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Factori i Procese Pedogenetice din Zona Temperat 10 S. nou (2011) 29-37

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CHARACTERIZATION OF SOIL TRICHODERMA

ISOLATES FOR POTENTIAL BIOCONTROL OF PLANT PATHOGENS

S. Matei1, Gabi-Mirela Matei

1, Petruta Cornea

2, Gabriela Popa

2

1National RD Institute for Soil Science, Agrochemistry and Environmental Protection2Center of Applied Biochemistry and Biotechnology

Caracterizarea tulpinilor de Trichoderma izolate din sol pentru potenialul de

biocontrol al agenilor patogeni ai plantelor

Abstract

Various fungal strains belonging to genus Trichoderma act as biological control

agents for soil born plant pathogens. Two new strains of Trichoderma harzianum (T.h.) and

Trichoderma viride (T.v.) were isolated from forest soils in Ilfov county and their morphologicalaspects, enzymatic and antagonistic activity were examined. Current chemical fungicides had

constantly, in time, less influence on pathogens due to their diversity, adaptability andincreasing resistance.

The paper present the morphological characterization of two strains of Trichodermaisolated from forest soils. Growth rate was higher in strain T.v.SP456 (0,675mm/h) than in

strain T.h.P8 (0,505mm/h) when fungi were grown on Czapek culture medium.Morphological description is completed with photographs of colonies in Petri plates

and microscopical aspects of fungal structures belonging to Trichoderma strains SP456 and P8.Comparative aspects concerning the level of main enzymes released by T.h. isolate P8

and T.v.SP456 in liquid culture media showed differences as a function of genetic structure of

each fungal isolate. The optimum culture media for inducing peroxidase, polyphenol-oxidase, -1,3-glucanase activity in T.v.SP456 isolate was Czapek and PDA for phenil-alanin-ammonium-oxidase and chitinase. T.v.SP456 was more efficient than T.h.P8 concerning enzymes activity.

The interaction between Trichoderma fungal strains SP456 and P8 and strawberry

plant pathogen strains, three belonging to Botrytis cinerea (S1, P1, P2) and one to Phytophtoraspp. were examined, also. Both Trichoderma strains act as mycoparasites for plant pathogens.The inhibition percent of radial growth was higher for T.v.SP456 when compared with T.h.P8for almost all pathogenic isolates.

Key words: Trichiderma, biocontrol, plant pathogens

INTRODUCTION

Trichoderma is a genus of fungi present in all soils as the most prevalent

cultivable fungi and can be characterized as opportunistic avirulent plant symbionts.

Several isolates of Trichoderma have been developed as biocontrol agents against

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fungal diseases of plants, most are from the species T. harzianum, T. viride and T.

hamatum. Trichoderma isolates can protect crops such as cauliflower, soybean, tomato,

potato, sunflower and are compatible with organic manure and bio-fertilizers or takes

part in the decomposition of plant residues in the soil.

Generally, in their natural habitat, the biocontrol agents grow on the root

surface and can also be effective against foliar diseases by various mechanisms which

include antibiosis, parasitism, inducing host-plant resistance, competition, tolerance to

stress and solubilization of inorganic nutrients. Trichoderma, being a saprophyteadapted to thrive in diverse situations, produces a wide array of enzymes.

The agricultural importance of the genus is that same isolates/species possess

good antagonistic abilities against plant pathogenic fungi, e.g. Botrytis, Fusarium,

Pythium species, abilities based on mechanisms of competition for space and nutrients

or mycoparasitism.

MATERIALS AND METHODS

Isolation of Trichoderma viride SP456 and Trichoderma harzianum P8 was

effected from a forest soil in Ilfov district.

Soil decimal dilutions were plated in Petri plates (10cm diameter) on PDA

(potato-dextrose-agar) and Czapek culture media. The plates were incubated in dark at

25oC for 7 days.

Consequently, pure cultures of Trichoderma have been isolated from

developed colonies and passed on agar slants in tests tubes.

Description of fungal isolates

Morphological aspects concerning colonies shape, diameter, density, pigment

color, conidiophores branching, phialides, conidia, stromata and chlamydospores were

analyzed under dissecting and optical microscope.

