Biological Control of Chili Anthracnose Disease using Talaromyces flavus Bodhi001

Plant-pathogenic fungi species of Colletotrichum that cause anthracnose disease can limit both the quality and quantity of harvest yield losses on numerous tropical crops worldwide, such as bananas, (Zakaria et al., 2009), tomatoes (Diao et al., 2014), mangoes (Lu et al., 2017; Li et al., 2020) and chili peppers (Oo and Oh, 2019). In tropical and subtropical regions, including Thailand and Asia, chili (Capsicum annuum L.), a member of the Solanaceae family, is a vital crop (Ratanacherdchai et al., 2007; Than et al., 2008). During the growth phase of chili production, numerous plant diseases, including fungi, bacteria, viruses and nematodes, attack the crop. Anthracnose, which is caused by a fungus pathogen, is the most serious disease threat to chili production (Sharma et al., 2005). The chili anthracnose disease in Thailand is mainly caused by Colletotrichum gloeosporiodes (Penz.) Sacc., Colletotrichum capsici (H.Syd.) E. Butl. and Bisby and Colletotrichum acutatum, which can cause severe losses at various stages of chili production, ranging from blossom stage to postharvest, causing 10-80% yield loss globally (Poonpolgul and Kumphai, 2007; Than et al., 2008; Kommula et al., 2017). Tiny black spots are commonly observed on the leaves, stems and both young and mature fruits as disease symptoms (Kim et al., 1999). In general, the disease outbreak occurs during the rainy season or a long rainy period and it has reduced chili yields at both the young and mature fruit stages during field trials. In addition, farmers primarily apply synthetic fungicides as curative and preventative measures against chili anthracnose. Despite the effectiveness of synthetic fungicides against the anthracnose pathogen, their repeated and ongoing use raises concerns not only for their impact on human health, consumers and the environment, but also for the pathogen resistance that may result (Saxena et al., 2016; Hawkins and Fraaije, 2018; Kongcharoen et al., 2020). Consequently, the use of biological control agents (BCAs) and plant extracts is safe and eco-friendly methods of controlling plant pathogens have replaced the use of systemic fungicides (Jantasorn et al., 2016a; Komhorm et al., 2021).
       
Currently, biological management strategies for plant disease control are highly recommended for disease management. Several BCAs and plant extracts have been developed as commercial biocontrol agent products to manage plant diseases, including Trichoderma spp., which are well-known as effective biocontrol agents for many economic crop diseases (Swain et al., 2018) and  T. asperellum, which exhibits strong potential to control rice diseases (Charoenrak and Chamswang, 2015). However, Trichoderma harzianum has been used to control Colletotrichum capsici, the fungus that causes chili pepper anthracnose (Ekefan et al., 2009). Interestingly, Imtiaj and Lee (2007) reported that Cordyceps sobolifera, an entomopathogenic fungus, exhibits potent antagonistic activity against C. gloeosporiodes and C. miyabeanus. Similarly, Ophiocordyceps sobolifera is a potent antagonist against anthracnose disease caused by Colletotrichum spp. (Jaihan et al., 2016). In addition, Talaromyces species have demonstrated potential antagonistic activity against plant pathogens in numerous crops via mycoparasitism, antibiosis and space and nutrient competition. Talaromyces spp. have recently been identified as a potential antagonist against crop diseases worldwide, such as rice disease (Dethoup et al., 2018), vascular wilt diseases in potatoes and tomatoes (Naraghi et al., 2012; Bahramian et al., 2016) and mango fruit rot (Suasa-ard et al., 2019). In our previous study, we investigated biological approaches to plant disease control (Jantasorn et al., 2016b; Jantasorn et al., 2016c; Jantasorn et al., 2017) and found that Talaromyces flavus Bodhi001 suppressed the development of black spot disease and reduced disease incidence by up to 32.56% under greenhouse conditions (Komhorm et al., 2021). Therefore, the objectives of this study were as follows: (a) to evaluate the antagonistic activity of Talaromyces flavus (Klöcker) Stolk and Samson Bodhi001, Talaromyces trachyspermus (Shear) Stolk and Samson Bodhi002, Talaromyces flavus (Klöcker) Stolk and Samson Bodhi003 and Neosartorya fischeri (Wehmer) Malloch and Cain Bodhi004 against chili anthracnose disease in vitro and (b) to test their potential use against Colletotrichum capsici in vivo.

