Antifungal Potential of Biocontrol Agents Against Phytophthora Capsici Causing Chili Fruit Rot
Muhammad Zohaib Anjum1,2*, Muhammad Adnan3, Syed Mukarram Ali4, Hafiz Muhammad Bilal5 and Hamza Javaid5
1Department of Plant Pathology, University of Sargodha, Pakistan
2Department of Plant and Environmental Sciences, University of Copenhagen, Denmark
3Department of Agronomy, University of Sargodha, Pakistan
4Department of Mathematics, University of Sargodha, Pakistan
5Department of Horticulture, University of Sargodha, Pakistan
Submission:July 29, 2019; Published: September 20, 2019
*Corresponding author: Muhammad Zohaib Anjum, Department of Plant Pathology, University of Sargodha, Pakistan
How to cite this article: Muhammad Zohaib Anjum, Muhammad Adnan, Syed Mukarram Ali, Hafiz Muhammad Bilal, Hamza Javaid. Antifungal Potential of Biocontrol Agents Against Phytophthora Capsici Causing Chili Fruit Rot. Depending on Different Managements.Agri Res& Tech: Open Access J. 2019; 22(4): 556209. DOI: 10.19080/ARTOAJ.2019.22.556209
Abstract
Biocontrol is an environmentally friendly and proficient way to manage the plant diseases which leads to comes true the dream of organic farming. In present study different antagonistic assays (dual culture, volatile and nonvolatile metabolite) were used to investigate the antifungal activity of three already molecular characterized isolates of Trichoderma viz, Trichoderma asperellum TH, Trichoderma harzianum HM, Trichoderma harzianum HK and two morphologically characterized isolates of Bacillus subtilis against Phytophthora capsici (Leonian) a threating pathogen of fruit rot of chili. Results showed that all the antagonist inhibited the radial growth of tested pathogen. In dual culture assay, T. asperellum showed maximum (61.6%) mycelial growth inhibition followed by Bacillus subtilis A (54.3%), T. harzianum HK (51.4%), T. harzianum HM (47.2%) and Bacillus subtilis B (41.5%). Culture filtrate (Extracted metabolites/ nonvolatile metabolites) were proved as more efficient inhibitor of pathogen as compared to volatile metabolites. Nonvolatile metabolites of T. asperellum TH showed maximum inhibition (44.5%) and minimum inhibition showed by Bacillus subtilis B (29.1%) while volatile metabolites of T. asperellum TH showed maximum inhibition (28.3%) and Bacillus subtilis B (11.5%) gave minimum inhibition as compared to other tested biocontrol agents against fruit rot pathogen. Based on results, it is concluded that biocontrol agents have great potential to manage the P. capsici in a better way.
Keywords:Chili; Fruit rot; Phytophthora capsica; Bacillus; Trichoderma; Antagonist
Introduction
Chili (Capsicum annuam) is one of the most growing crops in the world. Chili fruit is highly infected by Phytophthora capsici causing fruit rot. It was first described in 1922 by Leonian at Mexico on chili crop [1]. Phytophthora capsici, an oomycete, is a challenging pathogen at chili producing areas of the world. C. annuam & P. capsici have very complex pathosystem as compare to other Phytophthora pathosystems because this pathogen infects all the plant parts [2]. This pathogen causes not only fruit rot but also transmitted by seeds [3]. It has vast host rang and cause diseases also on pumpkin, melons, tomato, eggplant, lima beans and squash. High moisture is an important factor for disease development and pathogen reproduction. Use of synthetic fungicides known as best management strategy for this disease but continuous application of fungicides leads to resistance in pathogens and human health hazards [4]. Large scale adoption of organic farming has boosted up chili production in an eco-friendly way Insensitivity of P. capsici against metalaxyl and mefenoxam (Ridomil gold) is reported in Italy and United states [5]. So, there is a need of best alternative management practices that can minimize the disease development with no or less environmental and human health hazards. In this regard, currently usage of biological control agents stands first to suppress the induction of pathogen. Bacillus and Trichoderma species are extensively used as biological control agent against Phytophthora species [6]. Against fungal plant pathogens, Trichoderma is known as aggressive biological control agent [7]. It has different complex interaction mechanisms like antibiosis, mycoparasitism and competition to antagonist the plant pathogens [8]. Many species of Trichoderma produce volatile and non-volatile metabolites like massoilactone, viridine, furanone, trichodermine, peptaibols, harianic acid, gliovirin, heptelidic acid which are noxious to pathogens [9]. Use of Trichoderma also induces systemic resistance in some plants belonging to Poaceae, Solanaceae and cucurbitaceae family against some plant pathogens [10]. Two species of Bacillus reduced the mycelial growth of P. capsici [11]. Present study was designed to evaluate the antifungal ability of fungal and bacterial biocontrol agents against fruit rot disease of chili.
