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Trichoderma harzianum as a biocontrol agent against Alternaria alternata on tobacco.

Introduction

The presence of fungal diseases on tobacco and its economical consequences require the use of many pesticides. Besides chemical preparations, the Integrated Pest Management involves the use of biological control of pathogens. Modern production of tobacco in Macedonia is striving to involve this kind of disease control and limited chemical usage.

The greatest data in biological control literature, refers to genus Trichoderma. The activity of this useful species has been recognized from 1930 and today there are modern technologies for including them in biological control of various diseases.

For a long time, Trichoderma species have been known as biological agents for control of plant diseases (Ranasingh, Saturabh, Nedunchezhiyan, et al., 2006). They interact with root, soil and leafsurroundings. They produce and release many components, which induce local or systemic plant resistance to abiotic stress. According to Rosado et al. (2007), the main factor for ecological success of this genus is a combination of very active mycoparasitism mechanisms and an effective defensive strategy, induced in the plants.

These fungi are used in biological control against various plant pathogens. Similar to soil pathogens, they successfully suppress leaf pathogens as well.

Biological control offers an environmentally friendly approach to the management of plant disease and can be incorporated into cultural and physical controls and limited chemical usage for an effective integrated pest management (IPM) system (Monte, 2001).

The brown spot disease plays a significant role among some economically important diseases on tobacco in Republic of Macedonia. Its causing agent is the pathogenic fungus Alternaria alternata. The specific symptom (brown spots) causes biochemical changes that impair the quality of raw tobacco, and result in severe economic losses on tobacco production. The occurrence of brown spot disease depends on the climate conditions, but also on other factors such as plant age,

time of leaf harvesting, susceptibility of the variety, sugar content, etc.. The use of Trichoderma with all mechanisms of biological control will help suppress this pathogen.

According to Mandare et al. (2008), A. alternata development in laboratory conditions is inhibited by T. koningii, T. lingorum and T. virens. Six out of 16 isolates of Trichoderma species suppress the development of her mycelia more than 55%. Many of them are tolerant and the third isolates are compatible with more fungicide active ingredients (Umamaheswari, Thakore, and More, 2009). This species is known due to its ability to produce many lithic enzymes and/or antibiotics (Lieckfeldt, Samuels et al., 1999). Therefore, the pathogenicity of A. alternata is decreased when the solution of crude chitinase is applied in tobacco seedlings. The effect is significantly bigger in crude than purified chitinase (Gang, Xing Zhong et al., 2004).

T. harzianum is a biocontrol agent in control of A. alternata (Roco and Perez, 2001; Izzati and Abdulah, 2008; Monte, 2001; Sempere and Santamarina, 2007). According to Roco and Perez (2001), in vitro biocontrol of T. harzianum towards A. alternata is improved in the presence of the growth regulators or foliar nutrient at concentrations similar or higher than those used at the field level. So, their presence could benefit plant from the elicitor activity of this Trichoderma that induces plant defense mechanisms against the pathogen.

There is a not general solution and biocontrol system must be developed for each crop. Before the application of a commercial product, it is necessary to first do various studies. The first and quickest way was to screen for antibiotic production and/or mycoparasitism in petri dishes assays (Harman, 2006). Our aim, therefore, was to determine the impact of T. harzianum on the pathogen A. alternata, in laboratory conditions.

Materials and methods

Using ordinary laboratory method, the pathogen fungus A. alternata was isolated from transitional part between healthy tissue and brown spots on tobacco leaves. T. harzianum was isolated from the root zone of rhizosphere, by a method of dilution, using Czapek agar.

In-vitro biological activity of T. harzianum on A. alternata was investigated by double cultures on the potato dextrose agar.

Trials were set up in three replications, with five Petri dishes by each variant. They were incubated at 25[degrees]C and the diameter of the colonies was measured for 10 days.