Isolation of antagonistic fungi
 
The antagonistic fungi were isolated from soils collected from a riparian forest at College of Creative Agriculture for Society, Srinakharinwirot University Sakaeo Campus, Thailand, using the serial dilution and spread plate method on warm glucose ammonium nitrate agar containing 0.05% streptomycin sulfate, as described previously by Jantasorn et al., (2016b). The medium-grown mycelium was subcultured on potato dextrose agar (PDA), incubated at room temperature (28±3ºC) and maintained on slant PDA at 4ºC for further strain identification. The identification of antagonistic fungi was based on a molecular technique utilizing internal transcribed spacer (ITS) regions (Omid et al., 2017) and a morphology characterization technique.
 
Fungal pathogen
 
Colletotrichum capsici, the chili anthracnose pathogen examined in this study, was isolated from chili fruits exhibiting anthracnose symptoms as black spots using the tissue transplanting technique described by Yadav et al., (2021). After 7 days of the incubation period, the newly growing mycelium was subcultured on PDA medium at 28±3ºC. Under microscopic observation, the pathogen was identified based on conidial morphological and cultural characteristics. The chili fruits were tested for pathogenicity using Koch’s postulates prior to their use in this study.
 
Spore suspension preparations
 
Colletotrichum capsici and antagonistic fungi, T. flavus Bodhi001, T. trachyspermus Bodhi002, T. flavus Bodhi003 and N. fischeri Bodhi004, were cultured separately on PDA medium at 28±3ºC for 14 days. The spore suspensions were prepared in sterile water. Then, 10 mL of sterile water was poured onto a culture plate and a sterile loop was used to scrape the spores from the mycelium. The mycelial fragment of each fungus was extracted from the spore suspensions using three layers of sterile cheesecloth under aseptic conditions. The spore suspensions were diluted with sterile water and adjusted using a hemocytometer to achieve a final concentration of 10⁶ spores mL^-1.
 
Evaluation of antagonistic behavior
 
A dual culture method was used to determine the activity of antagonistic fungi against C. capsici on the PDA plate. C. capsici and antagonistic fungi were cultured on PDA medium for 7 days at 28±3ºC. Then, mycelial plugs of four antagonistic fungi and C. capsici with a diameter of 0.5 cm were created using a sterile cork borer obtained from 7-day-old active mycelium. The mycelial plugs of the four antagonistic fungi and C. capsici were placed 2 cm away from the edge of the 9 cm diameter plate on the opposite side and incubated at 28±3ºC for 14 days. The C. capsici-inoculated PDA medium served as a control and was placed on a separate plate. Five replications and four repetitions of the experiment were carried out. The percent inhibition of radial growth (%PIRG) was calculated using the following formula:
   
Where;
R1= The radial growth of C. capsici in the control treatment.
R2= The radial growth of C. capsici in the dual culture test.
 
The effectiveness of antagonistic fungi against  C. capsici on chili fruits, determined using a detached fruit assay
 
The mature fruits of the chili variety Jinda were purchased at a market in Bangkok, Thailand. Similar-sized and ripe chili fruits were selected, washed with tap water to remove dust and surface contaminations, rinsed with sterile distilled water and wiped with sterile tissue paper. The surface of chili fruits was disinfected by soaking them in 70% (v/v) ethanol for 2 min, then rinsing them three times with sterile distilled water and wiping them with sterile tissue paper before drying them in a flow cabinet. Chili fruits were wounded using sterile 200 mL tips (2 mm in diameter, one wound per chili fruit). Chili fruits were then soaked separately in a spore suspension of antagonistic T. flavus Bodhi001,  T. trachyspermus Bodhi002, T. flavus Bodhi003 and  N. fischeri Bodhi004 at 10⁶ spores mL^-1 for 5 min and placed in 15 × 2 × 25 cm (length × width ´× height) plastic boxes containing 10 fruits per box. The center of the treated chili fruits was inoculated with a mycelial plug of C. capsici. The control treatment consisted of chili fruits inoculated with a C. capsici mycelial plug and soaked in distilled water. The experiments were conducted with 40 replicates (40 chili fruits per treatment and one wound per fruit) and repeated three times. Regarding the boxes containing treated chili fruits, they were kept moist with wet paper for 5 days at room temperature. After 5 days of inoculation, the length of lesions on treated fruits was measured and recorded. Using the following formula, the severity of the disease was calculated as a percentage of the length of lesions on the treated fruits compared to the control treatment:
   
Where;
A= The average length of chili fruit lesions in the control treatment.
B= The average length of chili fruit lesions in the antagonistic fungi treatment.
 
