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ANTIFUNGAL ACTION OF NEW TRICHODERMA SPP. ROMANIAN

ISOLATES ON DIFFERENT PLANT PATHOGENS

C. P. Cornea1, A. Pop2, S. Matei3, M. Ciuca4, C. Voaides1,2, M.a Matei3, G. Popa1, A. Voicu5, M. Stefanescu5

1

USAMV Bucharest, Faculty of Biotechnology, Romania2BIOTEHNOL Bucharest, Romania3INCDPAPM Bucharest, Romania4National Agricultural Research and Development Institute Fundulea, Fundulea, Jud.Calarasi, Romania5Institute of Biology, Romanian Academy, Bucharest, Romania

Correspondence to: Calina Petruta Cornea and Mugur Stefanescu

E-mail:[email protected]; [email protected]

ABSTRACT

The genus Trichoderma comprises various fungal strains that can act as biological control agents against a large diversity of

plant pathogens. A number of commercial products are available but the diversity of plant pathogens, and their increased

resistance to the current control products (chemical or biological products) determined the search of new strains, potentially

useful for biological control. New strains of Trichoderma spp. were isolated from Romanian soils and their antagonistic

activity against Phytium spp and Rhizoctonia spp. was examined. The in vitro biocontrol activity of Trichoderma spp., as well

as of other antagonistic fungi (Penicillium chrysogenum Gliocladium roseum and Eppicoccum purpurescens) on the plant

pathogens was increased in the presence of FeCl3. The molecular analysis realized by ITS-RFLP and PCR with specific

primers allow the confirmation of previous taxonomic determination of T.harzianum and T.viride. However, an increased

intraspecific molecular polymorphism was observed using several arbitrary primers (RAPD analysis). The interactions

between fungal strains (plant pathogens and antagonistic strains) were also examined, in order to determine the mechanism of

action of the antifungal strains. It was observed that all of the Trichoderma strains were able to produce large amount of

hydrolytic enzymes (chitinase, cellulases and proteases) and to act as mycoparasites for pathogens. The involvement of fungal

lectins in the interactions was also examined. Fungal extracts obtained from the best antagonists were tested for the induction

of plant (soybean) resistance against pathogens. Higher levels of plant enzymes PAL, POX and chitinase were observed, and

correlated with an increased resistance to artificial infection. The results obtained could be used in further experiments to

establish new approaches of plant immunization with microbial products.

Keywords: antifungal activity, molecular tests, Trichoderma

Introduction

Plant diseases caused by soil-borne pathogens like Phytium,

Botrytis, Rhizoctonia, Fusarium and Phytophtora play an

important role in the destruction of natural resources inagriculture. Chemical pesticides have been extensively used

for control fungal plant disease but their employment favored

the selection of fungicides resistant strains as well as negative

effect on non-target organisms and environment (2). In this

respect, the development of alternative methods for plant

pathogens is of great interest not only for scientists but also

for agriculture. Biological control agents are risk free both for

environment and non-target organisms, and could reduce the

use of chemical products. Several commercial biological

products based on antagonistic microorganism are available

now on the market (13) but the interest for selection of new

antagonists is not diminished (4, 12, 16). The filamentous

fungi Trichoderma (Ascomycetes, Hyprocreales) have

attracted the attention because the inhibitory action against

various plants pathogens and the diversity of mechanisms ofaction (14).

The aim of the present study was the screening of some

new fungal Romanian isolates active in vitro against Pythium,

Botrytis and Rhizoctonia and the examination of the

mechanisms of action expressed by the antagonistic strains.

Molecular characterization of the fungal strains, using ITS-

PCR-RFLP and PCR with specific primers, was also

performed.

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Materials and methods

Trichoderma spp. cultures: The fungal antagonists were

isolated using dilution plate techniques on TSM medium (1)

from soil samples provided by ICDPAPM Bucharest,

Romania. The fungal isolates were purified and obtained

single spore cultures and their identification was based on

morphological characters. All cultures were maintained on

potato dextrose agar at 4oC.

Cultures of other fungal strains: In the experiments were

also used strains of Pythium spp., Rhizoctonia solani.,

Botrytis cinerea, Penicillium chrysogenum Gliocladium

roseum and Eppicoccum purpurescens kindly provided by dr

Maria Oprea from Institute of Plant Protection Bucharest.

In vitro antagonism test was performed by dual cultures

technique on Petri dishes containing PDA supplemented or

not, with 100 g/mL FeCl3. Petri plates were inoculated with

mycelia disc of 7-day-old culture of the pathogen and

antagonistic strains at equal distance from the periphery.

Inoculated plates were incubated for 3-7 days at 25oC and the

radial growth of the pathogen was measured. From the zone

of interaction between the antagonist and phytopathogen, the

mycelial mats were gently removed with a needle and

examined under microscope for hyphal interaction (4).

