Trichoderma asperellum (NST-009): A Possible Thai Native Antagonistic Fungus for Managing White Root Disease of Rubber Trees (Hevea brasiliensis)

The rubber tree (Hevea brasiliensis) is one of Thailand’s most produced agricultural plants because it provides important material for a variety of applications including the automobile industry. The latex sap from the rubber tree is used for the production of insulating handles, tires, balls, balloons, and shock absorbers. More than 92% of the world’s natural rubber is produced on plantations in Southeast Asia, including Thailand, Indonesia, and Malaysia (Diaby et al., 2011; Rukkhun et al., 2021). However, the white root disease, the most destructive root disease in rubber tree, caused by Rigidoporus microporus (Fr.) Overeem is a major problem in rubber plantations causing significant economic damage. It is the most destructive root disease in rubber plantations in Sri Lanka, India, Indonesia, Malaysia, Thailand, and West and Central Africa (Jayasuriya and Thennakoon, 2007). In the absence of woody substrates, R. microporus forms several white, flattened mycelial strands that expand and spread rapidly through the soil (Kaewchai and Soytong, 2010). Since the rhizomorph and infection sites are below ground level, early stages of infection through contact with a disease source, such as contaminated roots, dead stumps, or wood debris of root pathogens, are difficult to detect. Trees that die frequently go unnoticed for a long time, with no noticeable aerial (above-ground) signs (Ogbebor et al., 2013). This will ultimately lead to the death of many trees, as well as the destruction of the entire stand. The disease persists for a long time in dead or alive root debris, causing new infections in healthy plants (Kaewchai and Soytong, 2010). Cultural methods and chemical fungicides such as tridemorph, cyproconazole, hexaconazole, propiconazole, and fenicolonil are commonly used to treat this disease. These chemical fungicides, on the other hand, are known to affect human health, pollute the atmosphere and leave residues in agricultural soils and environments. Chemical fungicide resistance has also been observed for several plant pathogenic fungi. Furthermore, chemical fungicides are expensive. As a result, biological regulation is a viable option for reducing costs, environmental issues and health risks.
       
Trichoderma strains have been shown to be effective biological control agents against Alternaria, Botrytis, Bipolaris, Cercospora, Colletotrichum, Corynespora, Curvularia, Phomopsis, Pythium, Phytophthora, Ganoderma, Rhizoctonia, Fusarium, Sclerotium, Sclerotinia and Rosellinia. (Abo-Elyousr et al., 2014; Athul and Jisha, 2014; Fitrianingsih et al., 2019; Izzati and Abdullah, 2008; Nawrocka et al., 2018; Redda et al., 2018; Wonglom et al., 2019). T. asperellum NST-009 is a native fungus isolated from forest soil in Nakhon Si Thammarat, Thailand. This strain has been shown to be effective in controlling Phytophthora leaf fall in a rubber tree (Promwee et al., 2017). It is currently being recommended to farmers for plant disease control and growth promotion in a variety of plants, including rice, durian, oil palm and vegetables, by the Agricultural Microbial Production and Service Center at Walailak University and is being distributed to farmers throughout Thai provinces. There have been no records of native T. asperellum NST-009 used to manage rubber-tree white root disease. Hence, the objectives of this study were: 1) to determine the efficacy of T. asperellum NST-009 and its antifungal metabolite in inhibiting R. microporus mycelial development and 2) to determine the efficacy of T. asperellum NST-009 in controlling white root disease of rubber trees in an open-field house experiment.

Trichoderma strains and the Rigidoporus pathogen
 
Four native strains of T. asperellum (known as T. harzianum) from Nakhon Si Thammarat (T. asperellum NST-003, NST-009, NST-028 and NST-353) and the commercial strain of Thailand (T. asperellum CB-Pin-01) were used in this study (Promwee et al., 2017; Charoenrak et al., 2019; Unartngam et al., 2020). The pathogen (R. microporus Tha-01) was collected from the Walailak University’s Agricultural Microbial Production and Service Center. The Trichoderma strains and the Rigidoporus pathogen were subcultured on potato dextrose agar (PDA) for 5 days at room temperature (28±2°C). Koch’s postulates were used to confirm the symptoms of white root disease caused by R. microporus, and the morphological and reproductive characteristics of R. microporus were examined using a light compound microscope and a scanning electron microscope (SEM) (Kaewchai et al., 2010). This study was conducted at Agricultural Microbial Production and Service Center, Walailak University, Nakhon Si Thammarat, Thailand, during the period 2017-2020.
 
