In vitro antagonistic activity evaluation of some selected fungi isolated from burned soils in Mila region ( East of Algeria )

The present study was initiated to (i) determine burned forest-inhabiting fungi in Zouagha, TerriBeinène, Mila and (ii) study the antagonistic activities of Trichoderma sp against Fusarium sp, Penicillium sp, Rhizoctonia sp, and Alternaria sp. Eighteen fungal strains representing six genera were isolated from soil samples obtained from the burned forest of Zouagha in the Mila region: Trichoderma sp, Fusarium sp, Penicillium sp, Rhizoctonia sp, Alternaria sp, and Rhizopus sp. The direct antagonistic activity assays of Trichoderma sp on Potato Dextrose Agar medium (PDA) against the four fungi: Fusarium sp, Penicillium sp, Rhizoctonia sp, and Alternaria sp revealed that the fungus Trichoderma sp reduced the mycelium growth of Fusarium sp, Penicillium sp, Rhizoctonia sp and Alternaria sp to 23.13, 33.13, 33.75, and 38.31%, respectively, compared to the control after six days at room temperature. The results illustrated an inhibitory action of the antagonist Trichoderma sp characterized by slowing the mycelial growth of fungal strains. Strains of Fusarium sp, Penicillium sp, Rhizoctonia sp and Alternaria sp showed differences in the sensitivity to the antagonist. Because Trichoderma occurred more frequently in burned soils and were more antagonistic to phytopathogenic fungi in culture than isolates from unburned soils, the judicious use of fire may increase the abundance of Trichoderma isolates and their inhibitory action may be used for the control of fungal plant diseases.


INTRODUCTION
Forest ecosystems of cork and oak often present a balance of extreme complexity and their burning generate a cascade of degradations, which spread over many years and sometimes prove to be irreversible.Certainly, the trees which are weakened after a wildfire, present the ideal conditions for massive colonization by various species of fungi.Some of these are phytopathogenic fungi (Belhoucine and Bouhraoua, 2013).Some microorganisms are considered to be more sensitive to heat than others, for example fungi compared to bacteria *Corresponding author.E-mail: az_wided@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  (Gema et al., 2011).The forest fire is one of the most widespread disruption and potentially most destructive affecting fungi that inhabit the soil (Lygis et al., 2010).The genus Trichoderma includes a set of saprophytic imperfect fungi which are commonly found in soil, dead wood, plant debris and aerial parts of the plants.They are easily recognized in the culture through the generally greenish color of their spores and their typical phialides (bowling-like).
The antagonistic properties of Trichoderma have long been recognized since the first publication in 1887.However, the comprehensive study of the phenomenon of antagonism and its application as a means to fight crop pests started between the two world wars (Johanne, 2002).Trichoderma has a set of potentially usable mechanisms for attacking, but remain complex.It can use one or more modes of action simultaneously to control a pathogen.The deployment of modes of action also varies according to the partners involved and the physical and chemical conditions of the medium (temperature, humidity, etc).Trichoderma is effective when it is allowed to install before the pathogenic fungi.Therefore its action is preventive (Johanne, 2002).In this study, the effects of selected Trichoderma sp isolated from burned soils on the growth and development of Fusarium sp, Penicillium sp, Rhizoctonia sp and Alternaria sp were evaluated.

Study site and sample collection
The work focuses on the study of antagonistic fungi isolated from burned forests in Mila region.Soil, bark and leaves samples were collected from different sites in the forest Zouagha which is bound from the north by Djijal, the west by OuledRabeh, the south by BeniHaroune and the east by Grarem-Gouga.The study area is located on the topographical map of SidiMarouane scale 1/50.000,sheet N°50, between 804.8 to 805 altitude E and 366 à 366.2 longitude N. Z. Zouagha forest is spread over an area of 353, 50 ha and is entirely limited and divided into five regions: El Bahloul, Bouzourane, DjbelDaya, Arres and BeniAfek (Figure 1).
Sampling was conducted on February 19, 2014 at 11: 30 am.Three random sites of burned forest of Zouagha, BeniAfek region in Terri Beinène, Mila were selected.Samples were taken from tree bark and 1 kg of soil (after the removal of 3 cm of the upper layer of soil) of each site and as well as leaves from site 2 to evaluate the presence of fungi.After that, they were stored at 4°C in cooler, then transported to the laboratory until use.

Isolation and purification of fungi
Isolation of fungal strains was carried out according to the suspension dilution method (Davet and Rouxel, 1997).One gram of the soil sample, from each site, was aseptically added to 9 ml sterile saline water.The suspension was vortexed and diluted up to 10 -6 .The bark and leaves samples were washed first in bleach for 5 min, then with ethanol for 5 min to remove microorganisms from the surface.Purification of strains was done on PDA agar (pH =5.1).Plates were incubated at 25°C for 6 days.

Identification of fungi
Strain's identification was conducted following the conventional dichotomous identification scheme.The macromorphology identification was done on the basis of colonies' properties of the isolation media.The micromorphology of the isolates were determined by direct light microscopic examination at 10X and 40X (optical microscope EXACTA+OBTEC) according to the determination keys of Botton et al. (1990).

