Research Article

Comparative Evaluation of Different Microbial Biocontrol Agents against Rhizoctonia solani Kühn, the Causative Agent of Root Rot of Pepper Capsicum annuum L.

Yousra A. Swadi Al-Anzi*, Kadhim Z.K. Al-Karaawi and Iman Jawad Kadhim

Biological Control Technologies Department, Technical College, Al-Mussaib, Al-Furat Al-Awsat Technical University, Babylon, Iraq.

Received | December 06, 2024; Accepted | February 6, 2025; Published | April 30, 2025

*Correspondence | Yousra A. Swadi Al-Anzi, Biological Control Technologies Department, Technical College, Al-Mussaib, Al-Furat Al-Awsat Technical University, Babylon, Iraq; Email: [email protected]

Citation | Al-Anzi, Y.A.S., K.Z.K. Al-Karaawi and I.J. Kadhim. 2025. Comparative evaluation of different microbial biocontrol agents against Rhizoctonia solani Kühn, the causative agent of root rot of pepper Capsicum annuum L. Sarhad Journal of Agriculture, 41(2): 674-683.

DOI | https://dx.doi.org/10.17582/journal.sja/2025/41.2.674.683

Keywords | Phytopathogenic fungi, Rhizoctonia solani, Talaromyces oumae-annae, Bacillus paramycoides, Trichoderma longibrachiatum, Trichoderma harzianum, Bacillus subtilis

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Sweet pepper Capsicum annuum L. is one of the most important vegetable crops being cultivated in many countries around the world. Native to Central and South America, its production has been spread to Europe and Asia through Spain and Portugal in the 16th century, about 400 years ago (Chan, 2000; Katz, 2019). Green peppers are widely used as fresh or cooked in many food recipes. Dried peppers are used as spices in cooking and medicine. C. annuum fruits exhibit considerable stimulating and carminative effects which help maintaining health. In addition, it can prevent heart disease and thrombosis and has anti-inflammatory properties (Soare et al., 2017).

However, production of C. annuum is hampered by a number of factors including different fungal diseases. There are many fungal pathogens which infect vegetables including C. annuum. Particularly, soil-borne fungi such as Rhizoctonia solani Kühn has been a challenging disease of peppers. It infects the roots, stem base and canopy area of plants hindering the absorption of water, nutrients and salts from the rhizosphere (Mannai et al., 2018). Vegetable growers rely predominately on different synthetic fungicides which apart from giving unsatisfactory control manifest various side-effects including human health hazards and environmental contamination (Harris et al., 2001).

One of the alternate and environment-friendly solutions to these ecological consequences of synthetic fungicides is the utilization of different microbial biocontrol agents which can suppress the pathogenic organisms in the soil without affecting the beneficial flora of the rhizosphere (Madbouly and Abdelbacki, 2017). These beneficial microbes have a variety of mechanisms for controlling different phytopathogens, including direct parasitism, competition for nutrients, and production of secondary metabolites such as antibiotics and mycotoxins (Kumar and Kaushik, 2012; Aslam et al., 2023). One of the important biocontrol fungi is Trichoderma which are known for their ability to promote root growth and development, enhance resistance to various environmental conditions, and improve crop productivity and nutrients uptake (Ruangwong et al., 2021; Asad, 2022).

Among beneficial soil bacteria genus Bacillus, particularly B. subtilis, have received special attention, since these have been a crucial part in many biocontrol programs against a wide array of plant pathogens (Dimkić et al., 2022; Etesami et al., 2023). In addition, some species of fungi such as Penicillium and Talaromyces oumae-annae are known for their antagonistic activity against many plant pathogens by secreting certain antibiotic compounds and by inducing the systemic resistance in plants by activating several defense signaling pathways (Haque et al., 2021; Al-Karawi, 2022; Ren et al., 2024).

Keeping in view the ecological consequences of synthetic fungicides and biocontrol potential of above mentioned fungal and bacterial microbes, this study was aimed to compare the antagonistic efficiency of some selected microbial biological control agents against the pathogenic fungi R. solani, the causative agent of root rot of pepper C. annuum under laboratory conditions.