Dual culture test
 
All strains of T. asperellum were tested for their ability to inhibit the mycelial growth of R. microporus. A sterile cork borer (3 mm diameter) was used to cut a five-day-old T. asperellum on PDA and the agar plug of T. asperellum was mounted on one side of the PDA Petri dish (9 cm diameter). Then, on opposite sides of the PDA Petri dish, a 3-mm-diameter plug of 5-day-old R. microporus was mounted, and the plates were incubated for 5 days at 28±2°C. Using a completely randomized design (CRD) of four replications, the experiment was performed and replicated twice.
       
The per cent inhibition of mycelial growth was determined using formula (1):
 
Where,
RC: Represents R. microporus radial growth in the untreated regulation.
RT:  Represents R. microporus radial growth during the procedure.
 
Scanning electron microscopy analysis
 
Using a SEM (JEOL, JSM5600LV, England), the high-efficiency strain of T. asperellum, which was able to inhibit the mycelia of R. microporus in the dual culture test, was studied for its ability to parasitize the mycelia of R. microporus. T. asperellum and R. microporus were grown on PDA in a dual culture test. After a colony of R. microporus was targeted by T. asperellum mycelia, the activity zone samples were cut into small pieces (0.5×0.5 cm), fixed in 2.5% glutaraldehyde for 24 h at 4°C, rinsed with distilled water, and dehydrated in a 30-100% alcohol sequence. The samples were dried in a critical point dryer before being coated with gold using a sputter coater (Nanotech, Sempreps, England). SEM was used to analyse the coated samples right away.
 
Antifungal metabolite determination
 
Twenty-five mycelial agar plugs (7 mm diameter) obtained from the margins of developing colonies of T. asperellum grown on PDA were inoculated into a 3 L Erlenmeyer flask containing 1 L of 1/5 strength potato dextrose broth (PDB). The flask was then incubated for 28 d at 28±2°C. The spores and mycelia of T. asperellum were then separated from the broth culture using 0.45 m Whatman No.1 filtration. Ethyl acetate was used to extract the culture filtrates before evaporation at 40°C using a rotary vacuum evaporator (EYELA, Japan). The agar dilution method was used to assess antifungal metabolites (crude extracted substances) for their ability to inhibit the mycelial growth of R. microporus on PDA. Each antifungal metabolite was dissolved in 2% DMSO, combined with PDA to a final concentration of 500 mg/l and poured into a Petri dish. The R. microporus mycelial agar disc was then placed in the center of a solidified agar plate and incubated for 5 days at 28±2°C. The experiment was repeated twice with four replications using a CRD. R. microporus colony diameter was measured and the inhibition percentage of mycelial growth was calculated using formula (2):
 
Where
DC: Represents the mean mycelial diameter of R. microporus in the control treatment.
DT:  Represents the mean mycelial diameter of R. microporus in the tested treatment.
 
Disease control under open-field house experiment
 
Preparation of the R. microporus inoculum
 
R. microporus was subcultured on PDA for five days at 28±2°C and R. microporus inoculums were prepared using the modified technique described by Kaewchai and Soytong (2010). Ten R. microporus mycelial plugs were cultured in sterilized inoculum medium (100 g sawdust, 3 g rice bran, 2 g glucose and 100 mL distilled water) in each plastic bag for 30 days at 28±2°C.
 
T. asperellum fresh culture preparation
 
T. asperellum strains were subcultured on PDA for five days at 28±2°C. A simple technique was used to prepare fresh T. asperellum cultures. An electric rice cooker was used to cook rice and water (3:2 by volume). The hot cooked rice was placed in clear plastic bags (250 g/bag) and allowed to cool slightly above room temperature. In a plastic bag, ten mycelial plugs (0.8 cm diameter) of the T. asperellum colony growing on a PDA dish were inoculated and mixed with the cooked rice. Each bag’s open end was secured with a rubber band and a needle was used to puncture the attached area (25 holes/bag). Before use, all bags were incubated at 28±2°C for seven days under fluorescent light (12 h/day) (Charoenrak et al., 2019).
 
Disease severity assays
 
Fresh T. asperellum culture was added to the planting medium (10 kg of soil from a rubber plantation in Nakhon Si Thammarat mixed with 2 kg of cow manure) at 100 g per pot and incubated for seven days. The 6-month-old rubber tree cultivar RRIM 600 was then planted in a 15-inch-diameter plastic container and a bag of R. microporus inoculums was placed in a planting pot next to root system of the rubber tree. In this analysis, CRD with four replications per treatment and three plants per replication was compared to fungicide (cyproconazole 10% w/v), control 1 (with only pathogen) and control 2 (without pathogen). Six months after inoculation with R. microporus, the disease was observed on the rubber tree. The experiment was repeated twice.
       