Materials and Methods
Diseased chili fruit samples were collected from chili growing area of district Faisalabad, Punjab, Pakistan. Isolation was performed by using tissue segmented method on PARP medium and identification was done as described by [12]. Three molecular characterized isolates of Trichoderma namely Trichoderma asperellum TH, Trichoderma harzianum HM, Trichoderma harzianum HK were taken from Plant Pathology laboratory, College of Agriculture, University of Sargodha. For Bacillus isolation, soil samples from chili field were collected and isolation was done by using serial dilution technique. About 50μl solution was spread on PDA plates and incubated at 25+1oC for 36 hours [13]. Identification was performed based on morphological and cultural characters as described by [14].
Antagonistic Activity
Dual culture
For Trichoderma isolates:
Five days old, 6mm mycelial plug of pathogen and antagonist were placed opposite to each other aseptically on petri plates which contains sterilized PDA medium and sealed with parafilm.
For Bacillus isolates:
Five days old, 6mm mycelial plug of pathogen was placed at one side of sterilized PDA plate, then a loop full of two days old culture of Bacillus was streaked out on the opposite side of mycelial plug of pathogen and sealed with parafilm and incubated at 24+1oC. Petri plate containing only pathogen was served as control.
Metabolites
Volatile metabolites
To evaluate the antifungal activity of volatiles metabolites of Trichoderma against Phytophthora capsici, technique described by [15] was used. Five days old, 6mm mycelial plugs of Trichoderma isolates and P. capsici were placed at center of PDA petri plates aseptically, plates containing P. capsici were placed over on Trichoderma containing plates for three days. All petri plates were sealed with parafilm and incubated at 25+1oC. Same procedure was used for volatile metabolites of Bacillus isolates.
Culture filtrate / Nonvolatile metabolites
For Bacillus
To evaluate the antifungal activity of culture filtrates of two Bacillus isolates against P. capsici, technique described by (Abdulkareem et al., 2014) was used. A 6mm plug of Bacillus isolates were inoculated into 100ml nutrient broth agar and shaked at 150rmp for 48 hours at room temperature. The broth culture was filtered by Whatman No.1 filter paper and centrifuged at 6000rmp for 12min. To obtain cell free culture, liquid was refiltered by Millipore membrane filter (0.2μ). Twenty ml cell free culture was mixed in eighty ml sterilized PDA media by using food poison technique. After the solidification of media, 6mm mycelial plugs of P. capsici were placed at center of PDA petri plates aseptically. PDA petri plates without culture filtrate were served as control. All petri plates were sealed with parafilm and incubated at 25+1oC.
For Trichoderma
To evaluate the antifungal activity of Extract of metabolites (culture filtrates) of three Trichoderma isolates against P. capsici, technique described by [16] was used with minute modification. Five days old, sex mycelial plugs of Trichoderma isolates were inoculated in 100ml liquid potato dextrose broth (PDB) and shaked at 200rpm for 70 hours at room temperature. Then broth culture was centrifuged at 8000rpm for 25min. To obtaining cell free culture, broth culture was first filtered by filter paper (Whatman No. 1) and then passed through Millipore membrane (0.34μm). Twenty ml cell free culture was mixed in eighty ml sterilized PDA media by using food poison technique. After the solidification of media, 6mm mycelial plugs of P. capsici were placed at center of PDA petri plates aseptically. PDA petri plates mixed with distilled water instead of culture filtrate were served as control. All petri plates were sealed with parafilm and incubated at 25+1oC.