Materials and Methods

Isolation and pathogenicity test of R. solani

Infected pepper plants with root rot symptoms were randomly collected from different field sites located in Babil Governorate, Iraq. Infected plants were uprooted, placed in sterilized polythene bags and transported to the laboratory for study. The roots were washed with tap water for 3 min to remove attached soil particles. Root segments of approximately 1.0 cm in length were collected and sterilized with a chlorine free 1.0% sodium hypochlorite solution for 2–3 min. Cells are then transferred to sterile distilled water for 2 min, washed thoroughly and placed on sterile filter paper inside a laminar flow hood to remove excess water. Four plant segments were inoculated from Petri plate containing 15–20 ml of potato dextrose agar (PDA) supplemented with tetracycline @ 250 mg/L. The tool was sterilized in an autoclave at 121°C and 1.5 kg/cm² pressure for 15–20 min. The Petri plates were placed in an incubator at 25±1°C for four days. Fungal growth was examined from infected plant parts, and small portions of each fungal growth were transferred to Petri plates containing PDA and were incubated at 25±2°C for 7 days. The identification of fungi was carried out based on the morphological characteristics of the fungal colonies, the mycelial nature and using the taxonomic keys of Blazier (2004). The pathogenicity of isolated R. solani was tested according to Bolkan and Butler’s (1974). Seed germination percentage was calculated using the following equation:

Isolation of biocontrol agents

Soil samples were randomly collected from the rhizospheric profiles of a healthy maize crop located in Babil Governorate, Iraq, and these samples were transferred to the laboratory in sterile polythene bags where these were air dried for 24 h and then were sieved through a 1.0 mm mesh. Using serial dilutions protocol, soil mixtures were cultured on PDA medium enriched with tetracycline @ 250 mg/L. Petri dishes were gently shaken to ensure uniform sample distribution and then were incubated in a microbiological incubator at 25±1°C for 4 days. Fungal isolates of Trichoderma spp. were identified using taxonomic keys of Rifai (1969), while the Talaromyces oumae-annae isolate was identified according to the taxonomic key of Samson et al. (2001). Bacterial isolates were cultured using nutrients agar and were identified according to the standard taxonomic keys provided by Bergey et al. (1984).

Molecular diagnosis of isolated microbes by PCR

These diagnoses were performed at the DNA Genotek Laboratory® for molecular research. DNA of fungi Trichoderma longibrachiatum, T. harzianum, Talaromyces oumae-annae and of bacteria Bacillus paramycoides was extracted using ZR Fungal/Yeast/Bacterial DNA MiniPrep™ kit (Zymo Research, USA) following manufacturer’s instructions. Primers ITS1 and ITS4 (Martin and Rygiewicz, 2005) and 16S rRNA (Majeed et al., 2015) were used for PCR amplifications for fungal and bacterial detection, respectively. These primers were manufactured by Integrated DNA Technology®, as detailed in Table 1. Each PCR reaction mixture was of 25 µL volume containing Master Mix (iNtRON Biotechnology, Korea), 10 pmole of each primer and template DNA. PCR amplifications were performed in T3000 thermocycler (Biometra, Germany) using thermal protocols as detailed in Table 2. PCR products were visualized on an electrophoresis gel under UV light using gel documentation. In order to analyze the nucleotide sequence of the amplified DNA fragments, PCR amplification products from the isolates were forwarded to Macrogen Co., Ltd., Korea for DNA sequence analysis.

Regarding pathogenic bacteria, a previously identified isolate of B. subtilis was obtained from the Pathology Laboratory of the Department of Biotechnology, Technical College, Al-Musayyib, Iraq. B. paramycoides on the other hand was isolated from the soil as mentioned above. Both bacteria were mass-cultured in a nutrient broth medium in 500 mL sterile glass bottles in an autoclave at 121°C and a pressure of 1.5 kg/cm2 for 15 min. The medium was then inoculated with bacteria by taking a sterile sample of bacterial growth on a previously prepared agar medium. The contents of the vials were mixed well and then incubated at 32 ± 3°C for 3–4 days.

 

Table 1: DNA sequences of primer pairs used for the detection of ITS gene region of the biocontrol fungi Talaromyces oumae-annae, Trichoderma longibrachiatum and T. harzianum, and for detecting the 16S rRNA gene region in the bacterium Bacillus paramycoides.

Primer/ Sequence	Tm (ᵒC)	GC (%)	Product size
For fungi
F 5′- TCCGTAGGTGAACCTGCGG -3′	60.3	50	650 base pair
R 5′-TCCTCCGCTTATTGATATGC-3′	57.8	41
For bacteria
F 5'-AGAGTTTGATCCTGGCTCAG-3'	54.3	50.0	1250 base pair
R 5'-GGTTACCTTGTTACGACTT- 3’	49.4	42.1

 

Table 2: Thermal protocol used for the PCR amplification of fungal and bacterial DNA fragments.

