Download Phytopathologische Zeitschrift

Ph yrop3th . Z ., 107,168- 175 (1983)
© 1983 Vcrbg Paul Parcy, Berlin und H amburg
l SSN 0031 -948 1 I lntcrCodc: PHYZA3
The Hebrew University of Jerusalem,
Department of Plant Pathology and Microbiology,
Faculty of Agriculture, Rehovot, Israel
Ultrastructural Studies of the Interaction
Between Trichoderma spp. and Plant Pathogenic Fungi
By
Y.
ELAD, RINA BARAK,
I.
CHET
and Y.
HENIS
With 10 figures
Received October 18, 1982
Abstract
The ultrastructural changes during parasitism of the biocontrol agents Trichoderma harzianum and T. hamatum, were observed under a uansmission electron microscope. Electron
micrographs show that during the interaction of TridJoderma spp. with either Sclerotium
rolfsii or Rhizoctonia solani the hyphae of the paras ites contact their host, and then enzymatically digest their cell walls. Extracellular fibrillar material is deposited between the interacting cells. P arasite organelles, e.g. mitochondria, ves icles and dark osmiophilic inclusions,
accumula te in the parasitizing cells. In response to the invasion, the hoStt produces a sheath
matrix which encapasulates the penetrating hyph a and <the h ost cells become empty of cy.taplasm.
Z usammenfassung
Ultrastrukturelle Untersuchungen
der Wechselwirkung zwischen Trichoderma-Arten
und pflanzenpathogenen Pilzen
Die ultrastrukturellen .Anderungen wahrend der parasirtischen Phase der BiokontrolleAgen z ien Trichoderma harzianum und T. hamatum wurden unter einem Transmissionselektronenmikroskop beobachtet. Die Elektronenmikrogramme zeigen, daB wahrend der Wechselwirkung der Trichoderma-Arten entweder mit Sclerotium rolfsii oder mit Rhizoctonia solani
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Ultra.S1tructural Studies of the Interaction between Trichoderma spp.
169
die Hyphen der Parasiten mit ihrem Win in Konukt kommen und dann seine Zell wand e
enzymatisch vcrzehren. Ein exuazelluHirer fibrillenartiger Stoff wird zwischen den sich gegenseitig beeinflussenden Zellen eingelager.t. Parasi tische Organelle, z. B. Mitochondrien, Vesike l
und dunkle osmiumliebende EinschluBkorper, we rden in den parasiticrten Zellen angeha uft.
Gegen diese Invasion erzeugt der Win eine umhiillende Gewebeschicht, die die eindrangcnden
Hyphen einkapselt und die Wirtszellen sind Zytoplasma leer.
Several fungi are mycoparas1t1c on soil-borne plant pathogens. Among
these are Gliocladium spp. (Tu 1980), Trichoderma spp. (DENNIS and WEBSTER
1971 ), Pytbium acanthicum (HocH and FuLLER 1977) and S phaerotheca spp.
(HocH and PROVVIDENTI 1979). Work dealing with biological control of
pathogenic fungi demonstrates that T. harzianum and T. hamatum are effective biocontrol agents (ELAD et al. 1980, 1981 a, 1981 b) HENIS and CHET 1975,
WELLS et al. 1972 and HARMAN et al. 1980). Its mode of action in parasitism
was recently studied (ELAD et al. 1982 a, 1982 b) .
When a parasitic fungus reaches the host hyphae it usually coils around it
or produces appressorium-like structures and hook-shaped contact branches.
This was shown with Trichoderma hamatum vs. Rhizoctonia solani (CHET et
al. 1981); T. harzianum vs. Sclerotium rolfsii (ELAD et al. 1982 a) and Stephanorna phaeospora vs. Fusarium sp. (HocH 1978). Mechanical and enzymatic
penetration of the host fungi by the parasite are the next step of interaction
(HocH and FuLLER 1977, ELAD et al. 1982 a).
Trichoderma harzianum produced P-(1,3)-glucanase and chitinase when
grown on cell walls of its host - Sclerotium rolfsii or Rhiz octonia solani
(ELAD et al. 1982 b). The enzymatic activity detected in dual cultures of both
the mycoparasite and the soil-borne pathogens was suggested as the mechanism
by means of which this parasite attacks its host.
However, no ultrastructural studies by means of transmission electron
microscopy of the sequence of events, occurring during the interaction of
Trichoderma spp. with its host, have yet been made. In the present work we
used TEM to study the ultrastructural changes during the process of parasitism
in this fungal-fungal interaction.
Materials and Methods
Rhiz octonia solani Kuhn a nd Sclerotium rolfsii Sacc. were used as host fun gi to the
mycoparasires Trichoderma harzianum Rifai (ELAD et al. 1980, 1981a, 1981 b, l982a, 1982b)
and T. hamatum (Bon.) Bain (CHET et al. 1981 , ELAD et al. 1982a). Fungi were maintained on
a synthetic medium (SM) (OKON et al. 1973) at 30 ± 1 ° C .
