Smolinska 2007

2007 vol. 67, 81-93 DOI: 10.2478/v10032-007-0033-5 ________________________________________________________________________________________

THE EFFECTIVITY OF TRICHODERMA STRAINS IN THE PROTECTION OF CUCUMBER AND LETTUCE AGAINST RHIZOCTONIA SOLANI Urszula SMOLIŃSKA, Beata KOWALSKA, Michał OSKIERA Research Institute of Vegetable Crops Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland Received: July 30, 2007; Accepted: , 2007

Summary Antagonistic properties of four isolates of Trichoderma against fungal pathogen Rhizoctonia solani were investigated. The addition of T. harzianum strain PBG to the potground protected cucumber plants against root rot caused

by R. solani and increased the weight of cucumber plants growing in potground with pathogen by 19-26%, compared to infested control. All Trichoderma strains (T. harzianum strain PBG; T. longibrachiatum szczep T2; T. harzianum strain T4 and Trichoderma sp. strain T6) indicated positive effect on lettuce growth in the greenhouse experiment. They increased the number of germinated seeds and the average mass of seedlings. The highest growth stimulation of the lettuce was observed using Trichoderma strain T6 and T. harzianum strain PBG, respectively 49% and 33%, when compared to the control treatment.

Moreover, these isolates most efficiently protected lettuce seedlings against R. solani. The application of the combinations of four Trichoderma together did not increase the protective effect against R. solani. key words: biological control, antagonistic fungi, cucumber, lettuce, Trichoderma spp.

INTRODUCTION In order to protect the environment, effective alternatives to chemical control are investigated and the use of antagonistic microorganisms seems to be one of promising approaches. The interesting filamentous fungi Trichoderma (Deuteromycotina, Hyphomycetes) as a means of plant protection has not decreased for the last several decades, because of its biological properties and biocontrol applications (Grondona et al. 1997, Harman 2000, Wojtkowiak-Gębarowska 2006). In literature, many papers which deal with protection of various plants

by Trichoderma species against different pathogenic fungi can be found (Chet & Inbar 1994, Inbar et al. 1994, Bailey et al. 1998, Howell 2003). Among others, Rhizoctonia solani, a widely distributed soil–borne pathogen causing preCorresponding author: e-mail: usmolin@inwarz.skierniewice.pl © Copyright by RIVC

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and postemergence damping-off plants (Howell 1987, Howell & Stipanovic 1995, Howell et al. 2000, Shalini et al. 2006). Trichoderma strains are classified as imperfect fungi since they do not have a known sexual stage, however some Trichoderma species are morphologically similar to the anamorph Hypocrea (Chaverr 2003). Trichoderma fungi are worldwide and easily isolated from the soil. The interest on Trichoderma strains as biological control agents is due to their high reproductive capacity, efficiency in the utilization of nutrients, and strong aggressiveness against phytopathogenic fungi (Bailey & Lumsden 1998).

These fungi have an essential property of being able to colonize plant roots under field conditions. They grow on the surface of roots, where then may protect plants against pathogens and enhance growth. Knowledge concerning the behavior of these fungi as antagonists is essential for their effective use, because they can act against pathogen in several ways (Fravel 1988, Howell 1998, Altomare et al. 1999, Harman et al. 2004, Vinale et al. 2006, Degenkolb et al. 2006).

