Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

ABSTRACT

Background: Chickpeas are a prevalent and significant crop in the agricultural sectors of northern Iraq.  In recent years, production has significantly declined due to diseases affecting chickpea plants. Investigating the pathogenic fungi responsible for these diseases is essential to restore production levels to their prior and improved states.

Methods: Samples of diseased chickpea plants were collected from fields in the Erbil, Sulaymaniyah and Salah al-Din governorates in northern Iraq. Isolated associated fungi led to the identification of the dominant pathogenic species. A pathogenicity test was performed on the fungal isolates, showing  the most aggressive strain for subsequent antagonism testing against three rhizobacterial isolates: Azotobacter chroococcum, Bacillus cereus and B. pumilus. This testing was conducted both in vitro and in a greenhouse setting,  using  single treatments and combinations.

Result: The results showed that the pathogenic fungus Rhizoctonia solani was predominant, with the strain Srs-21 exhibiting the highest virulence in vitro. The three rhizobacterial isolates, A. chroococcum, B. cereus and B. pumilus, showed high efficacy in inhibiting the pathogenic fungus in vitro on potato dextrose agar (PDA) medium.  In the greenhouse, the triple bacterial interaction treatment showed a seed germination rate of 100% and diminished disease incidence and severity to 0%, in contrast to the positive control treatment, which recorded 90% and 70%, respectively. It also showed the highest rate of rise in the dry weight of plants, signifying enhanced growth metrics.

INTRODUCTION

Chickpea (Cicer arietinum L.) is recognized as an important leguminous crop on a global scale. This legume is recognized as the most productive in South Asia and is the third most produced globally, trailing only beans and peas (Gaur and Gowda, 2005). In 2003, Iraq’s total production was documented at 104,000 metric tons, followed by a steady decrease that culminated in an average of 203 metric tons by 2019 (FAOSTAT, 2021). The chickpea crop encounters several pathogens, including Fusarium solani, F. oxysporum, Macrophomina phaseolina and Rhizoctonia solani (Kuhn), which are considered  as major factors in seed rot, damping off in seedlings and the deterioration of roots and crowns (Agrios, 2005; Hussein et al., 2025c). Rhizoctonia solani is a facultative parasitic fungus categorized within the phylum Basidiomycota, subphylum Agaricomycotina, class Agaricomycetes, order  Cantharellales  and family  Ceratobasi- diaceae. The sexual basidiospores are generally generated on infected plants; nonetheless, the fungus primarily reproduces in its sterile anamorph stage (Alexopoulos et al., 1996; Agrios, 2005). This stage is marked by the emergence of rapidly expanding, brown-hued hyphae with significant diameters of 15-20 µm. These structures display distinct constrictions at their points of emergence and exhibit the formation of transverse septa in the branches close to the emergence area, characterized by double holes and multiple nuclei. The development of barrel cells or irregular shapes is a key feature, which amalgamate to form a mass known as sclerotia. This sclerotium functions as a mechanism for the fungus to endure adverse environmental conditions (Blazier and Conway, 2004; Howard et al., 2007). The fungus affects the plant during different growth stages, infecting seeds in the soil, which results in seed and seedling rot, leading to damping off both prior to and following emergence and impacting the roots and crown of the plant (Rivera et al., 2004; Agrios, 2005). There is an increasing focus on the use of environmentally friendly beneficial microorganisms for managing plant diseases (Hussein et al., 2025b). The rhizosphere-living bacteria that are antagonistic represent a significant and effective alternative approach for managing diseases in chickpea.  Rhizosphere-living bacteria function as biocontrol agents through various mechanisms, including the production of antibiotics, siderophores and enzymes, which effectively inhibit the proliferation of pathogenic microbes (Bhargavi et al., 2024). This study focused on identifying the fungus that causes rot root disease in chickpeas in northern Iraq, while also assessing various rhizobacterial isolates for their potential as biocontrol agents against the pathogen.

MATERIALS AND METHODS

Isolation and identification of the causal agents of the disease
 
Samples were collected from chickpea plants showing signs of root rot disease, which included yellowing and wilting leaves, along with rotting that displayed brown to black discoloration in the roots and crown region (Fig 1). The samples were collected from the fields in the Erbil, Sulaymaniyah and Salah al-Din governorates. Root samples underwent a washing procedure using running water for a duration of 15 minutes, after which they were cut into small segments of 0.5 cm in size. The segments underwent surface sterilization with a 2% sodium hypochlorite solution for 2 minutes, followed by two rinses with sterile distilled water and were subsequently dried using sterile filter paper in a laminar flow cabinet. Four samples were positioned on PDA medium within 9 cm petri dishes. The medium underwent sterilization in an autoclave at a temperature of 121oC, maintaining a pressure of 1.5 kg/cm2 for a duration of 20 minutes. The dishes were subsequently incubated at 25±1oC for a duration of 7 days.  The fungal isolates were subjected to examination with a light microscope, resulting in their identification at both the genus and species levels through morphological characteristics and alignment with established taxonomic keys (Parmeter and Whitney, 1970; Ellis, 1971; Booth, 1977; Domsch et al., 1980; Klich, 2002; McClenny, 2005). The proportion of appearance and frequency of the fungal isolates has been calculated utilizing the formulas established by Hussein (2024).




 

Fig 1: Symptoms of rot roots on infected chickpea plants.


 
Anastomosis interactions test of R. solani isolates
 
The number of nuclei per cell of R. solani isolates was counted by growing the isolates on the PDA and staining the hyphae with 1ug/ml of 42 -6 diamidino-2-phenylindole, petri plates examined under a fluorescent microscope (Gondal et al., 2019). Nine isolates of R. solani belong to nine AGs provided by Microbiology laboratory/ University of Mustansiriyah, used as tester strains of the respective AGs, the fifty-seven unknown strain of R. solani were paired with tester strains, by placing an agar disc (5 mm) of the hyphal growth of unknown isolate at the one side of sterile glass slide covered with a layer of the 1.5% water agar and at the other side of the slide a similar disc of the tester strain and incubated for 72 hours at a 25oC, the hyphal interactions between tester strain  and unknown strain was determined according to the method of Gondal et al., (2019).   
 
