Evaluation of Trichoderma as a biological control against different pathogenic bacteria and fungi

as a biological


Introduction
Bacterial and fungal pathogens cause infections in plant and animal hosts.These pathogens can cause disease on a narrow and also at wide ranges of hosts.However, some have a dual ability to cause diseases in different hosts [1].Use of traditional control methods are not eco-friendly approach and also produce different aromatic group or ethylated and methylated substances which have adverse effects on the environment and also pose a risk to human health via water contaminations, foodstuff or accidental exposure.Therefore, efforts were made to use biological control method for controlling the pathogens in the eco-friendly way which are safe both for humans and as well as for environment [2].The term biological control and its alternative expression biocontrol have been used in different fields of biology, most remarkably for pathology and entomology [3].In each field of biology, the use of animal, fungi and/or other microbes to feed upon, parasitize, suppresses or otherwise interfere with targeted pathogens is referred to as the biological control agents (BCAs) [4].Furthermore, these agents seek to renovate the beneficial balance of natural ecosystem [5].Biological control method is only wellknown when BCAs can effectively accomplish the interaction between the pathogen and host.Currently, several fungal and bacterial BCAs have been recognized.The

Sample collection
A total of 10 samples were collected to assure the presence of Trichoderma species from different collection sites of Pir Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS-AAUR) and from Zhob, Balochistan.Eight of the samples were collected from farmyard manure (FYM) and two from the rhizosphere of plants.The samples were collected in sterile plastic bags and were properly labelled to indicate the location sites and collection date.Then, the samples were brought into the department of Plant Pathology of PMAS-AAUR, Pakistan for further analysis.

Isolation of Trichoderma species
Serial dilution method was used to analyze the collected samples.First the collected samples were homogenized and then 1g of the samples were weighed and used to do serial dilution for the isolation of Trichoderma species.One gram of sample was properly blended into 10ml of sterile water then 1ml of suspension was taken into another tube containing 9ml of sterile water.The serial dilution technique was performed up to 10 -3 to 10 -4 .At that point, 100µl suspension of each diluted sample was transferred onto Potato Dextrose Agar (PDA) plates and spread carefully by using a spreader or sterilized glass.The plates were then stored at 28-30°C for a week.The plates were checked daily and observable colonies were transferred to new PDA plates and incubated at 28-30°C for one week.

Identification of Trichoderma isolates
The identification was performed by observing both microscopic and macroscopic features of the Trichoderma colonies.For microscopic study, mycelia from each colony were taken from PDA petri plate and spread on the top of a slide having a drop of sterile water and examined the slide under a microscope using 400X magnification power.The sizes and shapes of conidia, conidiophores and phialides were studied.For macroscopic study, the colour and growth rate of the fungal colonies were observed.The microscopic and macroscopic features were associated to the characteristics of Trichoderma as reported by Samuels et al.

In vitro evaluation of Trichoderma isolates against test pathogens by dual culture technique
Trichoderma isolates were evaluated in vitro for their biocontrol efficiency against fungal and bacterial pathogens viz., F. oxysporum, A. alternata, P. carotovorum and P. syringae by dual culture method.These pathogens were provided from the department of Plant Pathology of PMAS-AAUR, Pakistan.First of all, approximately 20 ml of PDA media for fungi and nutrient agar (NA) media for bacteria were poured in petri plates.When media were solidified, a 5mm diameter disc from the edge of 3-4 day old culture of Trichoderma colony and the test pathogens were positioned on the opposite side of the petri plate at equivalent space with the help of sterile cork borer.Later these plates were closed with Para film under sterile conditions.Petri plates having only test pathogen and without Trichoderma species served as control plate.There were three replications for each pathogen.The whole process was done in a sterile condition and both the test plates and control plates were stored at 28°C for one week.The colony of the test pathogen was measured after first, second, third and fourth days of the incubation.Finally, the percent (%) inhibition growth was measured in relation of growth in control plate Where, I = % inhibition of growth C = growth in control plate (cm) T = growth in test plate (cm) Results Isolation and identification of Trichoderma species Trichoderma isolates were successfully isolated from four samples out of 10 different samples.Three isolates (Ma1, Ma3 and Mz1) were from the FYM while one isolate (Ma6) from rhizosphere of plant.All the Trichoderma isolates grew faster and produced conidia within a week.Conidial growth of Ma1 was scattered, clustered, green colored and produced concentric rings.The dispersed, fast growth rate and yellowish colonies were observed in Ma3 and Ma6, respectively.While in Mz1, the colour appeared to be dark green.All the Trichoderma isolates except Mz1 fully covered the 9cm Petri plates in a week.The isolate Ma1 had branched conidiophore, paired phialides, broader and rough shaped conidia.Both Ma3 and Ma6 had minute conidiophore and rough shaped conidia.However, the isolate Mz1 had only branched conidiophore, phialides while conidia were not observed (Figure 1).Besides these characteristic, Samuels et al.
[18] reported that the colonies of Trichoderma produced coconut like odour which can be also the characteristic of that species.In present study isolate Ma1 produced an odour close to coconut.