Fungal isolates were identified according to Domsch & Gams (1970) and

Samson &Van Reenen-Hoekstra (1988).

Interaction between antagonistic Trichoderma isolates and pathogenic fungifrom genus Botrytis and Phytophtora was tested by dual culture technique (Phuoc,

1988).

Microphotographs were carried out to illustrate aspects of hiperparasitism

revealed by examination of cultures at optic microscope.

Enzymatic characterization

The characterization of enzymatic equipment of the two isolates of

Trichoderma was done by testing the activity of enzymes: peroxidases (POX), phenil-

alanin-ammonium liase (PAL) and polyphenol-oxidase (PPO) using

spectrophotometric methods for measuring the rate of conversion of specific reaction

mixtures by the development of color. -1.3-glucanase was determined by

spectrometric absorbtion at 500nm wave length in reaction mixture of enzymatic

extract and laminarin after incubation at 40o

C for 10 minutes.

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RESULTS AND DISCUSSIONS

Trichoderma viride SP 456 presents colonies with hyaline aspect initially, then

yellowish green more dense towards the margins and rarefied to the center (Fig.1) no

distinct odor and reverse pigmentation from white to reddish brown. Growth rate was

0.675 mm / h, the colony covering the Petri dish with Czapek medium in about six

days. Microscopic examination highlights the branching pattern of the conidiophoresthat form pustules.

From the thick central axis of 5.25 to 6.79 m, lateral branches come off at an

angle of 90C towards the growing branch.

Phialides appear singly or in groups of 2-4, swollen in the middle, with slightly

elongated neck, right, with dimensions of 7.04 to 8.75 m in length and thickness of

5.16 to 5.28 m in the swollen area.

Green thick-walled conidia have dimensions of 3.53 to 3.55 x 3.78 to 5.25 m

and subglobose to globose shape.

Fig. 1 Trichoderma viride SP456

(Czapek,14 days)

Solitary stromata is brown-reddish colored and intercalary chlamydospores,

hyaline in the center, present thick brown walls and have diameters of 9.78 to 11.08

m.

Trichoderma harzianum P8 colonies growing on PDA medium are lanose and

show the appearance of concentric circles of color from white to bright green, through

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yellow-green areas (Fig.2). Fungus growth is lax lanose on Czapek with uniform

colors in shades of yellow-green, reverse pigmented in yellow and odorless.

Fig. 2 Trichoderma harzianum P8

(PDA, 10 days)

Growth rate was 0.505 m / h on Czapek medium and 0.885 m / h on PDA

medium, the colony reached the edge of Petri plate after 7 and 4 days, respectivelly.

Microscopic examination revealed rough conidiophores, long, with thicknesses

from 3.06 to 4.55 m, branched with flask-shaped phialides that form groups of 2-4,

more frequently 3, usually on lateral branches that form straight angles of 90 withrespect to central axis.

Phialides have lengths of 5.75 to 8.30 m and 2.56 m thick at base, 3.53 m

in the swollen area and 1.26 m to elongated neck. Conidia green subglobose to ovoid,

smooth, shows the dimensions of 1.28 to 3.78 x 2.28 to 4.12 m. Intercalary hyaline

chlamydospores, subglobose to globose have diameters of 5.9 to 6.12 m.

In dual cultures (Fig. 3, 4, 5 and 6), the two fungal isolates grew with relatively

similar rates.

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Fig. 3 High antagonistic activity ofTrichoderma viride SP456in dual culture withBotrytis cinerea P2 (PDA, 14 days)

Fig. 4 Growth inhibition ofPhytophtora sp. by

Trichoderma viride SP456 (PDA, 6 days)

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Fig. 5 Interaction ofTrichoderma harzianum P8 with

Botrytis cinerea B1 in dual culture (PDA, 14 days)

Fig. 6 Growth inhibition ofPhytophtora sp. byTrichodermaharzianum P8 (PDA, 7 days)

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Pathogen development was restricted by both Trichoderma, the percent

inhibition radial growth % IRG ranging between 80-97%for Trichoderma viride and

46-94% forTrichoderma harzianum (Table 1)

The antagonism was illustrated by biochemical products released by both

species and by hiperparasitism.