The severity of the disease was divided into six levels (0-5): level 0 = stable, green leaves; level 1=1-25% yellow leaves; level 2=26-50% yellow leaves; level 3=51-75% yellow leaves; level 4=76-100% yellow leaves and level 5= dead tree (Kaewchai and Soytong, 2010). The disease severity index (DSI) was calculated using the formula (3):
   
Rubber root infected and covered by mycelia of R. microporus
 
Root infection and coverage by R. microporus mycelia were estimated six months after the plants were inoculated with R. microporus inocula and classified into five levels (0-4) as follows: level 0= no infection and colonization, level 1=1-25% of infection and colonization, level 2=26-50% of infection and colonization, level 3=51-75% of infection and colonization and level 4=76-100% of infection and colonization. The R. microporus root infected and covered by the mycelia index (RICI) was calculated using formula (4):
   
Root colonization of T. asperellum
 
Root colonization of T. asperellum was studied using Martin’s medium six months after inoculation with R. microporus. The rubber root was cut into ten pieces and soaked in 0.525 per cent sodium hypochlorite (Clorox®) for 5 min. The rubber root was then washed three times with sterilized distilled water. Five rubber root pieces were dried with sterilized paper and placed in a Petri dish on Martin’s medium. The dishes were sealed with paraffin film and incubated for four days at 28±2°C. The percentage of root colonization was then calculated. This experiment used a CRD with four replications and five rubber root per replication.
 
Population of T. asperellum in rhizosphere soil
 
The population of T. asperellum in rhizosphere soil was studied at six months after inoculation with R. microporus using the dilution plate technique and Martin’s medium. In a 250 mL flask containing 90 mL of sterile water, ten g of each rhizosphere soil was added and mixed for 30 min with shaking at 120 rpm. The soil suspension was then diluted 10^-2-10^-4 times before being placed on the surface of Martin’s medium in a Petri dish with 0.1 mL of the dilution. Before the plates were coated with paraffin film and incubated at 28±2°C for 4 days, the soil suspension was spread on the surface of the medium with a sterile glass rod. The growth of T. asperellum on agar was then estimated. This experiment used a CRD with four replications.
 
Statistical analysis
 
An analysis of variance (ANOVA) was performed on the results, followed by a comparison using Duncan’s multiple range test (P<0.05).

Effect of T. asperellum on growth of R. microporus
 
In a dual culture experiment, all strains of T. asperellum effectively inhibited R. microporus mycelia on PDA (70.82-77.07%), with T. asperellum NST-009 providing the highest percentage of mycelial growth inhibition (Table 1, Fig 1).
 

 

 
Parasitism of T. asperellum against R. microporus
 
Under SEM, the ability of T. asperellum to induce mycoparasitism on the mycelia of R. microporus revealed that the selected strain of T. asperellum NST-009 was able to parasitize the mycelia of R. microporus via Rigidoporus hypha colonization, Rigidoporus hypha drilling holes and conidia reproduction on Rigidoporus (Fig 2).
 

 
Effect of crude metabolites on R. microporus growth
 
The antifungal metabolites of all T. asperellum strains at 500 µg ml^-1 effectively inhibited R. microporus mycelial growth (71.15-92.31%), especially antifungal metabolites of T. asperellum strain NST-009, which showed the highest percentage of mycelial growth inhibition (Table 2 and Fig 3).
 

 

 
T. asperellum controls white root rot disease in open field conditions
 
Disease severity
 
The effectiveness of T. asperellum in controlling white root disease in rubber trees was investigated under open-field conditions. The results showed that 180 days after the rubber trees were inoculated with R. microporus inoculum, all T. asperellum strains had a high efficacy in controlling white root disease with a low disease severity index (0.00–19.44%), especially the T. asperellum strains NST-028, NST-009 and CB-Pin-01 (commercial strain), which had a disease severity index similar to that of the fungicide (cyproconazol). The control 1 treatment, which was only inoculated with R. microporus, had the highest disease severity index (76.38%) (Table 3).
 