Statistical analysis
The data was analyzed by using factorial test on Statistics software for the interpretations of results. Difference between means were calculated by using LSD test. Percentage inhibition was determined by using following formula (Vincent, 1927). Inhibition percentage (%) = C-T/C × 100
Where C = growth of pathogen in control plate, and T = mycelial growth of pathogen in test plate.
Results
Dual culture
In dual culture technique, treatment (F8, 170.75=149.30) effect was significant. T. asperellum TH showed highest inhibition (61.65%) among all other tested treatments. Bacillus A showed 54.36% inhibition followed by T. harzianum HK (51.43%), T. harzianum HM (47.26%) and Bacillus B (41.56%).
Metabolites
Volatile metabolites
In volatile metabolites, treatment (F4, 121.16=207.45) effect was significant. Volatile metabolites of T. asperellum TH showed maximum inhibition (28.34%) against tested pathogen followed by T. harzianum HK (22.89%), T. harzianum HM (19.72%), Bacillus A (16.56%) and Bacillus B (11.51%).
Culture filtrate/ extracted metabolites (Nonvolatile)
In nonvolatile metabolites treatment (F4,109.009= 445.7) effect was significant. Nonvolatile metabolites of T. asperellum TH showed maximum inhibition (44.58%) among all tested other treatments followed by Bacillus A (38.75%), T. harzianum HK (36.58%), T. harzianum HM (31.85%) and Bacillus B (29.16%) (Table 1).
Discussion
Management of phytopathogens through antagonistic biocontrol agents is more efficient, long lasting and nonchemical approach that is widely adopted in the world. Present study assessed the antifungal ability of different biocontrol agents by dual culture, volatile and nonvolatile metabolites. T. asperellum TH showed highest inhibition among all other tested antagonist in different assays. Bacillus isolates gave less inhibition percentage of radial growth of tested pathogen as compared to Trichoderma isolates. This may be due to microbial interaction like compatibility, antibiosis and stimulation levels of Trichoderma and Bacillus against P. capsici [17,18]. Antifungal activity of Bacillus against soil born phytopathogens was also reported [19]. Previously similar results about the antagonism of Trichoderma isolates against P. capsici was reported [20]. Lelay et al. (2007) reported the antifungal ability of six Trichoderma isolates that inhibited the mycelial growth of Rosellinia necatrix about 14 to 27%, it was accredited to the production of metabolites like viridine, glyotoxins, furanone, trichodermine, and 6-pentyl-α-pyrone. In present study, Bacillus isolates showed the inhibitory effect against tested pathogen as compared to control treatment [21]. Same isolates of Trichoderma and Bacilllus species which were used in present study, inhibited the growth of Phytophthora drechsleri under laboratory and field conditions [15], so our results also confirmed the antifungal ability of these biocontrol agents against P. capsici.
Conclusion
According to present study it is concluded Bacillus and Trichoderma isolates have great potential to manage the growth of phytopathogens. We can use them to obtain organic crops and manage the plant diseases safely as compared to synthetic chemicals that are harmful to human health and environment. Chemicals gave short duration control of any plant disease while bio control agents gave long lasting control of diseases. It is concluded that Trichoderma and Bacillus isolates and their metabolites have great potential to manage the Phytophthora capsici causal agent of fruit rot of chili with less or no residual effects.
References
- Leonian LH (1922) Stem and fruit blight of peppers caused by Phytophthora capsici sp nov. Phytopathology 12: 401-408.
- Ristaino JB, Parra G, Campbell CL (1997) Suppression of Phytophthora blight in bell pepper by a no-till wheat cover crop. Phytopathology 87(3): 242-249.
- Morales VG, Redondo JE, Covarrubias PJ, Cárdenas SE (2002) Detección y localización de Phytophthora capsici en semilla de chile. Revista Mexicana de Fitopatología 20: 94-97.