Pathogenicity testing assay
 
The pathogenicity of fifty- seven isolates of R. solani was evaluated on a sterile PDA. Plates were inoculated at the center with a 0.5 cm diameter disc of R. solani isolates that had been cultivated on PDA culture medium for seven days. The dishes were incubated at a temperature of 25 ±1oC for a duration of two days. Each plate was then sown with 10 seeds of the Marrakechy cultivar of chickpeas, which had undergone surface sterilization using a 2% sodium hypochlorite solution for two minutes, followed by two rinses with sterile distilled water. The seeds were then dried using sterile filter paper and arranged in a circular pattern near the edge of the plate. Four replications were conducted for each treatment, including the control treatment of  seedling only without the fungus. The dishes were incubated at a temperature of 25±1oC for a duration of 14 days, under a lighting schedule of 12 hours per day. The findings were documented and the germination percentage was determined using the equation provided by Hussein (2019).

 
Antagonistic activity in vitro
 
Evaluation of antagonism activity of three rhizobacterial isolates of A. chroococcum, B. cereus and B. pumilus which optioned from Mustansiriyah University, laboratory of microbiology, against the most virulent isolate of R. solani (Srs-21), these rhizobacterial isolates isolated from the rhizosphere of healthy chickpea plants in Iraq (Hussein, 2022). On the PDA a 0.5 cm diameter disc of the fungal isolate placed at the edge of the petri plate (about 2 cm out of the edge) and in the opposite side a loop full of each bacterial isolate 109 CFU/ml placed individually, plates incubated at 25±1 Co for 7 days, each treatment repeated 4 times, antagonism activity calculated using the following formula:

 
Where:
b = The distance between the bacterial line towards the fungal growth.
f = The distance of fungal growth towards the bacterial line.
 
Greenhouse evaluation
 
Inoculum of the fungal pathogen Srs-21 were prepared by growing it on the seeds of local millet, by adding 5 discs (0.5 cm diameter) of the pathogen in the culture medium (PDA) of 5 days old to a 250 ml glass beaker containing autoclaved millet seeds mixed with 10 ml of distilled and sterile water and incubated for 14 days. The inoculum was added to mixture of sterilized soil and compost in 1 kg plastic pots at a ratio of 1% (w/w). Each pot irrigated with 10 ml of the rhizobacterial inoculum of A. chroococcum, B. cereus and B. pumilus which were grown on the nutrient broth with concentration of 109 CFU/ml added in the same time of the seed planting, each treatment repeated 4 times and control treatments irrigated with distilled sterile water. Five seeds of chickpea (Marrakechy cultivar) were planted in each pot surface sterilized with 1% sodium hypochlorite solution. Pots were distributed according to a completely randomized design (CRD) and watered carefully. The percentage of germination was calculated after 14 days and the disease incidence was calculated after 40 days of planting and the disease severity index was measured according to the disease rating scale and the formulas described by Hussein, 2022). The dry weight of plants was also calculated to measure the effect of bacterial isolates on plant growth parameters.





RESULTS AND DISCUSSION

Fungi associated with infected chickpea plants
 
The findings from the isolation and diagnosis  showed that R. solani was the most prevalent fungus, appearing in 77% of the samples, with a frequency percentage of 59.5% (Table 1). All R. solani isolates exhibited fundamental characteristics, including branching near the terminal septum of cells within the mycelium, marked by a narrowing at the starting point of the branches. Additionally, barriers appeared near the starting point of the branches, along with barriers featuring double holes (Fig 2). The coloration of the fungal colonies transitioned from white initially to light brown, then  deep brown (Fig 2). All isolates produced sclerotia that diversified in size and form, with diameters ranging from 1-3 mm and with thick brown walls. Some isolates additionally produced barrel cells of varying sizes, consistent with the characteristics described by Parmeter and Whitney (1970). These results align with the findings of Ganeshamoorthi and Dubey (2013), who isolated 50 isolates of R. solani from chickpea fields across 10 various states within India.  Hussein (2022) demonstrated that within the soil-borne pathogenic fungi collected from infected chickpea plants in Iraq, R. solani emerged as the most prevalent, with an appearance rate of 85.5% and a frequency of 61.2%.

Table 1: Fungal isolates associated with the diseased chickpea plants.



Fig 2: Morphological characteristics of R. solani.


 
Anastomosis group test
 
The findings displayed in Table 2 regarding hyphal dyeing with DAPI and microscopic examinations of R. solani isolates indicated that all isolates were multinucleate. The interactions of hyphal anastomosis among the 57 isolates of R. solani and the tester strains of the respective AGs revealed that 40% of the isolates were classified under AG 4, which was the most prevalent. In contrast, 12%, 30%, 9%, 5% and 4% of the isolates were categorized into AG 1, AG 3, AG 5, AG 7 and AG 10, respectively (Fig 3).  Hussein (2022) discovered that 58% of the R. solani isolates isolated from infected chickpea plants in the Nineveh and Duhok governorates in northern Iraq were classified within the AG3 group and 3% belong to AG4.

Table 2: Pathogenic test of R. solani isolates using chickpea seeds.



Fig 3: Distribution of R. solani isolates categorized by anastomosis groups.



