relatively large, contained or were surrounded by a few cells of the mycoparasites. It should he noted that osmiophilic inclusions were observed in the Tri~~l~)ffi,r~If~ mycelium which interacted with the host cells.

Procedures

Specimens for TEM were fixed for I2 h in 3% glutaraidehyde and post-fixed for 2 h in aqueous I”/, 0~0, (Sigma). After dehydration in a graded ethanol series and a graded propylene oxide series, the specimens were embedded in a Epon 8 I2 medium (Polaron Equipment Ltd). Thin sections, prepared with an LKB Ultrotome III microtome, using glass knives, and stained with uranyl acetate and lead citrate, were examined with a Jeoi 100 CX ultramicroscope at 80 kV. RESULTS

The possible connection between the enzymatic activity of Trichodwnu and its ability to parasitize S. Table 1. Enzymatic activity of T. har:ianunt Enzymatic activity /f-( I ,3)-glucanase activity m soil @mot glucose mg protein-’ h-‘) (GU) Chitinase activity in soil (~~molGlcNAcmg protein-’ h--l) (CU) GU in sclerotiai extract CU in sclerotial extract

DISCUSSION

The enzymes a-(1,3)-glucanase and chitinase were shown by Elad et al. (1982a) to he excreted by T. harziunum when grown on cell walls of the soil-borne plant pathogens S. ro@ii and R. solani and hyphal cell wall degradation had been observed by SEM and TEM (Elad ef al., l983a, h). However, S. ro&ii sclerotia have melanin in their rind, which apparently increases resistance of the rind to biodegradation (Chet and Henis, 1969). Our data (Table I) show that T. harziunum 203 decomposed not only hyphal walls but also the contents of sclerotia of S. ro&Isii.These data show that significantly greater activity of hydrolytic enzymes was detected in the surrounding soil, isolates in the soil and in 5’. rolfkt sclerotia’ _

7‘. hur~ionurn isolate 203’ 250’

I IO’

0.05c4

4.36~1

2.96b

3.56ah

0.08~ 1.2%

5.88a 6.oOa 6.8Oa

2.12b 4.8Ob 4.2Ob

I .53c 4.4Ob 4.40b

1.34c

‘Extracts were made from soil and sclerotia. Enzymatic activity of fi-(1,3)-glucanase and chitinase was tested by following the release of glucose and N-acctylglucosaminc from laminarin and chitin, respectively. IT. hor:innum 203 is capable of parasitizing S. rolfsir and H. .\o/uni. ‘T. hrrrziunum 250 and I10 parasitize only R. .so/ani. ‘Numbers of each line followed by the same letters are not stgnifican!ly dilTcrenr [ P = 0.05).

T. harzianum

Fig.

I. Scanning

electron

micrograph

Fig. 2. SEM micrograph

(SEM)

of a sclerotium

383

on S. rolfsii

of non-parasitized 100 pm).

attacked

S. rolfsii sclerotium

by T. harzianum (Bar marker

(Bar

marker

= IOOpm).

=

Fi g. 3. SEM micrograph of T. hurzianum in between its cells (Bar marker = IOpm).

Fig. 4. SEM

Filg. 5. SEM

micrograph

micrograph

hyphae growing over a S. rolljii sclerotium and penett .ating Note the T. harzianum conidia over the sclerotium su rface.

of a broken sclcrotium showing many mycoparasitic sclerotium (Bar marker = IO pm).

of T. lrrrr%mrm

hyphac (arrows)

inside the

conidia growing inside a broken sclcrotium (Bar marker 384

= I pm).

385

T. hurziunumon S: roljkii

Fig.

6. TEM

micrographs of a thin section of an attacked sclerotium showing extracellular intercellular mycoparasitic hyphae of T. hurzianum(TH) (Bar marker = 0.2 pm).

and in the parasitized S. roffiii sclerotia in the presence of the pathogenic Trichoderma isolate 203, compared with the non-pathogenic isolates (Table 1). This phenomenon may explain the variability in the antagonistic activity of T. harziunum isolates. Baker and Cook (1974) suggested that hyperparasites should be most effective against survival structures of plant pathogens, if they are to be successful biological control agents. The antagonistic T. harziunum has indeed already proved its potential as a biological control agent (Elad ef al., 1982b). This fact is explained here by the high lytic activity of T. harziunurn associated with its ability to invade sclerotia. Sclerotia invasion and cell wall biodegradation was observed by both SEM and TEM (Figs l-6). Sclerotial cell wall degradation indicates the possible activity of extracellular lytic enzymes, which also cause the loss of cytoplasmic content from the attacked cells. Since T. harzianum excretes proteases and lipases (Elad et al., 1982a) it may be assumed to utilize the host cell contents, which probably enables

and

it to grow and sporulate intensively (Figs 3 and 5). However, the emptiness of host cells could have resulted from disintegration of cell walls and leakage of the cytoplasm. Enzymatic decomposition of host cell walls has been demonstrated with other mycoparasites such as Gliocludium virens, which attacks the mycelium and sclerotia of Sclerotiniu sclerotiorum (Tu, 1980) and T. hurziunum attacking hyphae of R. soluni and S. rolfii (Elad et al., 1983a, b). SEM and TEM techniques enabled us to demonstrate how the mycoparasite T. hurziunum attacks not only mycelium but also resistant resting structures of S. rolfsii.