The following system was used in orde r to obse rve •t he interaction sites: A cellophane
membrane, well washed in boiling, distilled water, was placed on t he surface of water agar
(CHET et al. 1981). Agar discs (SM) covered with mycelium of Tricho derma spp. were placed
on one end of the cellophane membrane and discs wi.t h one of the pathogenic fungi were
placed on the other. The cellophane plates were then incubaud a t 28 ± 1 °C for 4 days. The
mycoparasitc and its host grew towards each other and the hyphae in termin gled on th e cellophane plates. The cultures were fir st observed by a phase-conxrast microscope.
170
ELAD, B.ARAK, CHET
and
HENIS
Preparation for TEM microscopy
The mycelium fr om the in.tcraction site was removed and the organisms were fixed in
3 % glut::traldchyde (Sigma Chemicals Co., St. Louis, Mo. 63178, USA) buffered with 0.2 M
phosphate buffer, pH 7.0. After refri ge ration for 12 h, the specimens, in the same buffer
post-fi~ed in :tqueous 1 % O sO 4 (Sigma), a nd dehydrated via a graded e,t hanol series (and
l ate ~ wah a graded propylcneoxide seri es), were embedded in an Epon 812 medium (Polaron
Equtpmcnt Ltd., Watford, England, CHET and HENIS 1969). Thin sections, prepared wi.th an
LKB ultr ot oma III microtome, using glass knives, were stained with uranyl acetate and lead
citra t e and examined with a jeol 100 CX tra nsmiss ion electron microscope, at 80 kV.
Results and Discussion
Mycelial san1ples, taken from the interaction sites of dual cultures, of
either R. so/ani or S. rolfsii and either T. hamatum or T. harzianum, were
observed in a transmission electron microscope. In general, no significant differences were observed between the interaction of either of the mycoparasites
with either host. Ultrastructurally, the process of penetration and appressorium
development m ay b e associated with a variety of morphological alterations at
or near the host-parasite interface. A Trichoderma hypha first contacts the
host mycelium (Fig. 1). During this time a significant accumulation and deposition of intercellular fibrill ar material can be observed outside the host-parasi te interacting cells (Fig. 2). Our observations support the idea that there is
an increase of a mucilaginous substance, apparently polysaccharides, originating from either one of the interacting fungi. This was also found with Stephanorna phaeospora which parasitizes several species of Fusarium (HocH 1978)
and is simila r to the description of the nematode-capturing organs of Arthrobotrys ofigospora (NORDBRING-HERTZ and STALHAMMAR-CARLEMALM 1978).
Basically, there are two main types of interaction between Trichoderma
and either R. solani or S. rolfsii. The mycoparasite may produce and appressorium-like body (Fig. 1) or it may coil around the host hyphae (Fig. 3). That
Trichoderma spp. coil around the host hyphae, has been observed in earlier
works using light (DENNIS and WEBSTER 1971 )., Nomarski interference (CHET
et al. 1981) and scanning electron microscopy (ELAD et al. 1982 a). This, however_, is the first study of T richoderma-pathogenic fungi interactions, using
transmission electron microscopy. This method enabled us to detect the ultrastructural changes occurring during the process of parasitism, inside the cells
an d not only on their surface.
After the meeting of the two hyphal cells, the coiling hyphal branch of
Trichoderma constricts and partially digests the S. rolfsii cell wall in the interaction si te (Fig. 3 ). Trichoderma then begins to penetrate the host cell (Fig. 4).
Direct penetration of the host cell wall has been previously found with T. hamatum using SEM (ELAD et al. 1982 a). Penetration occurs at different locations of the host cell walls even when these are thick, melanized walls (Figs. 4
to 6 ). Melanin is known for its ability to confer resistance to biological and
Ultrastructural S.tudies of the Interaction between Trichoderma spp.
171
chetnical degradation in fungi (CHET and HEN IS 1969, HEN IS and CHET 1975) .
The digestion of the host cell walls indicates the activity of extracellular enzyn1es, e.g. P-(1 ,3 )-glucanases and chitinases, recently reported by ELAD et al.
(1982 b), with the same antagonistic fungus. The enzymatic decomposition of
the host cell wall was shown and discussed with other mycoparasites, such as
Piptocephalis virginiana (MANOCHA 1981 ), Gliocladium virens which attacks
Sclerotinia sclerotiorum (Tu 1980), Pythium acanthicum (HocH and FuLLER
1977) and Verticillium lecanii which parasitize uredospores of Puccinia graminis var. tritici (HANSSLER et al. 1982). On the other hand, our observations
also reveal that a physical pressure on the host hypha is exerted before penetration (Fig. 1).
After penetration, the host forms a layer associated with the penetrating
parasitic hyphae. This electronlucent sheath encapsulates the penetrating
hyphae in a relatively narrow layer (Figs. 6 and 7). It can be seen that the
host cell walls are partially dissolved (Figs. 4, 6 and 7). Similarly, JEFFERIES
and YouNG (1978), working with the mycoparasite Piptocephalis unispora
which attacks fungi from the Mucorales_, showed the formation of a sheath
matrix in the host cells, resulting from the invasion of the parasite. This effect
was also found by HocH (1978) and RAKVIDHYASASTRA and BUTLER (1973 ).