Pathogenicity assay
 
The findings from the pathogenicity test indicated that the germination rate varied from 0% to 65%, in contrast to the control group, which exhibited a 100% germination rate (Table 2). The Srs-21 isolate, classified as AG3 from the Sulaymaniyah governorate, demonstrated a complete inhibition of seed germination, achieving 0% germination and outperforming all other isolates in this regard (Fig 4). The variation in the pathogenic ability of these fungal isolates can be attributed to genetic differences among individuals of the same species, the response of the plant host varies depending on the isolates, which is also attributed to genetic variation, particularly since they are sourced from various regions.

Fig 4: Pathogenicity test of R. solani isolates in vitro.


 
Antagonistic activity in vitro
 
The findings from the antagonism test involving three rhizobacterial isolates against the fungal pathogen R. solani (Srs-21 isolate) indicated that the B. pumilus isolate demonstrated the highest level of antagonistic activity at 95.0%. This was followed by B. cereus, which showed an antagonism activity of 91.4%, while A. chroococcum exhibited a lower activity of 66.4% (Table 3) (Fig 5).

Table 3: Antagonistic activity of the rhizobacterial isolates.



Fig 5: Antagonism activities of rhizobacterial isolates against R. solani.


 
Greenhouse assay
 
The findings indicated that individual, dual and triple treatments of the rhizobacterial isolates enhanced seed germination percentages, ranging from 80% to 100%, in contrast to the positive control treatment, which recorded 70% (Table 4). The combination treatment of A. chroococcum, B. cereus and B. pumilus  proved a substantial efficacy in diminishing disease incidence and severity to 0%, in contrast to the positive control treatment, which recorded 90.0% and 70.0%, respectively (Table 4).  A combination treatment of B. cereus and B. pumilus resulted in disease incidence and severity rates of 35% and 28%, respectively.  Regarding individual treatments, while they resulted in a 70-80% reduction in disease incidence, they did not significantly decrease disease severity, which ranged from 61-65%. The triple treatment resulted in the most significant increase in the average dry weight of chickpea plants,  reaching 15 g/plant, in contrast to the positive and negative control treatments, which recorded 3.8 and 12.8 g/plant, respectively.  Bacteria in the rhizosphere of plants safeguard the roots by inhibiting fungal growth and invasion into the root cells and the adjacent stalk tissues near the soil surfaces (Hussein and Ibrahim, 2018). The inhibitory action of A. chroococcum on R. solani growth may be attributed to its capacity to synthesize metabolites and organic chemicals, including stearic acid, enzymes, antibiotics and hydrogen cyanide (Hussein et al., 2025a). The results aligned with those reported by Hussein (2019), which illustrated the efficacy of B. pumilus in managing cucumber root rot disease induced by F. solani. Aljuboori et al., (2022) illustrated that the Rhizoctonia solani - Meloidogyne javanica complex disease affecting chickpea roots can be biologically managed using a rhizobacterial isolate of Pseudomonas sp., which also enhanced both the dry and wet weight of the plants, significantly elevating the activity of polyphenol oxidase and peroxidase enzymes, as well as the total phenolic content. Agarwal et al., (2017) shown that B. pumilus significantly suppressed the development of R. solani and F. oxysporum through the synthesis of chitinolytic enzymes and the generation of antibiotics. These results align with the findings of Zarrin et al., (2009), who revealed the capacity of A. chroococcum to prevent the development of several pathogenic fungus, including R. solani. Al Isawy (2010) demonstrated the efficacy of A. chroococcum as a means of biological management in suppressing the growth of R. solani on PDA medium.  Hidayah and Yulianti (2015) discovered that B. cereus reduced growth of R. solani and Sclerotium rolfsii in vitro by 68.9% and 33%, respectively, on PDA medium. Additionally, B. cereus, obtained from the rhizosphere of mustard plants, prevented the growth of R. solani, Pythium ultimum and S. rolfsii (Pleban et al., 1995).  Huang et al., (2012) demonstrated  that B. pumilus altered the fungal hyphae of R. solani in vitro, resulting in the increase of cytoplasmic vacuoles and cytoplasmic exudation within the fungal cells.

Table 4: Biological control of chickpea root rot disease in greenhouse.

CONCLUSION

In vitro experiments demonstrated that the three bacterial isolates of A. chroococcum, B. cereus and B. pumilus, obtained from the rhizosphere of healthy chickpea plants, exhibited superior capabilities in inhibiting the growth of the highly aggressive isolate of the pathogenic fungus Rhizoctonia solani (Srs-21) on the PDA culture medium.  In controlled greenhouse conditions, the integration of the three bacterial isolates yielded the most favorable outcome in enhancing the germination rate of chickpea seeds, while also decreasing both the incidence and severity of the disease, alongside promoting the growth parameters of the plants as measured by dry weight.

ACKNOWLEDGEMENT

The author would like to acknowledge the role of Mustansiriyah University, Iraq, Baghdad in the completion of this research.

CONFLICT OF INTEREST

The author declares that they have no conflicts of interest.

REFERENCES


  1. Agarwal, M., Dheeman, S., Dubey, C., Kumar, P., Maheshwari, D. and Bajpai, V. (2017). Differential antagonistic responses of Bacillus pumilus MSUA3 against Rhizoctonia solani and Fusarium oxysporum causing fungal diseases in Fagopyrum esculentum Moench. Microbiological Research. 205: 40-47.

  2. Agrios, G.N. (2005). Plant Pathology (5th ed.). Academic Press.

  3. Al Isawy, J.M. (2010). Integrated management of eggplant’s damping off caused by the fungus Rhizoctonia solani Kuhn (Master’s thesis). University of Baghdad, College of Agriculture, Department of Plant Protection, Iraq.