No.	Phase	Tm (oC)	Time	No. of cycle
1	Initial denaturation	94ᵒC	3 min.	1 cycle
2	Denaturation	94oC	45sec	35 cycles
3	Annealing	52oC	1 min
4	Extension-1	72oC	1 min
5	Extension -2	72oC	7 min.	1 cycle

 

Evaluation of antagonistic potential of microbial isolates against R. solani

The antagonistic ability of B. paramycoides and B. subtilis was assessed against the pathogenic fungus R. solani on nutrients agar medium using the dilution method (1.0 × 101 - 1.0 × 108 cfu mL-1) post 72 h bacterial inoculations. In brief, 1.0 mL of the bacterial suspension was transferred to 9 cm Petri plate containing nutrient agar medium, with the plate gently shaken to evenly distribute the inoculum (1.0 × 104 - 1.0 × 108 cfu mL-1). Four replicates were maintained for each bacterial dilution. A 7-days old R. solani culture disc of 5.0 mm diameter was placed in the center of each plate for each dilution. Four plates were incubated without added bacteria as control. The Petri plates were incubated at 26 ± 1°C for 15 days. The growth rate of pathogenic fungi and the percentage of inhibition were calculated according to the following equation of Montealegre et al. (2003).

The antagonistic ability of the biocontrol fungi T. longibrachiatum, T. oumae-annae and T. harzianum was determined using double-culture technique (Miftahurrohmat et al., 2021) against R. solani in 9 cm Petri plates containing PDA medium. The medium was sterilized using an autoclave for 15–20 min and the Petri plate media was divided into two equal halves by drawing a line, with the central portion of one half inoculated with a 7 day-old R. solani culture disc, while the other half was inoculated with a fungal disc from the edge of the culture of the biocontrol fungi T. longibrachiatum. Same protocol was used for T. oumae-annae and T. harzianum. All Petri plates were then incubated at a temperature of 25±2 °C. Once the growth of the pathogenic fungus reached the edge of the plate in the control treatment, the percentage inhibition was calculated using the above-mentioned equation.

Statistical analysis

This research was carried out in a completely randomized design (CRD) and the statistical program GenStat (Payne, 2009) was used to analyze the data to study the effect of different treatments on the studied traits. Significant differences between means were compared using the least significant difference (LSD) test.

Results and Discussion

Isolation and identification of R. solani

Field samples collected from the diseased parts of pepper plants (i.e. from roots and areas adjacent to the trunk) revealed the presence of four R. solani fungal isolates. Microscopic examination showed a clear difference in their growth rate, sclerotia formation and fungal mycelial density, in addition to differences in the colony color ranging from brown to light brown with short and numerous cells. The fungal mycelium exhibited numerous ramifications at perpendicular and intersecting angles with the original fungal mycelium, as well as cellular constriction in the branching area and the formation of depressed barriers on the branches near the point of origin. It was observed that neither spores nor asexual conidia were formed as described by some previous studies (Stalpers and Andersen, 1996; Ajayi‐Oyetunde and Bradley, 2018).

Pathogenicity test with isolated R. solani on lettuce seedlings

According to pathogenicity test, R. solani isolate exhibited a differential effect on lettuce seed germination as shown in Figure 1. This antagonistic potential of R. solani corroborates the findings of previous studies (Adesina et al., 2009). Inhibition of lettuce seed germination would be due to the fact that fungus R. solani produces an array of enzymes that aid in the breakdown of host cell walls, such as pectinase, pectin methylesterase, cellulase and phosphatase (Dillard, 1987; Verwaaijen et al., 2017; Laevens et al., 2024).

 

 

Isolation and molecular identification of microbial biocontrol agents

PCR product of fungal DNA amplified utilizing ITS1 and ITS4 primers was electrophorized on sucrose gel and it exhibited a distinct band of molecular weight of 650 bp (Figure 2). This detection validates the efficacy of these primers in amplifying rDNA nucleic acid for fungal species as evidenced by literature (Korabečná et al., 2003). Similarly, for the bacterial strain examined, the electrophoresis on sucrose gel displayed a band of molecular weight of 1250 bp (Figure 3) when employing 16S rRNA cloning primers. This finding also affirms the capability of these primers to amplify rDNA nucleic acid of bacteria as demonstrated in various studies (Cardinale et al., 2004; Mirtalebi et al., 2019).