Acknowledgemenrs-We acknowledge the help and discussions with Dr E. Zamski, Aviva Ben-David, Baat and Zili Sadovski of the Hebrew University of lem, Faculty of Agriculture, Rehovot. This research was supported by a grant from the States-Israel Binational Agricultural Research and opment Fund (BARD).

useful Naomi JerusaUnited Devel-

Y. EL/W r/ ul.

3X6 REFERENCES

Adams P. B. and Ayers W. A. (1981) Sporidesmium scleroriorum distribution and function in natural biological control of sclerotial fungi. Phytopothology 71, 9Ck93. Baker K. F. and Cook R. J. (1974) Biological Control of flrmr Purhogens, Freeman, San Francisco. Chct I. (1975) Ultrastructural basis of sclerotial survival in soil. Micro&d Ecology 2, 194200. Chet I. and Henis Y. (1969) Effect of catechol and disodium EDTA on melanin content of hyphal and sclerotial walls of Sclerolium ro@ii Sacc. and the role of melanin in the susceptibility of these walls to /I-( 1,3)- glucanase and chitinase. Soil Biology & Biochemistry 1, 131-138. Chet I.. Henis Y. and Mitchell R. (1967) Chemical composition of hyphal and sclerotial walls of Sclerotium rolfsii. Cunadiun Journal of Microbiology 13, I37- I4 I. Chct I., Harman G. E. and Baker R. (1981) Trichodermu humurum: its hyphal interactions with Rhizoctoniu solani and Pyrhium sp. Microbial Ecology 7, 29-38. Davet P. (1979) Activite antagoniste et sensibilite aux pesticides de quelques champignons associex aux sclerotes du Sclerotiniu minor Jagger. Annals of Phylopaihology 11, 53-60. Elad Y., Chet I. and Hcnis Y. (1982a) Degradation of plant pathogenic fungi by Trichodermu harziunum. Cunudiun Journul qf Microbiology 28, 719-725. Elad Y., Hadar Y.. Chet I. and Henis Y. (1982b) Prevention with Trichoderma hurzianum Rifai aggr., of reinfestation by Sclerorium rolfsii Sacc. and Rhizoctoniu solani Kuhn of soil fumigated with methyl bromide, and improvement of disease control in tomatoes and peanuts. Crop Protection 11, 199-21 I. Elad Y.. Barak R., Chet I. and Henis Y. (1983a) Ultrastructural studies of the interaction between Trichodermu spp. and plant pathogenic fungi. Phytoparhologicshe Zeitschrifr 107, l68- 175. Elad Y.. Boyle P., Chct I. and Hcnis Y. (1983b) Parasitism of Trichodermu spp. on Rhizoclonia wluni and Sclerotium ro!/lsii-SEM studies and detection by FITC lectins. Phy/opu/hology 73, 85-88.

Gladders P. and Coley Smith J. R. (1980) Interacttons between Rhizocronia ruliparum sclerotia and soil microorganisms. Transactions ofthe Briiish Mywlogicul Sock/j. 74, 579-586. Huang H. R. (1980) Control of Sclerotinia wilt of sunflower by hyperparasites. Canadian Journal of Plant Pathology 7, 2632. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Okon Y., Chet I. and Henis Y. (1973) Effect of lactose. ethanol and cycloheximide on the translocation pattern of radioactive compound and on sclerotium formation in Sclerotium rorsii. Journal of General Microbiology 74. 251-258. Reissig J. L., Stominyer J. L. and Leloir L. F. (I 955) A modified calorimetric method for estimation of Nacetylamine sugars. Biological Chemis/ry 27, 259-266. Shigemitsu H., Hohno M. and Kunoh H. (1981) Microscopic observation of Corticium rolfsii sclerotia infected by Aspergillus terreus. Transactions of rhe Mycolog~cul Society of Japan 22, 283-291. Trutmann P., Keane P. J. and Merriman P. R. (1980) Reduction of sclerotia inoculum of Sclerorimu sclerotiorum with Coniothyrium minilans. Soil Biology & Biochemistry 12, 46 l-465. Tu J. C. (1980) Gliocludium oirens a destructive mycoparasite of Sclerotinia scleroriorum. Phytopulhology 70, 67&674. Uecker F. A., Ayers W. A. and Adams P. B. (1982) Lu/erispora breoirumu. a new hvphomvcete on sclcrotia of Sclero;inia minor. Myco~uxon -14, 49 i-496. Wells H. D.. Bell D. K. and Jaworski G. A. f 1972) EtJicac\ of Trichoderma harziunum as a biocontrol for Sclero/ium rolfkii. Phytoparhology 62, 442447. Willetts H. J. (1971) The survival of fungal sclerotia under adverse environmental conditions. Biologiwl Rwicwc 46. 387407.