  4. Alexopoulos, C.J., Mims, C.W. and Blackwell, M. (1996). Introductory Mycology (4th ed.). John Wiley and Sons.

  5. Aljuboori, F.K., Ibrahim, B.Y. and Mohamed, A.H.  (2022). Biological control of the complex disease of Rhizoctonia solani and root-knot nematode Meloidogyne javanica on chickpea by Glomus spp. and Pseudomonas sp. Iraqi Journal of Agricultural Sciences. 53(3): 669-676. https://doi.org/ 10.36103/ijas.v53i3.1577.

  6. Blazier, S.R. and Conway, K.E. (2004). Characterization of Rhizoctonia solani isolates associated with patch disease on turf grass. Proceedings of the Oklahoma Academy of Science. 84: 41-51.

  7. Bhargavi, G., Arya, M., Jambhulkar, P.P., Singh, A., Rout, A.K., Behera, B.K., Chaturvedi, S.K. and Singh, A.K. (2024). Evaluation of biocontrol efficacy of rhizosphere dwelling bacteria for management of Fusarium wilt and Botrytis gray mold of chickpea. BMC Genomic Data. 25(1): 7. https://doi.org/ 10.1186/s12863-023-01178-7. 

  8. Booth, C. (1977). Fusarium: Laboratory guide to the identification of the major species. Commonwealth Mycological Institute.

  9. Domsch, K.H. and Gams, W. (1980). Compendium of soil fungi. Academic Press.

  10. Ellis, M.B. (1971). Dematiaceous hyphomycetes. Commonwealth Mycological Institute.

  11. FAOSTAT. (2021, April 2). Retrieved from http://faostat3.fao.org/ faostat-gateway 

  12. Ganeshamoorthi, P. and Dubey, S.C. (2013). Morphological and pathogenic variability of Rhizoctonia solani isolates associated with wet root rot of chickpea in India. Legume Research: An International Journal. 38(3): 389-395. doi: 10. 5958/0976-0571.2015.00123.X.

  13. Gaur, P.M. and Gowda, C.L. (2005). Trends in World Chickpea Production, Research and Development. In E.J. Knights and R. Merril (Eds.), Chickpea in the Farming Systems. Queensland: Australia Publishers.

  14. Gondal, A. S., Rauf, A. and Naz, F. (2019). Anastomosis groups of Rhizoctonia solani associated with tomato foot rot in Pothohar Region of Pakistan. Scientific Reports. 9: 1-12. https://doi.org/10.1038/s41598-019-40043-5.

  15. Hidayah, N. and Yulianti, T. (2015). Bacillus cereus antagonism test against Rhizoctonia solani and Sclerotium rolfsii. Buletin Tanaman Tembakau, Serat dan Minyak Industri. 7(1): 1-8.

  16. Howard, F.S. and Gent, D.H. (2007). Damping-off and seedling blight (pp. 1-4).

  17. Huang, X., Zhang, N., Yong, X., Yang, X. and Shen, Q. (2012). Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43. Microbiological Research. 167(3): 135-143.

  18. Hussein, S.N. (2019). Biological control of root rot disease of cowpea (Vigna unguiculata) caused by the fungus Rhizoctonia solani using some bacterial and fungal species. Arab Journal of Plant Protection. 37(1): 31-39. https://doi.org/10. 22268/AJPP-037.1.031039. 

  19. Hussein, S.N. (2022). Detection of the causal agent of chickpea root and crown rot disease and its biological control. Indian Journal of Ecology. 49(18): 602-609.

  20. Hussein, S.N. (2024). Some chemical and biological methods to control pepper root rot disease in Iraq. Indian Journal of Agricultural Research. 58(4): 706-712. doi: 10.18805/IJARe.AF-831.

  21. Hussein, S.N. and Ibrahim, T.A. (2018). Biological control of the charcoal rot disease of pepper caused by Macrophomina phaseolina. Scientific Journal of King Faisal University. 19(2): 27-36.

  22. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025a). Biological control of tomato root rot disease by indigenous rhizobacteria in greenhouse setting. Indian Journal of Agricultural Research. 59(7): 1087-1094. doi: 10.18805/IJARe.AF-916.

  23. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025b). Eco-friendly management of Rhizoctonia solani, the cause of tomato root rot disease. Agricultural Science Digest. 45(4): 657-664. doi: 10.18805/ag.DF-709.

  24. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025c). Rhizobacteria from Iraq: A novel biocontrol approach for tomato root rot disease. Agricultural Science Digest. 45(4): 672-678. doi: 10.18805/ag.DF-707.

  25. Klich, M. A. (2002). Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures.

  26. McClenny, N. (2005). Laboratory detection and identification of Aspergillus species by microscopic observation and culture: The traditional approach. Journal of Medical Mycology. 43: S125-S128.

  27. Parmeter, J.R. and Whitney, H.S. (1970). Taxonomy and nomenclature of the imperfect stage. In J. R. Parmeter (Ed.), Rhizoctonia solani: Biology and pathology. University of California Press.

  28. Pleban, S., Ingel, F. and Chet, I. (1995). Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. European Journal of Plant Pathology. 101: 665-672. https://doi.org/10.1007/BF01874870. 

  29. Rivera, M.C., Wright, E.R., Lopez, M.V., Garda, D. and Barrague, M.Y. (2004). Promoting of growth and control of damping-off Rhizoctonia solani of greenhouse tomatoes amended with vermicompost. International Journal of Experimental Botany. 229-235.

  30. Zarrin, F.M., Saleem, M., Zia, T., Sultan, M., Slam, A., Rehman, R. and Chandhary, M.F. (2009). Antifungal activity of plant growth promoting rhizobacteria isolates against Rhizoctonia solani in wheat. African Journal of Biotechnology. 8(2): 219-225.
Published In
Agricultural Science Digest
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    ABSTRACT

    Background: Chickpeas are a prevalent and significant crop in the agricultural sectors of northern Iraq.  In recent years, production has significantly declined due to diseases affecting chickpea plants. Investigating the pathogenic fungi responsible for these diseases is essential to restore production levels to their prior and improved states.