 

Nucleotide sequencing study

Nucleotide sequence analysis of the fungus T. longibrachiatum showed 98% similarity with the global isolates available in GenBank NCBI. In addition, the phylogenetic tree of the fungus T. longibrachiatum (Figure 4) revealed that the Iraq isolate corresponded 98% with the isolates from Moldova and 99% with the isolates from China, Egypt, Mexico and Belgium. The genomic DNA sequences of fungal isolates have been deposited in the global repository GenBank (NCBI) with accession number PP564406 assigned, serving as a point of reference for Iraq, the Middle East, and worldwide (Figure 4). Nucleotide sequence results for the fungus T. oumae-annae showed that this isolate had a 100% similarity with the worldwide isolates available in GenBank database (NCBI). In addition, the phylogenetic tree of T. oumae-annae fungi (Figure 4) showed that the Iraq isolate corresponded 100% with the isolates from Iran, Ethiopia and Europe and 94% with isolates from China and Malaysia. These fungal nucleotide sequences are deposited in the GenBank database with the accession number PP576344, establishing itself as a key reference for Iraq, the Middle East, and globally (Figure 4). The results of the nucleotide sequences for the fungus Trichoderma harzianum revealed a 98% similarity with the diverse isolates available in GenBank NCBI.

 

Moreover, the phylogenetic tree analysis of T. harzianum (Figure 4) indicated that the Iraqi specimen displayed a 98% match with isolates from Egypt and a 97% resemblance with isolates from China, Brazil, India, and Thailand. Another set of fungal nucleotide sequences were deposited in GenBank under accession number PP564402, further solidifying it as a significant reference for Iraq, the Middle East and worldwide research (Figure 4). Regarding the nucleotide sequencing based phylogenetic analysis of bacterial isolates, it was found that bacterial isolate B. paramycoides exhibited a 98% match with the global bacterial isolates available in the NCBI GenBank. Additionally, the phylogenetic tree of the B. paramycoides bacteria (Figure 4) demonstrated a 98% alignment with the bacterial isolates from Pakistan and a 99% alignment with the isolates from USA, India, Tunisia and China. The nucleotide sequences of the bacteria were deposited in the global GenBank organization with the accession number PP567247, establishing it as a reference for Iraq and the Middle East (Figure 4).

 

Table 3: Inhibition of pathogenic fungus Rhizoctonia solani by some biocontrol agents (Bacillus paramycoides and B. subtilis, Talaromyces oumae-annae, Trichoderma longibrachiatum and T. harzianum) and synthetic fungicide Beltanol (hydroxyquinoline sulphate) under laboratory conditions.

S. No.	Treatments	Colony diameter (cm)	Scaling ratio (%)
1	Bacillus paramycoides + Rhizoctonia solani	0.3	95.3
2	Trichoderma longibrachiatum + Rhizoctonia solani	0.1	98.0
3	Rhizoctonia solani + Talaromyces oumae-annae	0.5	93.3
4	Pathogenic fungi alone	9.0	0.0
5	Trichoderma harzianum + Rhizoctonia solani	0.8	92.0
6	Bacillus subtilis + Rhizoctonia solani	0.6	91.0
7	Rhizoctonia solani +Beltanol	0.0	100
LSD (P ≤ 0.05) Colony diameter = 0.151		Scaling ratio = 1.779

 

In-vitro antagonistic potential of microbial biocontrol agents against R. solani

Laboratory bioassays carried out to assess the inhibition potential of the isolated fungal and bacterial strains against phytopathogenic fungus R. solani revealed a high antagonistic capacity of all these the biocontrol microbes. All these fungal and bacterial isolates exhibited a significant inhibition of the growth and proliferation of R. solani as compared to the control treatment (Table 3 and Figure 5). All tested fungal strains of Trichoderma and Talaromyces genera showed a high antagonistic potential and inhibition effect reached the edge of the plaque, after four days of the inoculation of pathogenic fungi (R. solani). The treatment with T. longibrachiatum showed the highest inhibition i.e. 98.0%. These results corroborate the fact that fungi such as Trichoderma and Talaromyces are important biological control agents against phytopathogens (Kakvan et al., 2013; Abbas et al., 2025). These fungi have various antagonistic properties against plant pathogens and are crucial for plant growth and production, in addition to their ability to stimulate plant resistance to different diseases and other pests (Harman and Uphoff, 2019; Thambugala et al., 2020; Xie et al., 2021; Abbas et al., 2025).