Trichoderma spp. may exert direct biocontrol by parasitizing many fungi (Chet et al. 1998). Mycoparasitism is a commonly observed phenomenon and this mode of action and its involvement in biological disease control has been reviewed extensively in the last years (Carsolio et al. 1999). It is supposed that biocontrol activity of Trichoderma sp. toward R. solani is based mainly on mycoparasitism, a very complex process that involves recognition, attack, penetration and killing of the host. During this process hyphae of Trichoderma have been found to grow towards hyphae of pathogen, presumably due to chemical signals originating from the host (pathogen). After attachment and coiling of the antagonists around the phytopathogenic fungus, Trichoderma produces enzymes which cause dissolution of pathogen hyphae and induce leakage of compounds (Benitez et al. 2004). The mycoparasitic activity of this organism is attributed to the production of cell wall degrading enzymes as 1,3-glucanase and chitynase (El-Katatny et al. 2000). During previous years the modified strains of Trichoderma carrying multiple copies of genes coding endochitynase were produced (Carsolio et al. 1999). Although evidence of fungicidal action of hydrolytic enzymes or selected antibiotics of Trichoderma spp. under in vitro and in vivo conditions are available, the concentrations required to protect plants against pathogens are far beyond those occurring in vivo. It is supposed, that a combination of hydrolytic enzymes and antibiotics results in a higher level of antagonism than obtained by either mechanism alone. A synergism between Trichoderma enzymes and antibiotics could occur in vivo (Schrimbock et al. 1994, Benitez et al. 2004). Trichoderma spp. can attack or inhibit the growth of plant pathogen directly, but the research of the previous years gave evidence that some Trichoderma strains could induce the production of defense related compounds in plants and enhance plant resistance. It is supposed that direct effect on plant pathogens is only one mechanism of control and perhaps less important than induced localized or systemic resistance (Yedidia et al. 2003, Harman et al. 2004, Khan et al. 2004, Viterbo et al. 2005, Hoitink et al. 2006).

Despite many profitable properties, biological control agents based on Trichoderma strains are living organisms. Their activities depend heavily on the

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different environmental conditions (Margolles-Clark et al. 1997, Bae & Knudsen 2004, Kaur et al. 2005, Li et al. 2005). Effective protection of plant against pathogen (biocontrol) exerted by Trichoderma strains is often unpredictable.

Although many researchers around the world are interested in these fungi and numerous commercial agents based on these microorganisms were produced, the use of these biological agents in agriculture practice is still very scare.

The objectives of this research were:1/ to determine whether the addition of Trichoderma isolates to the potground or application on the seeds of cucumber or lettuce can reduce severity of the root rot caused by Rhizoctonia solani; 2/ to study the effect of different isolates of Trichoderma on the growth of cucumber and lettuce; 3/ to obtain information about variability in protective property of different isolates of Trichoderma against R. solani in controlled conditions; 4/ to study the effect of combination four Trichoderma strains which show antagonistic activity in the control of R. solani.

MATERIALS AND METHODS Trichoderma fungi were isolated from soil samples taken from the fields near Skierniewice and maintained in the collection of Microbiology Laboratory of Research Institute of Vegetable Crops. The following strains were used: Trichoderma harzianum strain PBG; T. longibrachiatum strain T2; T. harzianum strain T4 and Trichoderma sp. strain T6. Their antagonistic properties towards pathogenic fungi were evaluated on potato dextrose agar (PDA-Merck) in laboratory tests.

The pathogenic fungus Rhizoctonia solani was isolated from a diseased plant and kept in the collection of Microbiology Lab. All fungi used in experiments were grown on PDA plates for 7-10 days or, in the case of R. solani in some of experiments, the fungus was grown in the liquid medium potato dextrose broth (PDB; 200g potato infusion, 20g glucose, 10g polypeptone, 1L distilled water, pH 5.6) (Szczech & Shoda 2005).

First part of the experiments was conducted in the greenhouse conditions.

The fungus R. solani was grown in liquid medium for 7 days. Then, the culture was filtered through filter paper to remove mycelial mats and mixed in a blender. The potground (Klasmann H) was infested with pathogen R. solani at the dose of 5g of wet mycellium mats per 1 L of substrate. Immediately the suspension of T. harzianum strain PBG was added. T. harzianum strain PBG was grown on PDA medium for 7-10 days, afterwards the mycelium with the spores were scraped from Petri plate and mixed with water (20 ml/plate) in a blender. The suspension of the fungus was added to the potground at a dose of 1 Petri plate/1 L of potground. The following treatments were prepared: 1/ control (without fungi); 2/ R. solani 3/ R. solani + Trichoderma strain PBG; 4/ T. harzianum strain PBG. After one week of incubation at the room temperature, the potground were placed to the plastic pots (500ml) and the seeds of cucumber cv. Iwa were placed. For each treatment 5 pots with 5 seeds of cucumber were prepared. The experiment was repeated twice.