    Methods: Samples of diseased chickpea plants were collected from fields in the Erbil, Sulaymaniyah and Salah al-Din governorates in northern Iraq. Isolated associated fungi led to the identification of the dominant pathogenic species. A pathogenicity test was performed on the fungal isolates, showing  the most aggressive strain for subsequent antagonism testing against three rhizobacterial isolates: Azotobacter chroococcum, Bacillus cereus and B. pumilus. This testing was conducted both in vitro and in a greenhouse setting,  using  single treatments and combinations.

    Result: The results showed that the pathogenic fungus Rhizoctonia solani was predominant, with the strain Srs-21 exhibiting the highest virulence in vitro. The three rhizobacterial isolates, A. chroococcum, B. cereus and B. pumilus, showed high efficacy in inhibiting the pathogenic fungus in vitro on potato dextrose agar (PDA) medium.  In the greenhouse, the triple bacterial interaction treatment showed a seed germination rate of 100% and diminished disease incidence and severity to 0%, in contrast to the positive control treatment, which recorded 90% and 70%, respectively. It also showed the highest rate of rise in the dry weight of plants, signifying enhanced growth metrics.

    INTRODUCTION

    Chickpea (Cicer arietinum L.) is recognized as an important leguminous crop on a global scale. This legume is recognized as the most productive in South Asia and is the third most produced globally, trailing only beans and peas (Gaur and Gowda, 2005). In 2003, Iraq’s total production was documented at 104,000 metric tons, followed by a steady decrease that culminated in an average of 203 metric tons by 2019 (FAOSTAT, 2021). The chickpea crop encounters several pathogens, including Fusarium solani, F. oxysporum, Macrophomina phaseolina and Rhizoctonia solani (Kuhn), which are considered  as major factors in seed rot, damping off in seedlings and the deterioration of roots and crowns (Agrios, 2005; Hussein et al., 2025c). Rhizoctonia solani is a facultative parasitic fungus categorized within the phylum Basidiomycota, subphylum Agaricomycotina, class Agaricomycetes, order  Cantharellales  and family  Ceratobasi- diaceae. The sexual basidiospores are generally generated on infected plants; nonetheless, the fungus primarily reproduces in its sterile anamorph stage (Alexopoulos et al., 1996; Agrios, 2005). This stage is marked by the emergence of rapidly expanding, brown-hued hyphae with significant diameters of 15-20 µm. These structures display distinct constrictions at their points of emergence and exhibit the formation of transverse septa in the branches close to the emergence area, characterized by double holes and multiple nuclei. The development of barrel cells or irregular shapes is a key feature, which amalgamate to form a mass known as sclerotia. This sclerotium functions as a mechanism for the fungus to endure adverse environmental conditions (Blazier and Conway, 2004; Howard et al., 2007). The fungus affects the plant during different growth stages, infecting seeds in the soil, which results in seed and seedling rot, leading to damping off both prior to and following emergence and impacting the roots and crown of the plant (Rivera et al., 2004; Agrios, 2005). There is an increasing focus on the use of environmentally friendly beneficial microorganisms for managing plant diseases (Hussein et al., 2025b). The rhizosphere-living bacteria that are antagonistic represent a significant and effective alternative approach for managing diseases in chickpea.  Rhizosphere-living bacteria function as biocontrol agents through various mechanisms, including the production of antibiotics, siderophores and enzymes, which effectively inhibit the proliferation of pathogenic microbes (Bhargavi et al., 2024). This study focused on identifying the fungus that causes rot root disease in chickpeas in northern Iraq, while also assessing various rhizobacterial isolates for their potential as biocontrol agents against the pathogen.

    MATERIALS AND METHODS

    Isolation and identification of the causal agents of the disease
     
    Samples were collected from chickpea plants showing signs of root rot disease, which included yellowing and wilting leaves, along with rotting that displayed brown to black discoloration in the roots and crown region (Fig 1). The samples were collected from the fields in the Erbil, Sulaymaniyah and Salah al-Din governorates. Root samples underwent a washing procedure using running water for a duration of 15 minutes, after which they were cut into small segments of 0.5 cm in size. The segments underwent surface sterilization with a 2% sodium hypochlorite solution for 2 minutes, followed by two rinses with sterile distilled water and were subsequently dried using sterile filter paper in a laminar flow cabinet. Four samples were positioned on PDA medium within 9 cm petri dishes. The medium underwent sterilization in an autoclave at a temperature of 121oC, maintaining a pressure of 1.5 kg/cm2 for a duration of 20 minutes. The dishes were subsequently incubated at 25±1oC for a duration of 7 days.  The fungal isolates were subjected to examination with a light microscope, resulting in their identification at both the genus and species levels through morphological characteristics and alignment with established taxonomic keys (Parmeter and Whitney, 1970; Ellis, 1971; Booth, 1977; Domsch et al., 1980; Klich, 2002; McClenny, 2005). The proportion of appearance and frequency of the fungal isolates has been calculated utilizing the formulas established by Hussein (2024).




     

    Fig 1: Symptoms of rot roots on infected chickpea plants.


     
    Anastomosis interactions test of R. solani isolates
     
    The number of nuclei per cell of R. solani isolates was counted by growing the isolates on the PDA and staining the hyphae with 1ug/ml of 42 -6 diamidino-2-phenylindole, petri plates examined under a fluorescent microscope (Gondal et al., 2019). Nine isolates of R. solani belong to nine AGs provided by Microbiology laboratory/ University of Mustansiriyah, used as tester strains of the respective AGs, the fifty-seven unknown strain of R. solani were paired with tester strains, by placing an agar disc (5 mm) of the hyphal growth of unknown isolate at the one side of sterile glass slide covered with a layer of the 1.5% water agar and at the other side of the slide a similar disc of the tester strain and incubated for 72 hours at a 25oC, the hyphal interactions between tester strain  and unknown strain was determined according to the method of Gondal et al., (2019).   
     