A high antagonistic effect of the two Trichoderma species against other

Botrytis, Phytophtora and Rhizoctonia pathogenic isolates was also reported in dualcultures on PDA supplemented or not with FeCl3 (Cornea et al., 2009).

Table 1

Percent inhibition of radial growth of pathogensBotrytis

andPhytophtora by T.v.SP456 and T.hz.P8

Fungal isolates B. c S1 B c. P1 B c. P2 Phy

T. v. SP456 83% 90% 80% 97%

T. hz. P8 85% 56% 46% 94%

The level of the five enzymes released by Trichoderma isolates T. viride

SP456 and T. harzianum P8 (Table 2) was measured at intervals of seven and fourteen

days.

The data analysis showed differences in enzymatic activity at 7 and 14 days for

Trichoderma isolates, as a function of genetic structure of each fungal isolate. In time,

enzymatic activity of both isolates increase for majority of enzymes analysed excepting

for POX activity. The level of POX activity decrease in time relatively equal at bothTrichoderma isolates. For all period, T.viride SP456 had an increased PAL and -1,3

glucanase (GLU) activity as compared with T.harzianum P8 and in the same manner,

T.hz.P8 determined a superior CHI and PPO enzymatic activity. The highest enzymatic

activity evolution in time was registered for PPO at T.hz.P8, that increased from 0,400

U/ml to 3,360 U/ml and for GLU at T.v.SP456 where 1,3 glucanase activity increased

from 11,167 to 17,042 U/ml.

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Table 2

The level of enzymatic activity ofT. viride SP456 and

T. harzianum P8 at 7 and 14 days

Fungal isolates POX U/ml PAL U/ml PPO U/ml GLU U/ml CHI U/ml

T. v. SP456

7 days

0,311 0,836 1,350 11,167 0,011

T. v. SP456

14 days

0,098 1,069 2,110 17,042 0,020

T. hz. P8

7 days

0,305 0,648 0,400 10,447 0,017

T. hz. P8

14 days

0,085 0,993 3,360 13,262 0,029

CONCLUSIONS

Two new fungal isolates of Trichoderma were identified and characterized

from morphological, antagonistic and enzymatic point of view.

Data analysis revealed that enzymatic activity of fungal isolates was influenced

in time by genetic structure.

The level of POX activity decreased relatively similar in both isolates.

The highest activity was registered after fourteen days for GLU activity in thecase ofT. viride SP456 and for PPO activity in the case ofT. harzianum P8.

The results showed the capacity of Trichoderma harzianum P8 to inhibit the

radial growth ofBotrytis cinerea F7 with 51%.

Biocontrol mechanism is firstly biochemical by releasing non-volatile

diffusible metabolites at contact zone.

Trichoderma harzianum P8 acts also as a hiperparasite by coiling and

penetrating pathogens hyphal structures with the tips and using their content as a

nutrient source for developing its branched pustules.

Further, Trichoderma harzianum P8 could be used in controlling Botrytis

cinerea effectively in strawberry.

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REFERENCES

1. CORNEA C.P., POP A., MATEI S., CIUCA M., VOAIDES C., MATE M.G.,

POPA G., VOICU A., STEFANESCU M. 2009 Antifungal action of new

Trichoderma spp.romanian isolates on different plant pathogens,

Bioehnol&Biotehnol.EQ, 23,/SE/ON-LINE, 766:7702. DOMSCH, K.H., GAMS, W., 1970 Fungi in agricultural soils. T&A Constable Ltd.

Edinburgh, London, 290 p.

3. PHUOC, N.T. 1988. Biological control of tomato root and stem rot caused by

Sclerotinium rolfsii Sacc. ARC Training, 1988 Report Tomato, P. 1 11.

4. SAMSON, A.R. and van REENEN-HOEKSTRA E. 1988 Introduction to food-

borne fungi, Ed. CBS Netherlands, p.1 209.

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