In diffusible metabolites, investigations were maintained with the variants:

--Placing the fragments of test fungus and pathogen on the separated half of a media

--Placing the fragments at the same time, with near-contact

--Temporal and spatial advantage of pathogen growth (up to filing the half of a medium) and then place the T. harzianum fragment.

In volatile metabolites investigations, fragments of test fungus and pathogen were placed in the center of two separate PDA plates sealed with parafilm, so, the test fungus was in the under plate and, the pathogen was in upper plate. There were two controls (T. harzianum and A. alternata) in an inverted position.

After the period of incubation, micro observations were made of the pathogen in the two kinds of double cultures.

Results

The colony of pathogen fungus A. alternata has mostly a grey or greenish color, with aerial mycelia (Figure 1). There are also differences among the isolates about color, and sporulation and conidia size. The investigated isolate has middle intensity of sporulation.

The first, T. harzianum forms a colony with white mycelia, but it became green when forming conidia and conidiophores. There are conidia formed densely over the center and undulating concentric rings toward the edge (Figure 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

In double cultures with fragments on separated half of a medium, A. alternata developed more slowly than T. harzianum, from first to the last day of incubation (Table.1). On the second day diameter of the colony is 22,60 mm, but that of T. harzianum is 58,60 mm, so, it is approaching to the pathogen (Figure 3).

A. alternata develops with a minimal amount (only 2-3mm) until the third or fourth day. The growth is seen only in some cases when it develops a lengthened deformed look as it makes an effort to develop towards the opposite site), until it is surrounded by T. harzianum. Also, it has very light color (Figures 3-6).

After the contact with a pathogen, T. harzianum continues to develop without obstacles. There are few differences between double cultures and check, the opposite than a pathogen (Table 1). A. alternata become captured by the colony of T. harzianum (Figures 4-6).

At the end of the investigation, it is noted that the whole plate is filled by Trichoderma, as it develops over the pathogen's colony, covering it (Figure 7).

In the case of near-contact pathogen and antagonist fragments, A. alternata has a very short developing time, but T harzianum develops more rapidly and surrounds the pathogen on the second day (Table 1; Figures 8, 9). The third day it is fully surrounded by the antagonist, when it stops developing. It has smaller dimensions, and like in previous case, it has irregular colony form. T. harzianum develops without obstacles, suppresses the A. alternata from developing. It is developing over the pathogen's colony at the same time (Figures 10, 11).

[FIGURES 3-6 OMITTED]

[FIGURE 7 OMITTED]

[FIGURES 8-11 OMITTED]

[FIGURE 12-14 OMITTED]

In temporal and spatial advantage of A. alternata growth, it continues to grow until it is in contact with the antagonist. From this moment, there is an attempt to grow towards the opposite side, which is visible in its deformed characteristic (Figure 12). At the same time, T. harzianum develops without obstacles (Figures 13, 14).

In investigations about Trichoderma's influence with volatile metabolites, it is noticeable that the pathogen grows more slowly than the antagonist does, as in the previous case. However, the growth slows and becomes stagnant about the fifth day, compared to check, which continues to grow and get up bigger diameter (Table 2). T. harzianum doesn't show a difference in dual cultures and check normal and inverted position, too (Table 2).

In the colony of A. alternata we can see wet zones of degradation. Also, it is a noticeably lighter circle in the later stages of pathogen incubation (Figures 15, 16).

[FIGURES 15-16 OMITTED]

Through the incubation period of dual cultures we see a change of the A. alternata colony look. From the beginning of incubation, there is obvious discoloring in diffusible metabolites (in volatile this is later noticeable), and a loss of sporulation.

Microscopic observations of the pathogen after the interval of incubation, from both of dual colonies cases showed deformations of hyphae, which exhibit a longer distance between septa and have empty ends. There is a small number or absence of conidia than in the check (Figures 17-19).

[FIGURE 17 OMITTED]

[FIGURE 18 OMITTED]

[FIGURE 19 OMITTED]

Discussion

One of the key elements of sustainable agriculture is the ecological approach to solving the problems with plant pathogens, by the application of biocontrol agents. The genus Trichoderma is most important in achieving that and, at the same time, sustaining a favourable environment, instead of using chemicals.