    Pathogenicity testing assay
     
    The pathogenicity of fifty- seven isolates of R. solani was evaluated on a sterile PDA. Plates were inoculated at the center with a 0.5 cm diameter disc of R. solani isolates that had been cultivated on PDA culture medium for seven days. The dishes were incubated at a temperature of 25 ±1oC for a duration of two days. Each plate was then sown with 10 seeds of the Marrakechy cultivar of chickpeas, which had undergone surface sterilization using a 2% sodium hypochlorite solution for two minutes, followed by two rinses with sterile distilled water. The seeds were then dried using sterile filter paper and arranged in a circular pattern near the edge of the plate. Four replications were conducted for each treatment, including the control treatment of  seedling only without the fungus. The dishes were incubated at a temperature of 25±1oC for a duration of 14 days, under a lighting schedule of 12 hours per day. The findings were documented and the germination percentage was determined using the equation provided by Hussein (2019).

     
    Antagonistic activity in vitro
     
    Evaluation of antagonism activity of three rhizobacterial isolates of A. chroococcum, B. cereus and B. pumilus which optioned from Mustansiriyah University, laboratory of microbiology, against the most virulent isolate of R. solani (Srs-21), these rhizobacterial isolates isolated from the rhizosphere of healthy chickpea plants in Iraq (Hussein, 2022). On the PDA a 0.5 cm diameter disc of the fungal isolate placed at the edge of the petri plate (about 2 cm out of the edge) and in the opposite side a loop full of each bacterial isolate 109 CFU/ml placed individually, plates incubated at 25±1 Co for 7 days, each treatment repeated 4 times, antagonism activity calculated using the following formula:

     
    Where:
    b = The distance between the bacterial line towards the fungal growth.
    f = The distance of fungal growth towards the bacterial line.
     
    Greenhouse evaluation
     
    Inoculum of the fungal pathogen Srs-21 were prepared by growing it on the seeds of local millet, by adding 5 discs (0.5 cm diameter) of the pathogen in the culture medium (PDA) of 5 days old to a 250 ml glass beaker containing autoclaved millet seeds mixed with 10 ml of distilled and sterile water and incubated for 14 days. The inoculum was added to mixture of sterilized soil and compost in 1 kg plastic pots at a ratio of 1% (w/w). Each pot irrigated with 10 ml of the rhizobacterial inoculum of A. chroococcum, B. cereus and B. pumilus which were grown on the nutrient broth with concentration of 109 CFU/ml added in the same time of the seed planting, each treatment repeated 4 times and control treatments irrigated with distilled sterile water. Five seeds of chickpea (Marrakechy cultivar) were planted in each pot surface sterilized with 1% sodium hypochlorite solution. Pots were distributed according to a completely randomized design (CRD) and watered carefully. The percentage of germination was calculated after 14 days and the disease incidence was calculated after 40 days of planting and the disease severity index was measured according to the disease rating scale and the formulas described by Hussein, 2022). The dry weight of plants was also calculated to measure the effect of bacterial isolates on plant growth parameters.





    RESULTS AND DISCUSSION

    Fungi associated with infected chickpea plants
     
    The findings from the isolation and diagnosis  showed that R. solani was the most prevalent fungus, appearing in 77% of the samples, with a frequency percentage of 59.5% (Table 1). All R. solani isolates exhibited fundamental characteristics, including branching near the terminal septum of cells within the mycelium, marked by a narrowing at the starting point of the branches. Additionally, barriers appeared near the starting point of the branches, along with barriers featuring double holes (Fig 2). The coloration of the fungal colonies transitioned from white initially to light brown, then  deep brown (Fig 2). All isolates produced sclerotia that diversified in size and form, with diameters ranging from 1-3 mm and with thick brown walls. Some isolates additionally produced barrel cells of varying sizes, consistent with the characteristics described by Parmeter and Whitney (1970). These results align with the findings of Ganeshamoorthi and Dubey (2013), who isolated 50 isolates of R. solani from chickpea fields across 10 various states within India.  Hussein (2022) demonstrated that within the soil-borne pathogenic fungi collected from infected chickpea plants in Iraq, R. solani emerged as the most prevalent, with an appearance rate of 85.5% and a frequency of 61.2%.

    Table 1: Fungal isolates associated with the diseased chickpea plants.



    Fig 2: Morphological characteristics of R. solani.


     
    Anastomosis group test
     
    The findings displayed in Table 2 regarding hyphal dyeing with DAPI and microscopic examinations of R. solani isolates indicated that all isolates were multinucleate. The interactions of hyphal anastomosis among the 57 isolates of R. solani and the tester strains of the respective AGs revealed that 40% of the isolates were classified under AG 4, which was the most prevalent. In contrast, 12%, 30%, 9%, 5% and 4% of the isolates were categorized into AG 1, AG 3, AG 5, AG 7 and AG 10, respectively (Fig 3).  Hussein (2022) discovered that 58% of the R. solani isolates isolated from infected chickpea plants in the Nineveh and Duhok governorates in northern Iraq were classified within the AG3 group and 3% belong to AG4.

    Table 2: Pathogenic test of R. solani isolates using chickpea seeds.



    Fig 3: Distribution of R. solani isolates categorized by anastomosis groups.



    Pathogenicity assay
     
    The findings from the pathogenicity test indicated that the germination rate varied from 0% to 65%, in contrast to the control group, which exhibited a 100% germination rate (Table 2). The Srs-21 isolate, classified as AG3 from the Sulaymaniyah governorate, demonstrated a complete inhibition of seed germination, achieving 0% germination and outperforming all other isolates in this regard (Fig 4). The variation in the pathogenic ability of these fungal isolates can be attributed to genetic differences among individuals of the same species, the response of the plant host varies depending on the isolates, which is also attributed to genetic variation, particularly since they are sourced from various regions.