Statistical analysis
 
The data in this experiment were analyzed using a one-way analysis of variance (ANOVA) and the means were compared using the least significant difference (LSD) with a 95% level of statistical significance (p<0.05). The statistical analysis was conducted with the aid of the statistical program 122 Statistix8 (Analytical Software, SXW, Tallahassee, FL, USA). At least three replications were utilized to calculate means and standard deviations.

Confirmation of antagonistic activity against the chili anthracnose disease
 
Four antagonistic fungi exhibited promising antagonistic activity against C. capsici. T. flavus Bodhi001 and N. fischeri Bodhi004 inhibited the mycelial growth of C. capsici by 68.99 and 70.76%, respectively, compared to the growth of   C. capsici alone in the control treatment (Fig 1 and 2). The biocontrol activities of T. flavus Bodhi001 and N. fischeri Bodhi004 included antibiosis production and nutrient and space competitions (Fig 2). T. trachyspermus Bodhi002 and T. flavus Bodhi003 inhibited the mycelial growth of the pathogen by 59.84 and 60.38%, respectively, in a dual culture test (Fig 1). During the confrontation between T. flavus Bodhi001 and C. capsici, antibiosis mechanisms with a distinct inhibition zone measuring between 0.6 and 0.7 cm in width were observed.
 

 

 
Effect of antagonistic activity on C. capsici severity, determined using a detached fruit assay
 
On chili fruits inoculated with 10⁶ spores mL^-1, the effect of antagonistic fungi T. flavus Bodhi001, T. trachyspermus Bodhi002, T. flavus Bodhi003 and N. fischeri Bodhi004 on controlling chili anthracnose in vivo was evaluated. Compared to the control treatment, chili fruit treated with a spore suspension of four antagonistic fungi suppressed anthracnose disease severity significantly. Chili fruits treated with T. flavus Bodhi001 demonstrated the greatest reduction in disease severity (0.98%) and anthracnose disease (80%) when compared to other antagonistic strains and the control treatment (Fig 3 and 4). However, fruits treated with  N. fischeri Bodhi004, T. trachyspermus Bodhi002 and T.  flavus Bodhi003 reduced the severity of anthracnose by 1.41, 1.46 and 1.53%, respectively (Fig 3). In addition, chili fruits treated with distilled water exhibited a pronounced anthracnose symptom (Fig 4).
 

 

       
Biological approaches to plant disease management, including biological control agents and plant extracts, have been isolated and evaluated (Poveda, 2021). The antagonistic microorganisms combat plant pathogens through multiple mechanisms, including parasitism, induction of host resistance, antibiosis and space and nutrient competition. This study revealed that T. flavus Bodhi001, T. trachyspermus Bodhi002, T.  flavus Bodhi003 and N. fischeri Bodhi004 inhibited the mycelial growth of   C. capsici in vitro by more than 50%. In vitro antibiosis produced by T. flavus Bodhi001 inhibited the mycelial growth of C. capsici significantly (Fig 2). Similar to previous reports, our findings showed that Talaromyces species exert antagonistic mechanisms against plant pathogens by producing antibiosis (Dethoup et al., 2018; Suasa-ard et.al., 2019; Komhorm et al., 2021). In a dual culture test, T. flavus Bodhi001 inhibited the mycelial growth of A. brassicicola by 64% and formed an inhibition zone between 0.8 and 0.9 cm wide. Under greenhouse conditions, the spore suspension containing 10⁶ spores mL^-1 of this antagonistic fungus inhibited the development of black spot disease in Chinese kale by up to 32.56% (Komhorm et al., 2021). Jantasorn et al., (2016c) also reported that the crude extract of T. flavus Bodhi001 completely inhibited the radial growth of Phytophthora palmivora, Pyricularia oryzae, Sclerotium rolfsii and Lasiodiplodia theobromae, including C. capsici and C. gloeosporioides, which cause chili anthracnose. The results of the current and previous studies indicate that  T. flavus Bodhi001 has effective antagonistic activity against phytopathogenic fungi that cause numerous economic crop diseases by producing antibiosis.
       