Fungi of the genus Trichoderma have long been recognized for their ability to act as biocontrol agents against plant pathogens. During this time, research has described their mechanisms of action and how they might be used for various purposes (Harman, 2006).

Antibiosis, mycoparasitism and food competition are the main mechanisms in biological control (Ghildyal and Pandey, 2008; Umamaheswari et al., 2009; Ranasingh et al., 2006).

Ghildyal and Pandey (2008) estimated that Trichoderma sp. is produced diffusible and volatile metabolites. The production of volatile and non-volatile antibiotics by the species of Trichoderma also was reported by Dennis and Webster (1971, loc cit. Ubalua and Oti, 2007).

Investigations into the biological activity of Trichoderma have shown it has a strong reducing effect towards A. alternata development, since it develops without obstacles. T. harzianum develops more rapidly than A. alternata in single as well as in double cultures. The intensive development of Trichoderma gives it a significant advantage in competition with pathogens for nutrient elements and space, even as it develops the system of mycotoxins (Barbosa, Rehm et al., 2001).

The contact between two fungi begins as early as at second day, with pathogen stagnation coming after, when Trichoderma is developing and spreading. According to Almeida et al. (2007), inhibition of pathogen in double cultures uegins soon after contact with the antagonist.

Trichoderma sp. develops exactly on other fungi's hyphae, coils around them and degrades the cell's walls. This action of parasitism restricts the development and activity of pathogenic fungi. Additionally, or together with mycoparasitism, some Trichoderma species release antibiotics (Harman, 1996).

It was concluded that Trichoderma sensed the presence of target fungi and appeared to grow tropically towards them. However, it was noticed that when they are together, endochitinase gene is activated before they come into contact, while activation of exochitinase occurs only after contact. Also, the degraded cell wall fragments of target fungi are highly potent inducers of enzymes, induction, an enhancement in Trichoderma growth (Harman, 2006).

According to Figure 7, the whole petri plate is filled with T. harzianum, thus, it develops even over the culture of A. alternata. In-vitro antagonistic activity of Trichoderma sp. is expressed by suppressing test-fungi growth and its rapid growth, which is multiplying over the pathogen's colony (Mirkova, 1982).

When the fragments are placed in near-contact, Trichoderma has a space advantage and bigger opportunities to stop pathogen development, and to develop its mechanisms of antagonistic action. In a short time, it significantly reduces A. alternata. The pathogen develops until the moment of contact with the fungus-antagonist, simultaneously it sporulates and changes the colony's color, acting as a super--parasite (Mirkova, 1983).

In the third case of double cultures, even when A.alternata has a significant space and time advantage, T. harzianum has shown to have an antagonistic influence. This effect is produced because of competition for food and space, mycoparasitism and possible antibiosis (Sempere and Santamarina, 2007).

Avoiding direct contact with an antagonist has given the pathogen an opportunity for greater development. However, it has also shown that T. harzianum expresses a reducing effect over both volatile and diffusible metabolites as in the previous cases. Generally, the diffusible metabolites have a bigger reducing effect than the volatile ones (Kucuk and Kivanc, 2003). Ghildyal and Pandey (2008), came to the same conclusion. According to them, three species of Trichoderma with their own diffusible and volatile metabolites have shown the greatest rate of inhibition on A. alternata among the investigated pathogens.

At the time of incubation of double cultures (in diffusible and volatile metabolites), discoloring of the colony and the loss of a sporulation both occured. This was confirmed by microscopic examination. In addition, hyphae deformations were noticeable, including greater distances between septa, and empty ends. The A. alternata conidia germination was strongly suppressed by chitinase derived from Trichoderma spp. for eight hours, resulting in the complete inhibition of germination, abnormalities, or breaking the germination tubes (Gang et al., 2008).