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The secondary trial was set up with lettuce cv. Syrena in 54 multipots, vol.100 ml each. 75 seeds of lettuce were used per one treatment (5 seeds were placed to one pot, and 15 pots were prepared for one treatment). Immediately after placement the seeds to the pots, 50 μl of Trichoderma suspension, prepared as described above, were dropped on each seed of lettuce. The suspension contained 1.0-0.6 x 108 cfu (colony forming units) of Trichoderma spp. propagules in one milliliter. Then, the same volume of R. solani suspension was placed on each seed. T. harzianum strain PBG and another Trichoderma isolates (T2, T4, T6) were used, separately or mixed in the proportion 1:1:1:1. The seeds inoculated only with water served as a control. After 20 days the upper part of plants were cut and weighed. The experiment was conducted in a greenhouse and repeated twice.

In a second set of experiments only the lettuce plants cv. Syrena were used. The experiments were conducted in the controlled conditions in a growth chamber and the plastic Phytotoxkit - growth -plates (Tigret, Poland) were used.

All strains of Trichoderma and R. solani were grown on PDA for 7 days. Into each Petri plate 20 ml of sterile distilled water was poured and the mycelium with the spores were scraped from Petri plate and mixed with water in a blender for 30 sec. The growth plates were filled with potground at 60% humidity. In each Phytotoxkit growth plate, about 0.5 cm from upper edge, 10 seeds of lettuce were placed. Afterwards, 50 μl of Trichoderma spore and mycelium suspension (contained 1.0-0.6 x 108 cfu/mL) were poured on each seed, and immediately, 50 μl of R. solani suspension. Trichoderma izolates were used separately or mixed in proportion 1:1:1:1. The growth plates without fungi, only with water, served as a control. Bioassays were conducted in a growth chamber at 25ºC (14 hour-light) and 20ºC (10 hour-night); humidity - 90%. Three growth plates were prepared for each treatment, and the experiment was repeated three times. After 7-10 days the number of seedlings was counted and the root growth and the fresh weight of seedlings were evaluated.

To enhance biocontrol efficacy of T. harzianum strain PBG, in the last set of experiments the carboxymethyl cellulose sodium salt (CMC ) (Sigma) was used. This compound is used to stabilize emulsions, and enhance the adhesive properties of the spore suspension. The experiments were prepared as described above, except that the fungus was kept 30 minutes in the 1% CMC, and than the lettuce seeds were inoculated with this suspension. The experiments were conducted in the growth chamber and repeated twice.

RESULTS The pathogen R. solani decreased the growth of cucumber plants by 46%, 42% and 68% (Fig. 1) when compared to the control treatment in three following experiments, respectively. The application of antagonistic fungus T. harzianum strain PBG to the potground protected the cucumber plants against root rot caused by R. solani. In two out of three experiments T. harzianum strain PBG increased the mass of cucumber plants, growing in potground with pathogen, by

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19% and 26% compared to infested control. However, when the disease was severe, the protection by Trichoderma was unsuccessful (Fig. 1 D3). In two experiments the 7-10% stimulation of growth of the cucumber plants in the presence of T. harzianum strain PBG was observed. The inoculation of cucumber seeds with T. harzianum strain PBG had no significant effect on the germination of seeds in the treatment without pathogen. However, in both cases, in the potground infested with R. solani, the number of germinated seeds was higher when the T. harzianum strain PBG was added (results not presented).