In vitro evaluation of Trichoderma against bacterial pathogens
The Trichoderma isolates were screened in vitro against P. carotovorum and P. syringae by dual culture (zig zag) technique on NA media for one week.Data were recorded every day and at the end of the week the antagonistic activity of Trichoderma isolates showed more influence against the growth of test pathogens.
Trichoderma isolates showed greatest performance against the growth of P. carotovorum which had 0.5cm growth as compared to 6cm in control plate.Trichoderma inhibited the growth of P. carotovorum by 91.7%.Whereas the growth of P. syringae was 0.5cm as compared to 6cm growth in control plate.The Trichoderma isolates inhibited the growth of P. syringae by 91.7%.The redial growth of both P. carotovorum and P. syringae, in the presence of Trichoderma isolates was 8.3% (Figure 3  b. Figure 4

. Inhibition and radial growth percentage of fungal and bacterial pathogens in the presence of Trichoderma isolates (a) Bar chart showing the inhibition percentage of tested pathogens (b) Bar chart showing the radial growth percentage of tested pathogens Discussion
Biocontrol is a promising and effective method to keep up the current level of the agricultural products by limiting the release of chemical pesticides to the environment and as well as making the plants free from pathogens.Biocontrol method is only well known when the BCAs can effectively accomplish the interaction among the pathogen and host.Plant pathogenic bacteria and fungi are the widespread problems and the utilization of synthetic chemical substances is not really effective.According to this study, biocontrol method has been found useful and is a better alternative against fungal and bacterial pathogens such as F. oxysporum, A. alternata, P. carotovorum and P. syringae.One of the main advantages of biocontrol method is that these alternatives are much safe, cheaper and environment friendly control method against pathogens and have no hazard to human and environment.Currently, several fungal and bacterial biocontrol agents have been recognized for controlling the pathogens such as Trichoderma, Candida, Rhizoctonia, Coniothyrium, Bacillus and Agrobacterium [6, 7].Among these, one of the fungal BCAs used in present study is Trichoderma species, they are saprophytic fungi that can commonly found everywhere including decaying plant material, rhizospheres of plants and as well as in animal manure.The reason for selecting Trichoderma species as BCA is their potential to decrease the incidence of diseases caused by the tested pathogens.The mechanisms of antagonism used by Trichoderma species include competition for nutrients and space, inhibitory compounds and antibiosis by processing non-volatile antibiotics and volatile components that suppress parasitism as well as for soilborne fungi.

Trichoderma species reduce the infections caused by the pathogens by various mechanisms such as antibiosis, secretion of enzymes, mycoparasitism, competition and hyphal interactions [20].
In this study, the results of in vitro inhibitory efficiency of Trichoderma isolates against F. oxysporum, A. alternata, P. carotovorum and P. syringae were studied by dual culture technique.Three replication of Trichoderma isolates were used during this technique for the calculation of inhibition and growth percentage of respective pathogen for a reliable result.The antagonistic activities of Trichoderma isolates were found to be very effective against these test pathogens.Significant results were obtained when Trichoderma isolates formed zone of inhibition against these fungal and bacterial pathogens though with variations.The maximum zone of inhibition was formed against bacteria P. carotovorum by 91.7% and P. syringae by 91.7% followed by fungus F. oxysporum by 80%.Whereas Trichoderma isolate formed the minimum inhibition zone against A. alternata by 75%.However, the results showed that the inhibition activity of Trichoderma isolate was higher against all tested pathogens but the gowth of P. carotovorum, and P. syringae were controlled the most.

Figure 1 .Figure 2 .
Macroscopic and microscopic observations of Trichoderma isolates.(a) Isolate Ma1 (b) Isolate Ma3 (c) Isolate Mz1 (d) Microscopic observation of isolate Ma1, branched conidiophore, paired phialides and rough conidia.In vitro evaluation of Trichoderma against fungal pathogensThe Trichoderma isolates were screened in vitro against F. Oxysporum and A. alternata by dual culture technique on PDA media for one week.Data were recorded every day and at the end of the week the antagonistic activity of Trichoderma isolates showed more influence against the growth of test pathogens.The Trichoderma isolates showed great inhibition activity against the growth of F. oxysporum which had 1cm growth as compared to 5cm in control plate.The Trichoderma isolates inhibited the growth of F. oxysporum by 80%.Whereas the growth of A. alternata was 1cm as compared to 4cm growth in control plate.The Trichoderma isolates inhibited the growth of A. alternata by 75 %.The redial growth of F. oxysporum and A. alternata in the presence of Trichoderma isolates were calculated as 20% and 25%, respectively (Figure2& 4).In vitro evaluation of Trichoderma isolates against fungal pathogens by dual culture technique.(a) Trichoderma isolate showing biocontrol efficiency against F. oxysporum.(b) Growth of F. oxysporum in control plate.(c) Trichoderma isolate showing biocontrol efficiency against A. alternata.(d) Growth of A. alternata in control plate.

Figure 3 .
In vitro evaluation of Trichoderma isolate against bacterial pathogens by dual culture (zig zag) technique.(a) Trichoderma isolate showing excellent biocontrol efficiency against P. carotovorum.(b) Growth of P. carotovorum in control plate.(c) Trichoderma isolate showing excellent biocontrol efficiency against P. syringae (d) Growth of P. syringae in control plate a.
There are several data reporting the biocontrol activity of Trichoderma species against A.