Hyphal shrinking, cell shortening and septa thickening were found by Barbosa et al. (2001) in Cladosporium herbarum, in the presence of Trichoderma. Exactly as we observed, spores of Trichoderma were visible in the microscopic preparations.

Morphological abnormalities in the pathogens's structure are confirmed by Ghildyal and Pandey (2008). Induction of deformities is causing by ability of Trichoderma to develop the direct interaction with a pathogen and produce antimicrobial substances as well as mycoparasitism involving a physical contact and production of hydrolytic enzymes, toxic components and antibiotics.

Conclusion

This study confirms in-vitro biological activity of T. harzianum towards A. alternata. It has shown strong reducing effect on the development of A.alternata with various mechanisms of antagonistic influence.

The diffusible metabolites have shown bigger reducing effect than volatile. This biocontrol agent caused abnormalities in the pathogens's morphology. The strong reducing effect of T. harzianum towards A. alternata can be applied in biological control of this pathogen.

References

Almeida, F., Cerqueira, F., Silva, R., Uhoa, C., Lima, A., 2007. "Mycoparatism studies of Trichoderma harzianum strains against Rhizoctonia solani: Evaluation of coiling and hydrolytic enzyme production," Biotechnol Lett., Vol.29(8), pp.1189-193

Barbosa, M., Rehm, K., Menezes, M., Mariano, R.L., 2001. "Antagonism of Trichoderma species on Cladosporium herbarum and their enzimatic characterization," Brazilian Journal of Microbiology, Vol.32, pp.98-104

Gang, T., Xing Zhong, L., Wang, G., Shidong, L., 2004. "Role of chitinase by Trichoderma sp. in biocontrol of tobacco brown spot caused by Alternaria alternate," Chinese Journal of Biological Control, Vol.20(4), pp.252-55

Ghildyal, A., Pandey, A., 2008. "Isolation of cold tolerant antifungal strains of Trichoderma sp. from glacial sites of Indian Nimalayan Region," Research Journal of Microbiology, Vol.3(8), pp.559-64

Harman, G.E., 1996. "Trichoderma for biocontrol of plant pathogens: From basic research to comercialized products," Conference on Biological Control, Cornell community, April 11-13

Harman, G.E., 2006. "Overview of mechanisms and uses of Trichoderma spp.," Phytopathology, Vol.96, pp.190-94

Izzati, N., Abdulah, F., 2008. "Disease suppression in Ganoderma-infected oil palm seedlings treated with Trichoderma harzianum," Plant Protect.Sci., Vol.44(3), pp.101-107

Kucuk, Q., Kivanc, M., 2003. "Isolation of Trichoderma Spp. and determination of their antifungal, biochemical and physiological fetures", Turk J Biol., Vol.27, pp.247-53

Lieckfeldt, E., Samuels, G., Nirenberg, H., Petrini, O., 1999. "A morphological and molecular perspective of Trichoderma viride: Is it one or two species," Appl Environ Microbiol., Vol.65(6), pp.2418-428

Mandare, V., Suryawanshi, A., Gawade, S., 2008. "Studies on Alternaria blight of chickpea," Agricultural Science Digest, Vol.28(3), pp.222-24

Mirkova, E., 1982. "Isledvanje in vitro na antagonistickata aktivnost na Trichoderma sp. km nekoi pocveni patogeni," Gradinarska i lozarska nauka, god. XIX, No.1, pp.73-81

Mirkova, E., 1983. "Prilozenie na Trichoderma harzianum Rifai za borba s fuzariinoto uvrhvane (Fusarium oxysporum sch.f. dianthi (Prill. Et Del.) Snyd. Et Hans.) pri oranzerinia karamfil," Selskostopanska akademia, Gradinarska i lozarska nauka, god. XX, No.1, pp.65-69

Monte, E., 2001. "Understanding Trichoderma: Between biotechnology and microbial ecology," Int. Microbiol., Vol.4, pp.1-4