Inhibition was evaluated as presence of inhibition zones prior

to any mycelial contact. The percent RI was calculated

according the indications of Grondona et al. (5). Similar

aspects were examined in different PDA variants: PDA, PDA

supplemented with 2% galactose or 2% raffinose.

Lectin activity in extracts from mycelium was assayed using

agglutination test with rabbit erythrocytes (11).

Hydrolytic enzymes analysis (chitinase, FP-ase and CMC-

ase) was performed in extracts from Trichoderma mycelia

(E3), from Botrytis mycelia (E2) or from mixtures of both

types of fungi (E4), using specific substrates (10).

DNA extraction: Total DNA was extracted by the method

described by Siddique et al. (12) and the DNA samples wereprepared in TE (10mM Tris-Hydrochloric acid and 1mM

EDTA, pH 8.0) and stored at -20oC in small aliquots.

PCR amplification: For amplification of Internal

Transcribed Spacer (ITS) region of rDNA ITS1/ITS4 primer

pair was used (15). For 5S rRNA IGS region, primers

IGS1/IGS2 were used (9). TharzF1/TharzR1 primer pair for

T.harzianum, and TviriF1/TviriR1 recommended for the

group Trichoderma viride/ atroviride/koningii were also used

(7). Primers were obtained from Biosearch Technologies. The

amplification reactions were performed as described

previously (3).

Results and Discussion

Antagonistic interactions between Trichoderma spp.isolates and fungal pathogens in vitro

Soil samples collected from different agricultural fields and

forests were inoculated on Petri plates with potato dextrose

agar (PDA) medium following dilution plate technique. After

7 days incubation period at 25oC, colonies determined to

belong to Trichoderma genus were purified. Seven distinct

strains with inhibitory action against other fungi present in

samples were selected in order to test them against plant

pathogens: strains Trichoderma spp.P8 and Trichoderma spp.

P456 were isolated from two forest soils (Ilfov), Trichoderma

spp.SB6 from soil under maize cultivated in biological

agriculture system (Arges), Trichoderma spp.S37 from an

agricultural soil fertilized with composted sewage sludge

(Caracal) and Trichoderma spp. TV1 and TV2 from garden

soil (Bucharest). The strain Trichoderma spp.UV was

selected after UV treatment of the strain TV1 (data not

shown).

The application of the molecular techniques allowed the

observation of an increased polymorphism among the

Trichoderma strains analyzed (Cornea et al, 2008). The use

of two primer pairs: TharzF1/ TharzR1 primer pair forT.harzianum, and TviriF1/TviriR1 for the group Trichoderma

viride/ atroviride/koningii (7) some interesting results were

obtained. With the primer pair TharzF1/TharzR1, various

products of amplification were obtained. Similar fragments

were observed in P8 and SP9, confirming the previous

results. Different profiles of amplicons were obtained with

these primers in the other strains. The results could be due,

probably, to a reduced sensitivity of these primers for

T.harzianum or, less probably, to the fact that none of the

strains belong to this species. The polymorphism of the

amplification products obtained by the use ofTviriF1/TviriR1 primer pair, recommended for the group

Trichoderma viride/atroviride/koningii, is more reduced,

comparing with that of previous primers: an unique fragment

of about 400 bp was observed in S37, SP9, TV2 and TV1

strains, and one fragment of 500 bp was detected in P8 and

TV.UV strains.

In vitro evaluation of antagonism of the new isolates and

collection fungal strains against Pythium spp. and

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Rhizoctonia solani was performed by dual culture techniques,

on Petri dishes containing PDA or PDA supplemented

with100 g/mL FeCl3. The results obtained showed that the

presence of FCl3 in culture medium increased significantly

the inhibitory action of some of the antagonist against both

pathogens (Table 1).The best results on PDA were obtained with the strains

TV1, TV2, P8 and P456 against R.solani and with strains

TV2, SB6 and S37 against Pythium spp. When FeCl3 was

added to culture medium the best results were obtained

TV.UV (with 89,9% increased action), Trichotecium roseum

(with 74,8% increased action) against R.solani and with

strains TV1 (+41,2%), Trichotecium roseum (+34,8%) andEpicoccum spp. (+55,5%) against Pythium spp.

TABLE 1

In vitro inhibition of plant pathogens by different fungal strains on PDA or PDA+ FeCl3

Rhizoctonia solani Pythium sp.Antagonist

PDA PDA+FeCl3 PDA PDA+FeCl3

Trichoderma spp. TV1 60,8% 78,3% 42,5% 60%

Trichoderma spp. TV2 71,7% 78,3% 55% 56,5%

Trichoderma spp. TV.UV 31,8% 60,4% NT NT

Trichoderma spp.P8 65,3% 77,9% 46,2% 59,7%

Trichoderma spp.P456 70,5% 76,6% 48% 54,2%

Trichoderma spp.SB6 58,6% 64,4% 52% 56,8%Trichoderma spp.S37 44,7% 58,2% 54,4% 60,1%

Trichotecium roseum 28,6% 50% 0% 34,8%

Gliocladium roseum 0% 33,3% 33,3% 34,8%

Eppicoccum purpurescens 0% 0% 0% 55,5%

These results suggest that the mechanism of action of

these strains didnt involve the competition for iron.