 

Most fungi including Trichoderma spp. show a high effectiveness against plant pathogens and have differential mechanisms of competition for space, nutrients and parasitism (Adnan et al., 2019). The antagonistic organisms directly remove nutrients from the pathogens, and the resistance mechanism involves the production of metabolic substances that prevent the pathogens from invading plant tissues (Baker and Paulitz, 1996; Stracquadanio et al., 2020; Adeleke et al., 2022). Apart from the above-mentioned fungal isolates, the bacteria B. paramycoides as well showed a high inhibition of 95.33%, followed by the treatment with the mold T. oumae-annae, which showed an inhibition of 93.33%. In addition, the treatment with the fungus T. harzianum showed a considerable inhibition (92.0%) of R. solani, and the treatment with the bacteria B. subtilis showed an inhibition of 91.0%. These results are in line with the findings of El-Sersawy et al. (2021) demonstrated the effectiveness of Bacillus isolates against R. solani and against other fungal diseases such as against Fusarium oxysporum-induced and R. solani-induced wilt in soybean plants (Jain et al., 2017; El-Sersawy et al., 2021; Maral-Gül and Eltem, 2024). Similarly, our results validate the results of Singh et al. (2021) and Lotfalinezhad et al. (2024) reporting the biocontrol efficiency of T. oumae-annae and T. harzianum against R. solani.

Conclusions and Recommendations

On the basis of overall study results, it is concluded that the microbial biocontrol agents tested in this in-vitro study could be effective and environment-friendly alternates for combating against the incidence of R. solani on plants. Particularly, fungal strain T. longibrachiatum and bacterial isolate B. paramycoides effectively reduced the severity of pathogenic fungus R. solani-induced root rot disease in plants. However, field evaluation of these microbial biocontrol agents and their interaction with other beneficial non-target fauna and flora constitute the future perspectives of this study.

Acknowledgements

Authors are thankful to Al-Mussaib Technical College, Iraq for providing technical support and equipment for the study. We are grateful to our colleague in the Lab who assisted us throughout the research work.

Novelty Statement

All the microbial biocontrol agents we tested in this in-vitro study, particularly fungal strain T. longibrachiatum and bacterial isolate B. paramycoides, could be used as effective and environment-friendly alternates against R. solani-induced root rot disease in plants.

Author’s Contribution

Yousra A. Swadi Al-Anzi , Kadhim Z.K. Al-Karaawi and Iman Jawad Kadhim: Conceived of the original idea, developed the theoretical and performed the statistical analysis for experimental data and discussed the results and contributed to manuscript writeup.

Yousra A. Swadi Al-Anzi and Kadhim Z.K. Al-Karaawi: Verified the analytical methods.

Kadhim Z.K. Al-Karaawi and Iman Jawad Kadhim: Worked for lab analysis and supervised the project.

Conflict of interest

The authors have declared no conflict of interest regarding the publication of this research work.

References

Abbas, A., S. Ali, M. Mubeen, A. Hussain, K.A. Gutumsary, B. Hussain and T. Chen. 2025. Talaromyces spp. are promising biocontrol agents for sustainable agriculture. In: Microbial Biocontrol Techniques: Importance in Ensuring Food Security, Singapore: Springer Nature Singapore. 54: 245-280. https://doi.org/10.1007/978-981-97-8739-5_13

Adeleke, B.S., M.S. Ayilara, S.A. Akinola and O.O. Babalola. 2022. Biocontrol mechanisms of endophytic fungi. Egypt. J. Biol. Pest Contr., 32(1): 46. https://doi.org/10.1186/s41938-022-00547-1

Adesina, M.F., R. Grosch, A. Lembke, T.D. Vatchev and K. Smalla. 2009. In vitro antagonists of Rhizoctonia solani tested on lettuce: Rhizosphere competence, biocontrol efficiency and rhizosphere microbial community response. FEMS Microbiol. Ecol., 69(1): 62-74. https://doi.org/10.1111/j.1574-6941.2009.00685.x

Adnan, M., W. Islam, A. Shabbir, K.A. Khan, H.A. Ghramh, Z. Huang and G.D. Lu. 2019. Plant defense against fungal pathogens by antagonistic fungi with Trichoderma in focus. Microb. Pathog., 129: 7-18. https://doi.org/10.1016/j.micpath.2019.01.042