    Fig 4: Pathogenicity test of R. solani isolates in vitro.


     
    Antagonistic activity in vitro
     
    The findings from the antagonism test involving three rhizobacterial isolates against the fungal pathogen R. solani (Srs-21 isolate) indicated that the B. pumilus isolate demonstrated the highest level of antagonistic activity at 95.0%. This was followed by B. cereus, which showed an antagonism activity of 91.4%, while A. chroococcum exhibited a lower activity of 66.4% (Table 3) (Fig 5).

    Table 3: Antagonistic activity of the rhizobacterial isolates.



    Fig 5: Antagonism activities of rhizobacterial isolates against R. solani.


     
    Greenhouse assay
     
    The findings indicated that individual, dual and triple treatments of the rhizobacterial isolates enhanced seed germination percentages, ranging from 80% to 100%, in contrast to the positive control treatment, which recorded 70% (Table 4). The combination treatment of A. chroococcum, B. cereus and B. pumilus  proved a substantial efficacy in diminishing disease incidence and severity to 0%, in contrast to the positive control treatment, which recorded 90.0% and 70.0%, respectively (Table 4).  A combination treatment of B. cereus and B. pumilus resulted in disease incidence and severity rates of 35% and 28%, respectively.  Regarding individual treatments, while they resulted in a 70-80% reduction in disease incidence, they did not significantly decrease disease severity, which ranged from 61-65%. The triple treatment resulted in the most significant increase in the average dry weight of chickpea plants,  reaching 15 g/plant, in contrast to the positive and negative control treatments, which recorded 3.8 and 12.8 g/plant, respectively.  Bacteria in the rhizosphere of plants safeguard the roots by inhibiting fungal growth and invasion into the root cells and the adjacent stalk tissues near the soil surfaces (Hussein and Ibrahim, 2018). The inhibitory action of A. chroococcum on R. solani growth may be attributed to its capacity to synthesize metabolites and organic chemicals, including stearic acid, enzymes, antibiotics and hydrogen cyanide (Hussein et al., 2025a). The results aligned with those reported by Hussein (2019), which illustrated the efficacy of B. pumilus in managing cucumber root rot disease induced by F. solani. Aljuboori et al., (2022) illustrated that the Rhizoctonia solani - Meloidogyne javanica complex disease affecting chickpea roots can be biologically managed using a rhizobacterial isolate of Pseudomonas sp., which also enhanced both the dry and wet weight of the plants, significantly elevating the activity of polyphenol oxidase and peroxidase enzymes, as well as the total phenolic content. Agarwal et al., (2017) shown that B. pumilus significantly suppressed the development of R. solani and F. oxysporum through the synthesis of chitinolytic enzymes and the generation of antibiotics. These results align with the findings of Zarrin et al., (2009), who revealed the capacity of A. chroococcum to prevent the development of several pathogenic fungus, including R. solani. Al Isawy (2010) demonstrated the efficacy of A. chroococcum as a means of biological management in suppressing the growth of R. solani on PDA medium.  Hidayah and Yulianti (2015) discovered that B. cereus reduced growth of R. solani and Sclerotium rolfsii in vitro by 68.9% and 33%, respectively, on PDA medium. Additionally, B. cereus, obtained from the rhizosphere of mustard plants, prevented the growth of R. solani, Pythium ultimum and S. rolfsii (Pleban et al., 1995).  Huang et al., (2012) demonstrated  that B. pumilus altered the fungal hyphae of R. solani in vitro, resulting in the increase of cytoplasmic vacuoles and cytoplasmic exudation within the fungal cells.

    Table 4: Biological control of chickpea root rot disease in greenhouse.

    CONCLUSION

    In vitro experiments demonstrated that the three bacterial isolates of A. chroococcum, B. cereus and B. pumilus, obtained from the rhizosphere of healthy chickpea plants, exhibited superior capabilities in inhibiting the growth of the highly aggressive isolate of the pathogenic fungus Rhizoctonia solani (Srs-21) on the PDA culture medium.  In controlled greenhouse conditions, the integration of the three bacterial isolates yielded the most favorable outcome in enhancing the germination rate of chickpea seeds, while also decreasing both the incidence and severity of the disease, alongside promoting the growth parameters of the plants as measured by dry weight.

    ACKNOWLEDGEMENT

    The author would like to acknowledge the role of Mustansiriyah University, Iraq, Baghdad in the completion of this research.

    CONFLICT OF INTEREST

    The author declares that they have no conflicts of interest.

    REFERENCES


    1. Agarwal, M., Dheeman, S., Dubey, C., Kumar, P., Maheshwari, D. and Bajpai, V. (2017). Differential antagonistic responses of Bacillus pumilus MSUA3 against Rhizoctonia solani and Fusarium oxysporum causing fungal diseases in Fagopyrum esculentum Moench. Microbiological Research. 205: 40-47.

    2. Agrios, G.N. (2005). Plant Pathology (5th ed.). Academic Press.

    3. Al Isawy, J.M. (2010). Integrated management of eggplant’s damping off caused by the fungus Rhizoctonia solani Kuhn (Master’s thesis). University of Baghdad, College of Agriculture, Department of Plant Protection, Iraq.

    4. Alexopoulos, C.J., Mims, C.W. and Blackwell, M. (1996). Introductory Mycology (4th ed.). John Wiley and Sons.

    5. Aljuboori, F.K., Ibrahim, B.Y. and Mohamed, A.H.  (2022). Biological control of the complex disease of Rhizoctonia solani and root-knot nematode Meloidogyne javanica on chickpea by Glomus spp. and Pseudomonas sp. Iraqi Journal of Agricultural Sciences. 53(3): 669-676. https://doi.org/ 10.36103/ijas.v53i3.1577.