Moreover, the presence of inhibition zones prior to any

mycelia contact indicates that the inhibition may be due to

the production of diffusible components by antagonistic

strains. Microscopic observation of the interactive zoneshowed vacuolization of the R.solani, Botrytis cinerea and

Pythium spp. hyphae, followed by cell disintegration. These

results suggest that Trichoderma spp. could inhibit the

development of pathogens not only by competition or direct

interaction (mycoparasitism) (observed mainly after a 5-7

days of incubation) but also by inhibitory compounds

diffusible in the culture medium that act in the first days of

incubation. No significant differences related to the type of

hyphal modification were observed on PDA or PDA

supplemented with FeCl3.

The possible involvement of lectin production in fungalinteractions was also examined. The presence of lectins in

extracts fron Trichoderma, Botrytis, Penicillium and

Rhizoctonia mycelium was determined. Only two fungi seem

to produce such compound: one strain of B.cinerea

designated P2 and a strain ofR.solani. Their agglutination

ability was inhibited by various carbohydrates, the best

inhibition being observed when galactose or raffinose was

used. The significance of the lectins in fungal pathogens is

still unclear, being purposed the idea of a possible role in

mycoparasitism or as a storage protein (6). In our

experiments was examined how the presence in culture media

of the specific carbohydrates (that inhibited in vitro the

agglutinating lectins activity) influenced the interactions with

antagonistic strains (Trichoderma spp.TV1, Trichodermaspp.P8 and Trichoderma spp.S37). Comparing with the

results on PDA, the inhibitory activity ofTrichoderma strains

was slightly increased when the carbohydrates were added. It

was observed that the inhibitory action was clearly detected

before any contact between fungi, he inhibition area being

larger when pathogen lectins were inhibited. The microscopic

examination of the interaction area shown increased aspects

of mycoparasitism on PDA supplemented with

carbohydrates: coiling, attachment to fungal cell wall and

overgrowth of Trichoderma on pathogen. These results

suggest that the presence of lectins in fungal pathogens isinvolved in slowing down the inhibitory activity of

antagonist, probably by reducing their enzymes activity

through interactions between soluble lectins and

oligosaccharide chains of glycoenzymes. This hypothesis is

supported by the previous demonstration of the glycoproteic

nature of some the -glucanases produced by Trichoderma

strains and their possible interaction with plant lectins (8).

Moreover, the level of three hydrolytic enzymes (chitinase,


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FP-ase and CMC-ase) in extracts from fungal mycelia

(extracts designated as E2, E3 and E4) was slightly reduced

in E4 variant that contains a mixture of pathogen and

antagonistic mycelia, comparing to E2 and E3 extracts, from

Botrytis mycelia or Trichoderma mycelia, respectively

(Fig.1).The increased inhibitory activity (mycoparasitic and lytic

action) of antagonistic Trichoderma spp.TV1 and

Trichoderma spp.S37againstRhizoctonia andBotrytis strains

when they are cultivated on PDA supplemented with

carbohydrates could be explained, both by the stimulation of

cell-wall degrading enzymes (in the absence of pathogen

lectins) and by the activation of some genes involved in

coiling response (by other signals than lectins), probably ofG-protein -subunit (14).

Fig.1. The level of hydrolytic enzymes activities from three different samples: E2 extracts from Botrytis mycelia; E3 extracts from Trichoderma mycelia;

E4 extract from a mixture of pathogen and antagonistic strains.

Conclusions1. Molecular characterization of the newly isolated fungal

strains emphasized that the primer pair TharzF1/TharzR1

resulted in the obtaining of various products of amplification,

while the use of TviriF1/TviriR1 primer pair led to a reduced

polymorphism of amplification products.

2. The inhibitory action ofTrichoderma spp may be due not

only to competition or direct interaction but also to the

production of diffusible compounds that induce the

appearance of inhibition zones prior to any mycelia contact.

3. Lectin presence in fungal pathogens is involved in

diminishing the inhibitory activity of the antagonist.

Acknowledgment

We thank dr.Maria Oprea for her help in the identification of

some of the fungal strains. The research was partially

supported by PNCDI II Research Program, grant

no.31078/2007(acronym PEFIMVAF) and by CEEX

Research Program, project no.52/2006 (acronym

BIOCOMB).

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