- Lamour KH, Hausbeck MK (2000) Mefenoxam insensitivity and the sexual stage of Phytophthora capsici in Michigan cucurbit fields. Phytopathology 90(4): 396-400.
- Parra G, Ristaino JB (2001) Resistance to mefenoxam and metalaxyl among field isolates of Phytophthora capsici causing Phytophthora blight of bell pepper. Plant Disease 85(10): 1069-1075.
- Wang HK, Xiao RF (2013) Antifungal Activity of Bacillus coagulans TQ33, Isolated from Skimmed Milk Powder, against Botrytis cinerea. Food Technology and Biotechnology 51(1): 78-83.
- Keswani C, Mishra S, Sarma BK, Singh SP, Singh HB ( ) Unraveling the efficient applications of secondary metabolites of various Trichoderma Applied Microbiology and Biotechnology 98(2): 533-544.
- Pal KK, Gardener BM (2006) Biological control of plant pathogens. The Plant Health Instructor 2: 1117-1142.
- Qualhato TF, Lopes FAC, Steindorff AS, Brandao RS, Jesuino RSA, et al. (2013) Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production. Biotechnology letters 35(9): 1461-1468.
- Tondje PR, Roberts DP, Bon MC, Widmer T, Samuels GJ, Ismaiel A, Begoude AD, Tchana T, Nyemb-Tshomb E, Ndoumbe-Nkeng M, Bateman R, Fonten D, Hebbar KP (2007) Isolation and identification of mycoparasitic isolates of Trichoderma asperellum wich potential for suppression of black pod disease of cacao in Cameroon. Biological Control 43: 202-212.
- Lim, Jong H, Sang DK (2010) Biocontrol of Phytophthora blight of red pepper caused by Phytophthora capsici using Bacillus subtilis AH18 and B. licheniformis K11 formulations. Journal of the Korean Society for Applied Biological Chemistry 53(6): 766-773.
- Granke LL, Lina QO, Kurt L, Mary Kh (2012) Advances in research on Phytophthora capsici on vegetable crops in the United States. Plant Disease 96(11): 1588-1600.
- Maleki M, Mokhtarnejad L, Mostafaee S (2011) Screening of rhizobacteria for biological control of cucumber root and crown rot caused by Phytophthora drechsleri. Plant Pathology Journal 27(1): 78-84.
- Schaad NW, Jones JB, Chun W (2001) Laboratory Guide for Identification of Plant Pathogenic Bacteria. American Phytopathological Society (APS Press).
- Anjum, MZ, Muhammad UG, Imtiaz H (2019) Bio-efficacy of Trichoderma isolates and Bacillus subtilis against root rot of muskmelon cucumis melo l caused by Phytophthora drechsleri under controlled and field conditions. Pakistan journal of botany 51(5): 1877-1882.
- Jeyaseelan EC, Tharmila S, Niranjan K (2012) Antagonistic activity of Trichoderma and Bacillus spp. against Pythium aphanidermatum isolated from tomato damping off. Archives of Applied Science Research 4(4): 1623-1627.
- Akter MK, Hossain MD, Nahar K, Meah MB, Hossain MA (2007) Pathogenicity of Phytophthora capsici and possibilities of its biological and chemical control. Journal of Agroforesty and Environment 1(2): 81-84.
- Raza W, Faheem M, Yousaf S, Rajer FU, Yameen, M (2013) Volatile and non-volatile antifungal compounds produced by Trichoderma harzianum SQR-T037 suppressed the growth of Fusarium oxysporum f sp niveum. Science Letters 1: 21-24.
- Filippi C, Bagnoli G, Pices G (1989) Antagonistic effect of soil bacteria on Fusarium oxysporum f sp dianthi. Agricoltura Mediterrania 119: 327-336.
- Akgül DS, Mirik M (2008) Biocontrol of Phytophthora capsici on pepper plants by Bacillus megaterium Journal of Plant Pathology 29-34.
- Lelay Y, Ruano RD, López HC (2007) In vitro compatibility studies of monoconidia Trichoderma Isolates with potential to be used as biocontrol agent against white avocado decay. Proceedings VI World Avocado Congress (Actas VI Congreso Mundial del Aguacate).