Ajayi-Oyetunde, O.O. and C.A. Bradley. 2018. Rhizoctonia solani: Taxonomy, population biology and management of Rhizoctonia seedling disease of soybean. Plant Pathol., 67(1): 3-17. https://doi.org/10.1111/ppa.12733

Al-Karaawi, K.Z. and D.S. Al-Waily. 2022. Morphological and molecular diagnosis of bio-fungus isolate Penicillium commune DSKZ and its laboratory use in biological control of pathogenic fungus Sclerotinia sclerotiorum (Lib) DeBary causing white mold on eggplant. NQ., 20(10): 1612. https://doi.org/10.51470/bca.2022.22.2.3613

Al-Shujairi, K.A., H.K. Albehadlli, Z.N. Kamaluddin, A.N. Al-Abedy and D.K. Al-Taey. 2022. Genetic variation among some Sclerotinia sclerotiorum isolates causing white mold disease in eggplants (Solanum melongena). Int. J. Agric. Stat. Sci., 18(1): 399-407.

Asad, S.A., 2022. Mechanisms of action and biocontrol potential of Trichoderma against fungal plant diseases. A review. Ecol. Complex., 49: 100978. https://doi.org/10.1016/j.ecocom.2021.100978

Aslam, S., M.I. Hamid, M.Z. Majeed, S. Sayed, A. Mahmood and M.A. Javed. 2023. Antifungal activity of indigenous microbiota for the suppression of red rot of sugarcane under field conditions. Ges. Pfl., 75(5): 1497-1505. https://doi.org/10.1007/s10343-023-00844-1

Baker, R. and T.C. Paulitz. 1996. Theoretical basis for microbial interactions leading to biological control of soilborne plant pathogens. Principles and practice of managing soilborne plant pathogens, R. Hall, ed. St. Paul, MN: Am. Phytopathol. Soc., pp. 50-79.

Bergey, D.H., D. Hendricks, J.G. Holt and P.H. Sneath. 1984. Bergey’s manual of systematic bacteriology. Vol. 2. Williams & Wilkins.

Blazier, S.R., 2004. Characterization of Rhizoctonia solani isolates associated with patch diseases on turfgrass. Proc. Okla. Acad. Sci., 84: 41-51.

Bolkan, H.A. and D.F. Bulter. 1974. Studies on heterokaryosis and virulence of Rhizoctonia solani. Phytopathology, 64: 513-522. https://doi.org/10.1094/Phyto-64-513

Cardinale, M., L. Brusetti, P. Quatrini, S. Borin, A.M. Puglia, A. Rizzi and D. Daffonchio. 2004. Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl. Environ. Microbiol., 70(10): 6147-6156. https://doi.org/10.1128/AEM.70.10.6147-6156.2004

Chan, S., 2000. Vegetable breeding (2nd Edition). Agricultural Press, Asia, pp. 257.

da Silva, M.R.B.L., 2018. Gliotoxin and Bis-methyl-glitoxin production by Trichoderma spp. as biocontrol agents. Master’s thesis, Instituto Politecnico do Porto (Portugal). pp. 28991868.

Dillard, H.R., 1987. Characterization of isolates of Rhizoctonia solani from lima bean grown in New York state. Phytopathology, 77: 748-751. https://doi.org/10.1094/Phyto-77-748

Dimkić, I., T. Janakiev, M. Petrović, G. Degrassi and D. Fira. 2022. Plant-associated Bacillus and Pseudomonas antimicrobial activities in plant disease suppression via biological control mechanisms. A review. Physiol. Mol. Plant Pathol., 117: 101754. https://doi.org/10.1016/j.pmpp.2021.101754

El-Sersawy, M.M., S.E.D. Hassan, A.A. El-Ghamry, A.M.A. El-Gwad and A. Fouda. 2021. Implication of plant growth-promoting rhizobacteria of Bacillus spp. as biocontrol agents against wilt disease caused by Fusarium oxysporum Schlecht. in Vicia faba L. Biomol. Concepts, 12(1): 197-214. https://doi.org/10.1515/bmc-2021-0020

Etesami, H., B.R. Jeong and B.R. Glick. 2023. Biocontrol of plant diseases by Bacillus spp. Physiol. Mol. Plant Pathol., 126: 102048. https://doi.org/10.1016/j.pmpp.2023.102048