120 a a

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Fig. 1. The effect of T. harzianum strain PBG on the growth of cucumber in the potground infested with R. solani (in percentage of weight of plant tops compared with the control) Results from three repetitions of the experiment are presented.

Number with the same letter are not significantly different according to Newman-Keuls test (P<0.05)

In the experiments with lettuce cv. Syrena four isolates of Trichoderma spp. were used (Fig. 2).

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Fig. 2. The effect of Trichoderma strains on the germination (A) and growth of lettuce (B) in the potground infested with R. solani - greenhouse experimernts (D1experiment I; D2â&#x20AC;&#x201C;experiment II).

The fresh weight of lettuce plants was presented as a percentage compare to control treatment. Numbers with the same letter are not significantly different according to Newman-Keuls test (<0.05)

All of them showed antagonistic properties toward R. solani in the laboratory tests on Petri plates with potato dextrose agar (results not presented). The experiments conducted in the greenhouse conditions showed that often, but not

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always, Trichoderma strains (PBG, T2, T4, T6) indicated positive effect on lettuce growth (Fig. 2B). The highest growth stimulation of the lettuce was observed with Trichoderma sp. strain T6 and T. harzianum strain PBG, respectively 49% (D2) and 33% (D1), compare to the control treatment. Moreover, Trichoderma sp. strain T6 and PBG, in almost all combinations, protected lettuce seedlings against R. solani. The best results were obtained with T. harzianum strain PBG. The weight of the fresh plant was 70% higher when compared to the treatment with R. solani without this antagonistic strain (Fig. 2B, Photo 1B). However, in the combination with T. longibrachiatum strain T2, the seed germination of lettuce growing in the potground with patogen was lower when compared to infested control (Fig. 1A). The application of mixture of different Trichoderma isolates did not increase significantly the growth of seedlings or protective properties of these fungi to the effect of single strain.

In spite of general positive effect of Trichoderma isolates on the growth of lettuce plants, the differences between experiments were noticeable large. For this reason following set of experiments was conducted in controlled conditions in growth chamber using Phytotoxkit growth plates (Table 1). Similarly as in the greenhouse experiments, the best results were obtained with T. harzianum strain PBG (Photo 1A). The fungus showed positive influence on the growth of lettuce seedlings in two out of three experiments. The antagonistic fungi protected lettuce against R. solani and the isolates T. harzianum strain PBG and Trichoderma sp. strain T6 were the most effective. In spite of stable temperature, air humidity and light intensity, the effectiveness was variable in each experiment. The inoculation of lettuce seeds with T. harzianum strain PBG and carboxymethyl cellulose increased the protection of these plants against R. solani about 34%, when compared to infested control (results not presented).

Table 1. The effect of Trichoderma strains on the growth of lettuce plants in the potground infested with R. solani (growth chamber experiment ) Fresh plant biomass Exp. I Exp. II Exp. III Treatment g/growth % g/growth % g/growth % plate of control plate of control plate of control Control 0.55 ab (100) 0.49 a (100) 0.31 a (100) R. solani 0.27 def (49) 0.17 b (35) 0.15 b (48) Trich. PBG 0.63 a (115) 0.42 a (86) 0.37 a (119) Trich. PBG+R. sol. 0.41 bcd (75) 0.23 b (47) 0.29 a (94) T2 0.55 ab (100) 0.47 a (96) 0.37 a (119) T2+R. sol.

0.14 f (26) 0.18 b (37) 0.25 ab (81) T4 0.55 ab (100) 0.54 a (110) 0.32 a (103) T4+R. sol.

0.23 ef (42) 0.11 b (22) 0.23 ab (74) T6 0.59 ab (107) 0.41 a (84) 0.31 a (100) T6+R. sol.

0.46 abc (84) 0.17 b (35) 0.29 a (93) Tmix 0.55 ab (100) 0.44 a (90) 0.33 a (106) Tmix+R. sol.