Ranasingh, N., Saturabh, A., Nedunchezhiyan, M., 2006. "Use of Trichoderma in disease managemant," Orissa Review, September-October, pp.68-70

Roco, A., Perez, L., 2001. "In vitro biocontrol activity of Trichoderma harzianum on A. alternata in the presence of growth regulators," Plant Biotechnology, Vol.4(2), pp.68-73

Rosado, I., Rey, M., Codon, A., Gonavites, J., Moreno-Mateos, M.A., Benitez, T., 2007. "QID74 Cell wall protein of Trichoderma harzianum is involved in cell protection and adherence to hydrophobic surfaces," Fungal Genetics and Biology, Vol.44(10), pp.950-64

Sempere, F., Santamarina, M., 2007. "In vitro biocontrol analysis of Alternaria alternata(Fr.) Keissler under different environmental conditions," Mycopathologia, Vol.163(3), pp.183-90

Ubalua, A., Oti, E., 2007. "Antagonistic properties of Trichoderma viride on post harvest cassava root rot pathogens," African Journal of Biotechnology, Vol.6,(21), pp.2447-450

Umamaheswari, B., Thakore, B., More, T., 2009. "Post-harvest management of ber (Ziziphus mauritiana Lamk) fruit rot (Alternaria alternata (Fr.) Keissler) using Trichoderma species, fungicides and their combinations," Crop Protection, Vol.28(6), pp.525-32

Biljana Gveroska (1), Jugoslav Ziberoski (2)

(1) Scienticic Tobacco Institute-Prilep, University St.Kliment Ohridski, Republic of Macedonia

(2) Faculty of Agriculture science and food, University St. Cyril and Methodius, Republic of Macedonia

e-mail: bgveros@yahoo.com
TABLE 1. EFFECT OF T. HARZIANUM ON DEVELOPMENT OF A. ALTERNATA
(DIFFUSIBLE METABOLITES)

Diameter (mm)                                   Days

                                     1       2       3        4

Fragments on        A. alternata   12.40   22.60   27.80    29.20
half-medium         T. harzianum   18.60   58.60   103.40   110.00
Near-contact        A. alternata   11.60   18.60   22.80
fragments           T. harzianum   15.20   54.80   104.60   110.00
Check [empty set]   A. alternata   12.40   26.40   39.60    49.20
                    T. harzianum   17.60   59.40   106.20   110.00

Diameter (mm)                                   Days

                                     5       6       7       8

Fragments on        A. alternata
half-medium         T. harzianum
Near-contact        A. alternata
fragments           T. harzianum
Check [empty set]   A. alternata   59.60   70.40   83.00   100.20
                    T. harzianum

Diameter (mm)                           Days

                                     9        10

Fragments on        A. alternata
half-medium         T. harzianum
Near-contact        A. alternata
fragments           T. harzianum
Check [empty set]   A. alternata   104.00   107.40
                    T. harzianum

TABLE 2. EFFECT OF T. HARZIANUM ON DEVELOPMENT OF A. ALTERNATA
(VOLATILE METABOLITES)

Diameter (mm)                                   Days

                                      1       2       3        4

A. alternata                        9.62    22.22   30.84    38.89
T. harzianum                        29.96   67.11   106.67   110.00
Check [empty set]    A. alternata   9.53    16.67   24.00    32.47
(inverted position)  T. harzianum   19.67   61.53   104.73   110.00

Diameter (mm)                                   Days

                                      5       6       7       8

A. alternata                        45.02   50.02   54.02   56.49
T. harzianum
Check [empty set]    A. alternata   40.87   49.33   59.47   65.40
(inverted position)  T. harzianum

Diameter (mm)                           Days

                                      9      10

A. alternata                        57.47   58.38
T. harzianum
Check [empty set]    A. alternata   72.73   79.87
(inverted position)  T. harzianum
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Author:Gveroska, Biljana; Ziberoski, Jugoslav
Publication:Applied Technologies and Innovations
Article Type:Report
Date:Jun 1, 2012
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