Treatment with T. flavus Bodhi001 inhibits the development of the anthracnose disease, as determined by the detached fruit assay. Chili fruits treated with this antagonistic fungus demonstrated a 0.98% reduction in lesion length and disease severity of C. capsici, as well as an 80% reduction in anthracnose disease (Fig 3 and 4). These results indicate that T. flavus Bodhi001 suppressed the severity of the disease more effectively than other antagonistic fungi examined in the current study. Our results also revealed that T. flavus Bodhi003 and T. trachyspermus Bodhi002 had the lowest antagonistic activity against C. capsici, the pathogen responsible for chili anthracnose disease, compared to other strains tested in vitro and in vivo in this study. In addition, it has been demonstrated that the antifungal effect of Talaromyces and Neosartorya species against plant pathogens is dependent on the conditions of the plant materials and experimental methods. For example, in vitro tests revealed that N. fischeri Bodhi004, T. trachyspermus Bodhi002 and T. flavus Bodhi003 effectively inhibited the mycelial growth of C. capsici. However, in vivo testing revealed that these three antagonistic fungi had a minimal effect on anthracnose disease severity. These findings imply that antagonistic strains and species isolated from different habitats can produce a variety of bioactive compounds (Suay et al., 2000). Our study confirmed the efficacy of the T. flavus Bodhi001 isolate against C. capsici, the causal agent of chili anthracnose, both in vitro and in vivo. In addition, there are few reports of Talaromyces species demonstrating a potent effect on preventing the development of chili anthracnose. Talaromyces species demonstrated significant antagonistic activity against plant diseases in numerous economic crops, including vascular wilt disease of potato and tomato (Naraghi et al., 2012; Bahramian et al., 2016), rice disease (Dethoup et al., 2018; Dethoup et al., 2022), Chinese kale black spot disease (Komhorm et al., 2021) and Lasiodiplodia theobromae, which causes mango fruit rot (Suasa-ard et al., 2019). Many other studies have reported the in vitro and in vivo effectiveness of antagonistic fungi, Trichoderma species, against chili anthracnose disease (Ruangwong et al., 2021; Yadav et al., 2021). According to Jaihan et al., (2016), the entomopathogenic fungi strain Ophiocordyceps sobolifera inhibited the mycelial growth and conidial formation of Colletotrichum spp. in vitro. Similarly, the mushroom culture filtrate, Clitocybe nuda (LA82), effectively reduced the severity of anthracnose disease (Chen and Huang, 2010). Suprapta (2022) found that the formulation of Paenibacillus polymyxa C1 effectively controlled the anthracnose disease under field conditions. 
       
Our results revealed that the antagonistic fungi T. flavus Bodhi001 isolated from riparian forest soils exhibited the greatest biocontrol effect in controlling chili anthracnose disease in vivo and significantly inhibited the mycelial growth of C. capsici in vitro. It has been demonstrated that their antagonistic mechanism prevents plant pathogen infection by producing antibiosis, thereby reducing the severity of chili anthracnose. However, there are no reports of human or environmental toxicity associated with the use of spore suspensions of Talaromyces species for the control of plant diseases. This study demonstrated that T. flavus Bodhi001 is a promising biocontrol agent for chili anthracnose disease caused by C. capsici and could be developed as an alternative to synthetic fungicides for disease management in organic and sustainable cropping systems.

The results of this study confirmed the biocontrol potential of T. flavus Bodhi001 against C. capsici, the pathogen responsible for chili anthracnose. By producing antibiosis in dual culture tests, this strain has demonstrated its high antagonistic activity. T. flavus Bodhi001 exhibited the most potent antagonistic activity against the chili anthracnose disease in vivo. These results indicate that T. flavus Bodhi001 has the potential to be developed as a biological control agent for chili anthracnose disease management. However, additional research is necessary to investigate the efficacy of T. flavus Bodhi001 under field conditions.

This research was financially supported by the Strategic Wisdom and Research Institute, Srinakharinwirot University, under the project “Application of antagonistic microorganisms isolated from riparian forest soils for biological control of Colletotrichum spp. causing anthracnose disease in chili (project no. 618/2564).”

The authors declare that they have no conflict of interest.