0.34 cde (62) 0.08 b (16) 0.24 ab (77) Numbers with the same letter in columns are not significantly different according to Newman-Keuls test (P<0.05)

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DISCUSSION The results obtained in this work showed positive effect of Trichoderma strains: T. harzianum strain PBG; T. longibrachiatum strain T2; T. harzianum strain T4 and Trichoderma sp. strain T6 on the growth of cucumber and lettuce plants. T. harzianum strain PBG increased the weight of cucumber plants by 710% and lettuce by 33% (Fig. 1,2; Photo 1). Positive influence on plant growth and development of some Trichoderma strains has been known for many years, however the precise mechanism is unknown. It is supposed that this beneficial effect may be the results of many factors. Among others, these fungi may improve mineral uptake efficiency, increase resistance to abiotic stresses, exhibit adverse effect towards deleterious microorganisms or induce systemic resistance in plants ( Harman et al. 2004, Yedidia et al. 2004).

In the experiments conducted in a greenhouse, T. harzianum strain PBG decreased incidence of damping-off of cucumber caused by R. solani and increased growth of these plants in two out of three experiments. The stimulation of cucumber growth after application of T. harzianum T-203 was observed also

by Yedidia et al. (2004). In our study, the level of plant protection was influenced by the severity of root rot caused by R. solani. Low antagonistic effectiveness of T. harzianum strain PBG has been observed in the experiment in which disease development in the infested control was high. Similar results were obtained by Coventry et al. (2006) with T. viride S17A. In his experiments, the fungus protected onion against Sclerotium sclerotiorum only in areas with lower disease pressure.

R. solani is a very fast growing fungus. In appropriate conditions it can overgrow soil and seeds during several hours. It is supposed that mycoparasitism is the main mechanism of biocontrol by Trichoderma spp. against R. solani (Chet et al. 1998). This phenomenon occurred regardless of the supply of external nutrients to the pathogen or mycoparasite (Howell 2003). Although under certain circumstances Trichoderma strains might act as a competitor for nutrients with R. solani, it is possible that this mechanism of the antagonistic action occurs only in specific, nutrients poor conditions.

In the experiments with lettuce four isolates of Trichoderma were used: T. harzianum strain PBG and T4, T. langibrachiatum strain T2 and Trichoderma sp. strain T6. In previous study, all of them showed antagonistic properties towards soil-borne fungal pathogens in in vitro laboratory tests. The results obtained in this work showed that the best antagonistic properties were showed by T. harzianum strain PBG and Trichoderma sp. strain T6. These fungi protected lettuce against R. solani almost in all experiments, although their effectiveness was variable. T. langibrachiatum strain T2 and T. harzianum strain T4 also protected plants against R. solani in the greenhouse experiments, but their effect was much weaker when compared to T. harzianum strain PBG and Trichoderma sp. strain T6. It has been demonstrated that biocontrol efficacy was en-

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hanced after the addition of carboxymethyl cellulose (CMC) to the suspension of T. harzianum strain PBG propagules. CMC is used in food science as a viscosity modifier or thickener and also used to stabilize emulsion.

The application of the mixture of four Trichoderma spp. isolates together did not increase the protective effect against R. solani. The low effectiveness of the mixture of Trichoderma spp. in reducing root rot caused by R. solani suggests that these fungi did not act synergistically, and ineffective protection of seeds or seedlings was the effect of decreasing of amount T. harzianum strain PBG and Trichoderma sp. strain T6 in overall volume of mixtures. Adams (1990) supposed that to obtain disease control with Trichoderma species one needs a soil population of at least 105 propagules/g of soil, because soil microorganisms have an adverse affect on potential saprophytic abilities of Trichoderma spp. In a naturally suppressive soil to R. solani in Columbia, the population of Trichoderma fungi was 8x105 cfu/g soil (Chet 1990). As its supposed, biocontrol activity of Trichoderma sp. against R. solani is based mainly on mycoparasitism. Mycoparasitism occurs under appropriate ecological conditions. Interaction of two fungi appeared to be a unique result of the properties of these fungi in a particular environment at a particular stage of growth and behavior in interaction with other organisms. It is known that activity of fungi strongly depends on pH, temperature and humidity of soil (Knudsen & Bin 1990), and can be increased by relatively specific substances like for example, chitin (Howell 1987).