 
Rubber root infected and covered by mycelia of Rigidoporus microporus
 
Rubber roots infected and covered with mycelia of R. microporus were studied 180 days after inoculation with the inoculum of R. microporus. The results showed that treatment with T. asperellum caused significant root infection and was covered by mycelia of R. microporus index (RICI) as compared with the control 1 (with only pathogen). In particular, the treatments with T. asperellum strain NST-009, NST-028 and CB-Pin-01 provided the lowest RICI (0.00%), while control 1 had the highest RICI (64.58%) (Table 3).
 
Trichoderma root colonization
 
Root colonization of T. asperellum was determined after application of T. asperellum for 180 days. Treatment with T. asperellum strains NST-028 and NST-009 resulted in the highest rate of root colonization (100.00%), followed by NST-353 (95.00%), CB-Pin-01 (85.00%) and NST-003 (75.00%), which showed significant differences compared to control 1, control 2 and cyproconazole treatments, which did not find Trichoderma strains colonize the roots (Table 3).
 
Trichoderma population
 
The population of Trichoderma strains in the planting medium was studied using the dilution plate technique with Martin’s medium. The results showed that the treatments with Trichoderma strain provided the population at 1.25×10⁶-3.25×10⁶, 4.00×10⁵-12.75×10⁵ and 2.00-4.50×10⁵ CFU/g of the planting medium at 60, 120 and 180 days, respectively, after inoculation with a fresh culture of T. asperellum. On the other hand, treatments with fungicide (cyproconazole), control 1 (with the pathogen) and control 2 (without pathogen) were not found in the population of Trichoderma strains in the planting medium (Table 4).
 

       
In this study, T. asperellum NST-009 displayed strong antifungal activity against R. microporus in a dual culture assay. The dual culture assay revealed competition and mycoparasitism of T. asperellum. Crude metabolites of T. asperellum NST-009 showed fungicidal activity against R. microporus, revealing antibiosis. Application of fresh T. asperellum NST-009 reduced the disease severity index caused by R. microporus under open-field conditions and colonization of the rubber tree rhizosphere.
       
Trichoderma species are known as potent biocontrol agents against several plant diseases because of their capacity to compete for nutrients and space and emit volatile antifungal compounds against plant pathogens (Gangwar and Singh, 2018; Baiyee et al., 2019b; Wonglom et al., 2020). Based on the results of this study, T. asperellum NST-009 competed for nutrients and space when co-cultured with R. microporus. These results support the findings of Jayasuriya and Thennakoon (2007) and Kaewchai and Soytong (2010) that Trichoderma strains had a competitive ability to inhibit mycelial growth of R. microporus. Moreover, Trichoderma strains can inhibit the mycelial growth of several plant pathogens, such as Alternaria alternata, Botrytis cinerea, Corynespora cassiicola, Curvularia oryzae, Fusarium solani, F. oxysporum, Pythium aphanidermatum, Rhizoctonia solani, Rosellinia necatrix and Sclerotium rolfsii (Intana et al., 2003; Rosa and Herrera, 2009; Nallathambi et al., 2009; Sunpapao et al., 2018; Baiyee et al., 2019b, Wonglom et al., 2019; Wonglom et al., 2020). This dual culture test suggested that the two mechanisms of T. asperellum were competition for nutrients and space, as well as mycoparasitism. Trichoderma spp. can produce antifungal metabolites that restrict fungal growth. The results of this study demonstrated that crude metabolites of T. asperellum NST-009 inhibited the fungal growth of R. microporus.

The Trichoderma asperellum strain NST-009 is native to southern Thailand. T. asperellum NST-009 effectively inhibited R. microporus mycelial growth more than the Thai commercial strain T. asperellum CB-Pin-01 in both dual culture and crude extract tests. T. asperellum NST-009 exhibited mycoparasitism under a scanning electron microscope. T. asperellum NST-009 exhibited disease severity indexes comparable to those of cyproconazole fungicide in an open-field house experiment. Furthermore, T. asperellum NST-009 showed root colonization that was protective against R. microporus infection of rubber trees and could survive in soil.

We thank Assoc. Prof. Dr. Anurag Sunpapao from the Agricultural Innovation and Management Division, Faculty of Natural Resources, Price of Songkla University, Songkhla, and Assist. Prof. Gerd Katzenmeier from School of Allied Health Sciences, Walailak University, Nakhon Si Thammarat, Thailand for his valuable help in improving the manuscript for this paper. This work was supported by the Office of the Higher Education Commission, Thailand Fund, Grant No. 10/2553 and the Walailak University Fund, Thailand, Grant No. WU62259.