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Suppression of Fusarium wilt of banana with an application of Trichoderma asperellum inoculants

Fusarium wilt is one of the most serious diseases of banana plants in Malaysia that now becomes a worldwide problem. It is caused by soil-borne pathogen Fusarium oxysporum Schlecht f. sp. cubense (E.F. Smith) Snyder & H.N. Hansen. In this study, in vitro and in vivo experiments were conducted to evaluate the effects of Trichoderma asperellum B01 inoculants on disease incidence of Fusarium wilt of banana. Results indicated that percentage inhibition of radial growth (PIRG) was observed in dual culture plates of Trichoderma isolates B01 by 84.85%. In addition, under plant house condition, the application of conidial suspension of T. asperellum B01 has significantly decreased the incidence rate of Fusarium wilt by up to 94.4% compared to control. In general, the application of T. asperellum B01 inoculants revealed a great potential for the control of Fusarium wilt disease in banana plants under plant house condition, which a good approach to start in agricultural field. For future study, new method must be focusing on optimize the production of Trichoderma spores to be using for agricultural purposes.


Introduction
Disease management of Fusarium wilt of banana caused by F. oxysporum f. sp. cubense (Foc) in the industry using synthetic fungicides. It is linked to the increase of frequency of some types of cancer and poses serious risks of health within the human food chain [1]. In Malaysia, to minimize the disease from spreading and making damage to the plantation, some approaches have been practiced including shifting cultivation practiced by farmers, intercropping with oil palm or rubber to achieve 3-4 harvests and the use of chemical fungicides [2]. Many types of fungicide have been applied worldwide to treat the plant disease [3]; however, they can be unsafe to the ecosystem.
Alternative approach in managing Fusarium wilt disease can be done by using biocontrol, which has no negative effect on plant, animal, human and environment as it is eco-friendly and can be obtained from the surrounding; for instance, microorganisms. Based on a study by [4], Trichoderma spp. and Pseudomonas spp. can act as potential biological controls of Fusarium wilt disease of banana. Trichoderma spp. are interactive in a soil, root and foliar environments. In the host, they produce various metabolites that induce localized or systemic resistance responses. Many researchers have used Trichoderma species as biological control agent in substituting chemical pesticides. Based on [5], the application of T. harzianum T73s has successful inhibited Fusarium ear rot of maize under plant house condition. Additionally, a soil borne-pathogen of basal stem rot in oil palm, Ganoderma boninense can also be suppressed by T. harzianum FA1132 [6]. In order to ensure an eco-friendly cure of the disease, this study has focused on screening the natural enemies agent specifically Trichoderma species against pathogen.

Dual culture test
Trichoderma asperellum isolate B01 was challenged for its antagonistic properties against the pathogenic isolate of Foc under in vitro condition. The isolate was cultured on Potato Dextrose Agar (PDA) and incubated at 28±2 o C for seven days. A disk of young mycelial of pathogen sized 0.5 cm was placed by 1 cm from the end plate and a disk of Trichoderma isolate sized 0.5 cm was placed also by 1 cm from the other opposite end side in PDA culture plate. For the control plate, a disk of pathogen sized 0.5 cm was placed in a similar position which was 1 cm apart from the plate end but without Trichoderma isolate. The inoculated plates were incubated at 28±2 o C for five days and completed in four replicates.
The competency of Trichoderma isolate B01 to inhibit pathogen's growth was assessed by measuring the radial growth of pathogen in the presence of Trichoderma and its growth on the control plate. The fungal growth was converted into percentage inhibition of radial growth (PIRG) [8].

In-vivo study on Trichoderma isolates against F. oxysporum f.sp. cubense
About 1.5 kg of the mixture topsoil, peat and sand in ratio 3:2:1 (v/v) was used as a medium. The medium was put into each polyethylene bag sized 10 x 10 cm covering half of the banana seedling (Cavendish variety) at age 4 weeks-old plant's depth. The experiment was conducted under plant house condition in which the space between each plant was 25 cm apart with 25 cm between rows. Nitrogen, phosphorus and potassium (NPK) fertilizer was given at week 0 -6 after acclimatization following the manufacturer's recommendations. All other factors were controlled manually, and the plants were watered manually using tap water once a day [5].
The preparation of conidial suspension in concentration 1x10 7 spore/ml of Trichoderma isolate was completed following the method by Nur Ain Izzati and Abdullah [6]. Conidia of seven days-old Trichoderma isolate were harvested. The mycelia debris was removed by filtration using sterile doublelayered muslin cloth. The conidial suspension was watered on the soil 500 ml/polybag for every two weeks after acclimatization. The inoculation of Foc isolate 9888 was introduced in week 3. The preparation of conidial suspension procedure was done similar to that mentioned above; nevertheless, the concentration was adjusted to 1x10 6 conidia/ml [7].
After seven days of inoculation, the plants' conditions were recorded and scored for the discoloration of the leaves and height of the plant. The scoring was conducted every week until week 20 using disease scale from 0 -4 following the scoring scheme proposed by [9] with slight modifications for visible symptoms recorded.

Data analysis
Data of PIRG and disease severity index (DSI) of plants under plant house condition were analysed using IBM SPSS Statistics version 21.

Results and Discussion
Trichoderma asperellum isolate B01 inhibited the growth of the pathogen with the PIRG at 84.85%. Dual culture test is one of the conventionally quantitative methods in screening the efficacy of Trichoderma species to be used as biocontrol agent in managing plant diseases. This in vitro test can display complete antagonistic potential of agent [10] as it is mainly a predictive tool to determine growth inhibition capability before carrying out time-consuming and more expensive study [11]. Prolonged incubation, the mycelia of all Trichoderma isolates were fully overgrown on the pathogen colony and sporulated on the pathogen colony for space to live.
For in-vivo study on Trichoderma isolates against Foc, after 20 weeks of pathogen inoculation, the symptoms of Fusarium wilt disease were observed and recorded. Figure 1 shows the symptoms of Foc experienced by basal parts of banana plants treated with T. asperellum isolate B01 on week 20 of postinoculation. The suppression of T. asperellum against Foc isolate 9888 for this plant house condition was employed by direct mechanism as both T. asperellum and Foc as pathogens were directly applied on soil. Studies showed that many Trichoderma strains are very efficient in the biocontrol of several pathogens or in plant growth promotion via rhizosphere colonization [12] or in providing nutrients to the plant. Fusarium oxysporum f.sp. cubense is known as one of invasive and lethal diseases of banana that become a major problem for the present banana crop plantation and industry [13]. Even though there are many holistic approaches that have been completed to overcome the threat of banana wilt disease [14], the circumstance did not show much firm promising results in treating diseases. Due to this condition, the local farmers decided to use extreme solution by using synthetic chemical pesticides or synthetic fungicides as a main tool to control the plant disease [11]. From this study, T. asperellum can be used as promising biological agent substituting chemical reagent in controlling plant disease. Using Trichoderma as biocontrol for plant disease is a good approach to start in agricultural field. However, to substitute Trichoderma as bio fungicide in controlling plant disease in plantation needs lots of budget for formulating their spores. For future study, new method must be focusing on optimize the production of Trichoderma spores that can be used for agricultural purposes.

Conclusions
As a conclusion, T. asperellum isolate B01 in this study has showed its effectiveness and ability in inhibiting Fusarium wilt pathogen, Foc under in vitro and in vivo conditions.