In all experiments conducted in this work the efficiency of cucumber or lettuce protection by Trichoderma spp. against rhizoctonia root rot was variable.

To taking into consideration the variation in temperature and humidity it is possible to explain the results obtained from experiments conducted in a greenhouse. However, it is much more difficult to explain discrepancies in diseases incidence obtained in a growth chamber, where the conditions of lettuce growth were much more stable.

Several biotic and abiotic factors affect the extent and patterns of growth of Trichoderma spp. and R. solani in the vicinity of plant roots and it makes the biocontrol in the greenhouse and field conditions unpredictable. Also, different mechanisms, other than mycoparasitism and competition may play the role in the interaction between those organisms. Researchers suggested that isolation of active mycoparsites and their genetic manipulation would improve the efficiency of the plant protection (Lorito et al. 1998, Brunner et al. 2005). However, it is possible that even the use of genetically modified strains which can produce high amount of hydrolytic enzymes, would not change stability of plant protection, because the biotic and abiotic conditions would never equally stimulate the growth and activity of antagonistic and pathogenic fungi. Presumably, it would be most promising to use antagonistic organisms along with pesticides (Howell 2007). The commercial fungicides, even applied in lower than recommended dose, can decrease the level of pathogens to the level acceptable to control by an antagonistic organism. It is possible, because fungi from Trichoderma genera showed low sensitivity towards many fungicides and herbicides (Roberti et al. 2006). This feature makes it possible to use those organisms in

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integrated agriculture along with chemical pesticides. Furthermore, the use of antagonistic microorganisms would lead, with time, to a reduction of inoculum density of the pathogen and, after application to several consecutive diseased crops, the soil could become more suppressive to one or more pathogens.

Acknowledgements The authors thank Dr Magdalena Szczech for identification of Trichoderma spp. isolates.

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U. SMOLIŃSKA et al. – THE EFFECTIVITY OF TRICHODERMA STRAINS ...

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[DOI: 10.1128/AEM.71.10.6241-6246.2005] Wojtkowiak-Gębarowska E. 2006. Mechanizmy zwalczania fitopatogenów glebowych przez grzyby z rodzaju Trichoderma. Post. Mikrobiol. 45, 4, 261-273. [in Polish] SKUTECZNOŚĆ DZIAŁANIA SZCZEPÓW TRICHODERMA W OCHRONIE OGÓRKA I SAŁATY PRZED RHIZOCTONIA SOLANI Streszczenie Celem badań było określenie antagonistycznych właściwości czterech izolatów Trichoderma w stosunku do grzybowego patogena Rhizoctonia solani. Otrzymane wyniki wykazały, że grzyb T. harzianum strain PBG w znacznym stopniu chronił rośliny ogórka przed R. solani i podwyższał masę roślin o 19-26% w stosunku do zakażonej kontroli.

Wszystkie cztery szczepy Trichoderma wpływały także korzystnie na wzrost sałaty w doświadczeniach przeprowadzanych w warunkach szklarniowych. Wzrost siewek najsilniej stymulowały T. harzianum szczep PBG and Trichoderma sp. szczep T6. Masa roślin z tych kombinacji była odpowiednio o 49% i 33% większa niż masa sałaty w kontroli.

Także te dwa szczepy, T. harzianum szczep PBG oraz Trichoderma sp. T6, najskuteczniej

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chroniły sałatę przed patogenem R. solani. Zastosowanie czterech szczepów Trichoderma łącznie, nie podwyższyło skuteczności ochronnego działania antagonistów.

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