    6. Blazier, S.R. and Conway, K.E. (2004). Characterization of Rhizoctonia solani isolates associated with patch disease on turf grass. Proceedings of the Oklahoma Academy of Science. 84: 41-51.

    7. Bhargavi, G., Arya, M., Jambhulkar, P.P., Singh, A., Rout, A.K., Behera, B.K., Chaturvedi, S.K. and Singh, A.K. (2024). Evaluation of biocontrol efficacy of rhizosphere dwelling bacteria for management of Fusarium wilt and Botrytis gray mold of chickpea. BMC Genomic Data. 25(1): 7. https://doi.org/ 10.1186/s12863-023-01178-7. 

    8. Booth, C. (1977). Fusarium: Laboratory guide to the identification of the major species. Commonwealth Mycological Institute.

    9. Domsch, K.H. and Gams, W. (1980). Compendium of soil fungi. Academic Press.

    10. Ellis, M.B. (1971). Dematiaceous hyphomycetes. Commonwealth Mycological Institute.

    11. FAOSTAT. (2021, April 2). Retrieved from http://faostat3.fao.org/ faostat-gateway 

    12. Ganeshamoorthi, P. and Dubey, S.C. (2013). Morphological and pathogenic variability of Rhizoctonia solani isolates associated with wet root rot of chickpea in India. Legume Research: An International Journal. 38(3): 389-395. doi: 10. 5958/0976-0571.2015.00123.X.

    13. Gaur, P.M. and Gowda, C.L. (2005). Trends in World Chickpea Production, Research and Development. In E.J. Knights and R. Merril (Eds.), Chickpea in the Farming Systems. Queensland: Australia Publishers.

    14. Gondal, A. S., Rauf, A. and Naz, F. (2019). Anastomosis groups of Rhizoctonia solani associated with tomato foot rot in Pothohar Region of Pakistan. Scientific Reports. 9: 1-12. https://doi.org/10.1038/s41598-019-40043-5.

    15. Hidayah, N. and Yulianti, T. (2015). Bacillus cereus antagonism test against Rhizoctonia solani and Sclerotium rolfsii. Buletin Tanaman Tembakau, Serat dan Minyak Industri. 7(1): 1-8.

    16. Howard, F.S. and Gent, D.H. (2007). Damping-off and seedling blight (pp. 1-4).

    17. Huang, X., Zhang, N., Yong, X., Yang, X. and Shen, Q. (2012). Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43. Microbiological Research. 167(3): 135-143.

    18. Hussein, S.N. (2019). Biological control of root rot disease of cowpea (Vigna unguiculata) caused by the fungus Rhizoctonia solani using some bacterial and fungal species. Arab Journal of Plant Protection. 37(1): 31-39. https://doi.org/10. 22268/AJPP-037.1.031039. 

    19. Hussein, S.N. (2022). Detection of the causal agent of chickpea root and crown rot disease and its biological control. Indian Journal of Ecology. 49(18): 602-609.

    20. Hussein, S.N. (2024). Some chemical and biological methods to control pepper root rot disease in Iraq. Indian Journal of Agricultural Research. 58(4): 706-712. doi: 10.18805/IJARe.AF-831.

    21. Hussein, S.N. and Ibrahim, T.A. (2018). Biological control of the charcoal rot disease of pepper caused by Macrophomina phaseolina. Scientific Journal of King Faisal University. 19(2): 27-36.

    22. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025a). Biological control of tomato root rot disease by indigenous rhizobacteria in greenhouse setting. Indian Journal of Agricultural Research. 59(7): 1087-1094. doi: 10.18805/IJARe.AF-916.

    23. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025b). Eco-friendly management of Rhizoctonia solani, the cause of tomato root rot disease. Agricultural Science Digest. 45(4): 657-664. doi: 10.18805/ag.DF-709.

    24. Hussein, S.N., Safaie, N., Shamsbakhsh, M. and Al-Juboory, H.H. (2025c). Rhizobacteria from Iraq: A novel biocontrol approach for tomato root rot disease. Agricultural Science Digest. 45(4): 672-678. doi: 10.18805/ag.DF-707.

    25. Klich, M. A. (2002). Identification of common Aspergillus species. Centraalbureau voor Schimmelcultures.

    26. McClenny, N. (2005). Laboratory detection and identification of Aspergillus species by microscopic observation and culture: The traditional approach. Journal of Medical Mycology. 43: S125-S128.

    27. Parmeter, J.R. and Whitney, H.S. (1970). Taxonomy and nomenclature of the imperfect stage. In J. R. Parmeter (Ed.), Rhizoctonia solani: Biology and pathology. University of California Press.

    28. Pleban, S., Ingel, F. and Chet, I. (1995). Control of Rhizoctonia solani and Sclerotium rolfsii in the greenhouse using endophytic Bacillus spp. European Journal of Plant Pathology. 101: 665-672. https://doi.org/10.1007/BF01874870. 

    29. Rivera, M.C., Wright, E.R., Lopez, M.V., Garda, D. and Barrague, M.Y. (2004). Promoting of growth and control of damping-off Rhizoctonia solani of greenhouse tomatoes amended with vermicompost. International Journal of Experimental Botany. 229-235.

    30. Zarrin, F.M., Saleem, M., Zia, T., Sultan, M., Slam, A., Rehman, R. and Chandhary, M.F. (2009). Antifungal activity of plant growth promoting rhizobacteria isolates against Rhizoctonia solani in wheat. African Journal of Biotechnology. 8(2): 219-225.
    In this Article
      Contact us
      Follow us
      Published In
      Agricultural Science Digest

      Editorial Board

      View all (0)