Haque, M.E., D.K. Lakshman, A. Qi and M.F. Khan. 2021. Penicillium pinophilum has the potential to reduce damping-off caused by Rhizoctonia solani in sugar beet. Sugar Tech., 23(4): 872-880. https://doi.org/10.1007/s12355-021-00958-8

Harman, G.E. and N. Uphoff. 2019. Symbiotic root-endophytic soil microbes improve crop productivity and provide environmental benefits. Scientifica, 2019: 106395. https://doi.org/10.1155/2019/9106395

Harris, C.A., M.J. Renfrew and M.W. Woolridge. 2001. Assessing the risks of pesticide residues to consumers: recent and future developments. Food Addit. Contam., 18(12): 1124-1129. https://doi.org/10.1080/02652030110050122

Jain, S., A. Vaishnav, S. Kumari, A. Varma, N. Tuteja and D.K. Choudhary. 2017. Chitinolytic Bacillus-mediated induction of jasmonic acid and defense-related proteins in soybean (Glycine max L. Merrill) plant against Rhizoctonia solani and Fusarium oxysporum. J. Plant Growth Regul., 36: 200-214. https://doi.org/10.1007/s00344-016-9630-1

Kakvan, N., A. Heydari, H.R. Zamanizadeh, S. Rezaee and L. Naraghi. 2013. Development of new bioformulations using Trichoderma and Talaromyces fungal antagonists for biological control of sugar beet damping-off disease. Crop Prot., 53: 80-84. https://doi.org/10.1016/j.cropro.2013.06.009

Katz, E., 2019. Flavors and colors: The chili pepper in Europe. In: Transatlantic Trade and Global Cultural Transfers Since, 1492: 30-53. https://doi.org/10.4324/9780429427305-3

Korabečná, M., V. Liška and K. Fajfrlík. 2003. Primers ITS1, ITS2 and ITS4 detect the intraspecies variability in the internal transcribed spacers and 5.8 S rRNA gene region in clinical isolates of fungi. Folia Microbiol., 48(2): 233-238. https://doi.org/10.1007/BF02930961

Kumar, S. and N. Kaushik. 2012. Metabolites of endophytic fungi as novel source of biofungicide: A review. Phytochem. Rev., 11: 507-522. https://doi.org/10.1007/s11101-013-9271-y

Laevens, G.C., W.C. Dolson, M.M. Drapeau, S. Telhig, S.E. Ruffell, D.M. Rose and A.A. Stegelmeier. 2024. The good, the bad, and the fungus: Insights into the relationship between plants, fungi, and oomycetes in hydroponics. Biol., 13(12): 1014. https://doi.org/10.3390/biology13121014

Lotfalinezhad, E., A. Taheri, S.E. Razavi and S.J. Sanei. 2024. Preparation and assessment of alginate-microencapsulated Trichoderma harzianum for controlling Sclerotinia sclerotiorum and Rhizoctonia solani on tomato. Int. J. Biol. Macromol., 259: 129278. https://doi.org/10.1016/j.ijbiomac.2024.129278

Madbouly, A.K. and A.M. Abdelbacki. 2017. Biocontrol of certain soilborne diseases and promotion of growth of Capsicum annuum using biofungicides. Pak. J. Bot., 49(1): 371-378.

Mannai, S., H. Jabnoun-Khiareddine, B. Nasraoui and M. Daami-Remadi. 2018. Rhizoctonia root rot of pepper (Capsicum annuum): Comparative pathogenicity of causal agent and biocontrol attempt using fungal and bacterial agents. J. Plant Pathol. Microbiol., 9(2): 431-439.

Maral-Gül, D. and R. Eltem. 2024. Evaluation of Bacillus isolates as biological control agents against soilborne phytopathogenic fungi. Int. Microbiol., 1-15. https://doi.org/10.1007/s10123-024-00490-1

Martin, K.J. and P.T. Rygiewicz. 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol., 5: 1-11.