Antagonism test
Direct confrontation method also called opposite cultures technique was used to determine antagonism activity.In a Petri dish containing 15 ml of PDA medium, two agar pellets (8 mm in diameter) of antagonist and pathogen were placed 4 cm from each other.Petri dishes containing pathogenic fungi were used as control.The plates were incubated at a temperature of 25°C and continuous light as an activation factor of certain enzymes.The development of mycelia growth was monitored every 24 h by measuring diameters of mycelial colony in millimeter.The percentage of mycelial growth inhibition was determined using the following formula (Hmouni et al., 1996): I(%): is the percentage of mycelia growth inhibition Cn: is the average diameter of the colonies in the presence of the antagonist Co: average diameter of the control colonies.

Isolation and identification of fungi
Fungi were isolated from almost all analyzed samples and were identified.The percentages of fungal isolates varied; 16.66% for Fusarium sp, 5.55% for Alternaria sp, 55.55% for Trichoderma sp, 5.55% for Rhizopus sp, 11.11% for Rhizoctonia sp and 5.55% for Penicillium sp (Tables 2  and 3).

Antagonism
The results of antagonism test of Trichoderma against four fungal isolates (Alternaria sp, Fusarium sp, Penicillium sp, and Rhizoctonia sp) show a medium reduction of mycelial growth of colonies of different fungal isolates compared to control (Figure 2 and Table 4).The tested colonies of Trichoderma sp inhibited the germination of Fusarium sp conidia by 23.13%, followed by Penicillium sp by 33.13%, Rhizoctonia sp by 33.75% and Alternaria sp by 38.31% (Figure 3).

DISCUSSION
In the study, the fungus flora of the burned forest soil of Zouagha, Mila region was determined.Among the genera obtained Trichoderma sp, Fusarium sp, Penicillium sp, Rhizoctonia sp, Alternaria sp, and Rhizopus sp.Lucarotti (1981) obtained higher frequencies of Trichoderma, Penicillium, MucorMich ex Fr. and      Mortierella Coemans at in soil of burned forest in Canada.It can be postulated that these species do not show much sensitivity to ecological extremes and are more resistant to negative conditions.Also, Reaves et al. (1990) reported that they obtained Trichoderma citrinoviride Bissett most frequently in burned forest soil.Chwalinski (1989) determined that the variety of species following a fire was renewed within a year but the fungal density was not renewed completely in this period.Many researchers reported that the soil humidity, soil pH (RamaRao, 1970), salt amount (Hasenekoglu and Sulun, 1990), and organic matter content (Behera and Mukerji, 1985) influence the activity of soil microorganisms.The fact that the amount of organic matter is very high in all soils proves that the rapidly spread fire, did not do much harm underground and the fire was only on the surface (Hasenekoglu and Sulun, 1990).
In addition, 20% of organic matter is nitrogen, and thus these soils are considered to be very rich in nitrogen.This may have a positive effect on microorganism activity in the soil (Table 2).The fact that the soil has a low rate of salt and lime (Ca +2 ) could exclude their negative effect on the activity of soil microorganisms.Suciatmih (2006) found that a significant positive correlation existed between the fungal population and the total organic carbon content.Waid (1960), listed temperature, humidity, CO 2 , oxygen concentration, size of the soil pores, longevity of fungal mycelium, interaction between soil fauna and soil fungi and soil reaction as According to Suciatmih (2006), the forest fire leads to a reduction and possibly an elimination of soil fungi.Chet (1984) reported studies on mode of action of Trichoderma used for biological control against Rhizoctonia solani in the case of cotton and strawberry cultivation, the results highlighted the importance of mycoparasitism phenomenon in the effectiveness of Trichoderma.
In the case of direct confrontation between Alternria alternata and Trichoderma harzianum.A. alternria has a very short development time but T. harzianum grows faster and surrounds the pathogen on the second day.T. harzianum develops without obstacles, and it has opportunities to stop the development of A. alternate.It grows over the colony of the pathogen at the same time (Biljana and Jugosslave, 2011;Gveroska and Ziberosk, 2011).Rajendiran et al. (2010) demonstrated the inhibitory effect of Trichoderma viride against Fusarium sp., Penicillium sp.And Aspergillus sp.Growth inhibition of these fungi is due to its rapid growth nature, extracellular secretion of harmful compounds such as antibiotics, enzymes that can degrade cell wall such as gluconases, endochitinases, chitinases and mycoparasitism.Harman et al. (2004) described the mycoparasitic action of Trichoderma sp against pathogens.It coils around the hyphae of pathogen and produced peptaible which facilitate the entry of hyphae of Trichoderma sp into the lumen of parasitic mold.

Figure 2 .
Figure 2. Representative images of controls of five fungal genera after six days.

Figure 2 :
Figure 2 : Representative images of controls of five fungal genera after six days.

Figure 3 .
Figure 3. Colony diameter of Fusarium sp, Penicillium sp, Rhizoctonia sp and Alternaria sp in the presence of Trichoderma sp after 6 days.

Table 1 .
Source of isolates and their frequencies.

Table 2 .
Samples analysis of unburned soil from Zouagha forest.

Table 3 .
Samples analysis of soil obtained from Zouagha forest.

Table 4 .
The effect of Trichoderma sp on Fusarium sp, Penicillium sp, Rhizoctonia sp and Alternaria sp.