The Trichoderma isolates tested were found to be ''destructive mycoparasites" (HASHIOKA 1973, Tu 1980). Mycoparasitism finally causes the host cells
to be empty of cytoplasmic contents. The remaining cytopslasm_, mainly surrounding the invading hyphae, also show disintegration of the remaining host
cytoplasm. Decomposition of the host cytoplasm seems to proceed rapidly
after the wall is pierced by the mycoparasite (Figs. 2 and 4-1 0).
In general, the hyphal cells of the host, R. solani, are large enough to
contain a few parasitic cells (Fig. 8). The parasitic cells also invade from cell
to cell, via the host's septum (Fig. 10). In some cases, the invading hypha! tips
of T. hamatum expand inside the host (Fig. 9).
Invaded R. solani cells were found to be empty or with little cytoplasmic
residue. This emptiness results from the disintegration of the cell wall and
leakage of the cytoplasm. The leakage could, however, occur during specimen
preparation. Lack of cell contents may result from translocation into intact
cells and autolysis (OKON et al. 1973 ). The host cytoplasm is, apparently,
utilized by the parasite, which is capable of degrading proteins and lipids
(ELAD et al. 1982 b) .
Cytoplasm of the parasite accumulates in the cells associated with the
host mycelium (Figs. 1 and 3). An accumulation of vesicles and mitochondria
can be observed in the penetrating hyphae (Figs. 4, 7_, 8 and 9). This may
indicate a high metabolic activity in the infecting tip. The presence of vesicles
in metabolically active tip growing cells has often been connected with wall
synthesis and softening activity (BARTNICKI-GARCIA 1973 ), at the tips of
which, layers of newly formed cell wall material can be observed (Figs. 6
and 7).
172
ELAD, BARAK , CHET
and HENIS
.·c . . . ..
Fig. 1. Adjacent cells of T. harzianum and S. rolfsii. Appressorium-like structure of T. harzianum is attached to S. rolfsii cell, Y 7875. • Fig. 2. A ssociation between T. harzianum and
R. solani. Note fibrillar materi al deposited in between the hyphae, X 5250. • Fig. 3. Electron
micrograph of :1 thin section across T. harzianum coiling around an S. rolfsii cell. Note the
constriction an d the partial digestion of the host's cell wall, X 6825. • fjg. 4. T. hamatum
hypha l branches penetrating R . solani cell. Lysed host cell walls and remaining cytoplasm are
observed, X 7500
Abbrevia.rions: HR = T. harzianum; HM = T. hamatum; m = mi-tochondria; s = sheath;
v = vesicles; = osmiophilic inclusion; cw = cell wall; mew = melanized cw; fm =
UltraSitructural Studies of the Interaction between Trichoderma spp.
173
7
0
•
6
,
...
....-.
..
.)
.
."
~
Fig. 5. Penetration of T. hamatum hyphal tip through melanized R. solani cell wall, X 11 ,250.
• Fig. 6. Formation of shea.th matrix by R . solani in response to T. hamatum hypha l penet ration, X 8625. • Fig. 7. P enetration of T. hamatum hypha! branches into R . so /ani cell. Note
the lysed R. solani cell wall, the accumulation of i.ts cytoplasm around the T . hamatum cell
and ·the formation of sheMh matrix, X 6450. • Fig. 8. Different stages in t he interact ion
between T. hamatum and R. so/ani, X 3975
fibrillar material; ph = penetrating hyph ae; c
appressori urn.
= cytoplasm; 1 = layers of cell wall ; ap =
174
ELAD, BARAK, CHET and HENIS
Fig. 9. Penetration of T. hamatum into R. so/ani hyphae. Note the accumulation of mucilaginous material o utside the host cell and widening of the parasi.te's tip inside the R. solani
cell, X 4875. • Fig. 10. Penetration of T. hamatum through R. solani septum. Note the aggregation of host cytoplasm around the parasitic tip, X 3825
In several cases osmiophilic inclusions were observed in Trichoderma
mycelium which interacted with its host. These are more abundant in the
infecting tips than in normal cells. Similar organelles were found in A. oligospora during nematode trapping (NoRDBRING-HERTZ and STALHAMMARCARLEMALM 1978) . Apparently, these organelles have a function in the parasitic activity of Trichoderma . Mycoparasitism, as evidenced by the interactions
described herewith, is the mode of action by which the potential biocontrol
agents, Trichoderma spp., attack the soil-borne plant pathogens, R. solani and
S. rolfsii.
This r esearch was supported by a grant from the United Sta.te s-Israel Binational Agricultural Research and Development Fund (BARD). We thank E. HATAV, E. ABRAHAM, N.
BAHAT and Z. SADOVSKI of the E lectron Microscopy and Photography Department in the
Hebrew University of Jerusalem for their technical assistance.
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Authors' address: Depafltment of Plant Pathology and Micr obiology, The H ebrew
University of Jerusalem, Faculty of Agriculture, R ehovot 76100 (Israel).
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