Miftahurrohmat, A., I.R. Nurmalasari and A.E. Prihatinnigrum. 2021. In vitro evaluation of the inhibitory power of Trichoderma harzianum against pathogens that cause anthracnose in chili. J. Phys. Conf. Ser., 1764: 012026. https://doi.org/10.1088/1742-6596/1764/1/012026

Mirtalebi, M., F. Sabahi and Z. Banihashemi. 2019. Fruit rot caused by Neoscytalum hyalinum on melon in Iran. Australas. Plant Dis. Notes, 14: 2-4. https://doi.org/10.1007/s13314-019-0338-5

Montealegre, J.R., R. Reyes, L.M. Pérez, R. Herrera, P. Silva and X. Besoain. 2003. Selection of bioantagonistic bacteria to be used in biological control of Rhizoctonia solani in tomato. Electron. J. Biotechnol., 6(2): 115-127. https://doi.org/10.2225/vol6-issue2-fulltext-8

Murray, R.G.E. and J.G. Holt. 2005. The history of Bergey’s Manual. Bergey’s Manual® of Systematic Bacteriology, springer nature link, pp. 1-14. https://doi.org/10.1007/0-387-28021-9_1

Payne, R.W., 2009. GenStat. Wiley Interdisciplinary Reviews: Comput. Stat., 1(2): 255-258. https://doi.org/10.1002/wics.32

Ren, X.T., S. Li, Y. Ruan and L. Wang. 2024. Three new species of Talaromyces sect. Talaromyces discovered in China. PeerJ, 12: 18253. https://doi.org/10.7717/peerj.18253

Rifai, M.A. 1969. A revision of the genus Trichoderma. Mycol. Mem., 116: 56-69.

Ruangwong, O.U., P. Wonglom, N. Suwannarach, J. Kumla, N. Thaochan, P. Chomnunti and A. Sunpapao. 2021. Volatile Organic Compound from Trichoderma asperelloides TSU1: Impact on plant pathogenic fungi. J. Fungi, 7(3): 187. https://doi.org/10.3390/jof7030187

Samson, R.A. and Houbraken, J., 2001. Laboratory isolation and identification of fungi. In: (eds.Flannigan B., R.A. Samson, J.D. Miller) Microorganisms in home and indoor work environments: Diversity, health impacts, investigation and control. CRC Press. pp. 247.

Singh, S., R. Balodi, P.N. Meena and S. Singhal. 2021. Biocontrol activity of Trichoderma harzianum, Bacillus subtilis and Pseudomonas fluorescens against Meloidogyne incognita, Fusarium oxysporum and Rhizoctonia solani. Indian Phytopathol., 74(3): 703-714. https://doi.org/10.1007/s42360-021-00368-6

Soare, R., M. Dinu, C. Băbeanu, M. Popescu and A. Popescu. 2017. Nutritional value and antioxidant activities in fruit of some cultivars of pepper (Capsicum annuum L.). J. Agroaliment. Process. Technol., 23(4): 217-222.

Stalpers, J.A. and T.F. Andersen. 1996. A synopsis of the taxonomy of teleomorphs connected with Rhizoctonia sl. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology, and Disease Control, Dordrecht: Springer Netherlands. pp. 49-63. https://doi.org/10.1007/978-94-017-2901-7_4

Stracquadanio, C., J.M. Quiles, G. Meca and S.O. Cacciola. 2020. Antifungal activity of bioactive metabolites produced by Trichoderma asperellum and Trichoderma atroviride in liquid medium. J. Fungi, 6(4): 263. https://doi.org/10.3390/jof6040263

Thambugala, K.M., D.A. Daranagama, A.J. Phillips, S.D. Kannangara and I. Promputtha. 2020. Fungi vs. fungi in biocontrol: An overview of fungal antagonists applied against fungal plant pathogens. Front. Cell. Infect. Microbiol., 10: 604923. https://doi.org/10.3389/fcimb.2020.604923

Verwaaijen, B., D. Wibberg, M. Kröber, A. Winkler, R. Zrenner, H. Bednarz and A. Schlüter. 2017. The Rhizoctonia solani AG1-IB (isolate 7/3/14) transcriptome during interaction with the host plant lettuce (Lactuca sativa L.). PLoS One, 12(5): 0177278. https://doi.org/10.1371/journal.pone.0177278

Xie, L., X. Zang, W. Cheng, Z. Zhang, J. Zhou, M. Chen and Y. Tang. 2021. Harzianic acid from Trichoderma afroharzianum is a natural product inhibitor of acetohydroxyacid synthase. J. Am. Chem. Soc., 143(25): 9575–9584. https://doi.org/10.1021/jacs.1c03988

ZK, Y.A.S.A.K., 2024. Evaluation of the efficiency of biocontrol agents, including Bacillus paramycoides, Bacillus subtilis, Trichoderma longibrachiatum, Talaromyces oumae-annae and Trichoderma harzianum, to control pepper root rot disease caused by Rhizoctonia solani in Babil Province, Iraq. Euphrates J. Agric. Sci., 16(2): 447-466.