Isolation and diagnosis of some associated fungi with cowpea root rot disease and testing its pathogenicity
Ahmed Jassim Awad1 and Theyab A.Farhan1
1 Department of Plant Protection, College of Agriculture, University of Anbar, Anbar, Iraq; [email protected]
* Corresponding author, [email protected];
Available from: http://dx.doi.org/10.21931/RB/2023.08.02.91
Execute search by date 1/4/2021, The results of collecting samples from the regions of Anbar, Baghdad, Salah al-Din and Wasit showed that cowpea root rot disease is widespread in all studied areas, and The results of isolation phenotypic and molecular diagnosis showed the presence of different isolation of fungi that infected cowpea root, such as Fusarium nygamai (Fu1), F. nygamai (Fu2), F. solani (Fu3), F. solani(Fu4), Rhizoctonia solani (Rh5), and Fusarium solani (Fu6), The results of the pathogenicity test on radish seeds showed that tested isolates were significantly decreased germination percentage of radish seeds of water Ager, and the most effective isolation was F. solani (Fu4). The infection rate was 90% compared to 0.00% of the control media treatment, which was uncontaminated by the pathogenic fungus. Isolated fungi showed a difference in the percentage and severity of infection on cowpea seedlings and seedlings, as the isolate of F.nygamai (Fu1) achieved the highest infection rate of 66.67 % and the severity of disease at 75%. All fungal isolates significantly increased the rate and severity of infection on seed radish compared with the control treatment not contaminated with pathogenic fungi by 0.0%.
Keywords: Vegan unguiculata; Rhizoctonia solani; and Fusarium solani; PCR.
Cowpea (Vegan unguiculata) is a multi-purpose crop belonging to the legume family, and it is grown in dry and semi-dry tropical regions1. The global cowpea production is estimated at 4.5-6.5 million tons, and 80% of production is from Africa,2. In Iraq, the cultivated area of cowpeas reached 72,507 m2, and the output of one dunam ranged between 2600-2768 kg,3. Copwea is affected by many diseases, such as root rot and damping off, as these diseases cause losses that may reach 55% of the cowpea production,4-5. Moreover, the losses can get 100% yield in the appropriate environmental conditions,6-7. Root and stem rot disease is caused by fungi such as Fusarium solani and Rhizoctonia solani, the most dangerous fungi in cowpea, 8-9, because they contain toxins and enzymes (fludarabine, fusaric acid, javanicine, pectin methyl esterase and galacturonic enzymes), as well as their ability to tolerate toxins and antibiotics secreted by other organisms, 10-11. Fusarium solani and Rhizoctonia solani fungi are classified based on their phenotypic characteristics,12,13-14. However, several studies have been using Polymerase Chain Reaction technology (PCR) as a modern application in diagnosing fungi based on the nitrogenous bases sequence traits in a single DNA strand, 15, 16-17. Found 18-19 that the root rot disease and damping off of cowpea seedlings, caused by Fusarium solani and Rhizoctonia solani, have caused significant losses in yield, and they are the most common pathogens that infect cowpea.
MATERIALS AND METHODS
Samples of infected cowpea plants by fungi were collected from different governorates of Iraq, Anbar, Baghdad and Wasit and Salah Al-Din, in June, July, August and September 2021. that showed symptoms of root rot diseases were collected, such as leaves wilting, burning of the leaves edges, discoloration of the roots and plant death,20.
Isolation of associated fungi with cowpea root rot disease
Infected parts of cowpea plants were cut into small pieces 0.5-1 cm in length and superficially sterilized using a solution of 6% sodium hypochlorite (Naocl) free chlorine, then washed with sterilized distilled water to remove the remnants of the sterilizing solution and placed on a filter paper, then transferred into Petri dishes 9 cm containing Potato Dextrose Agar (PDA) and incubated at 25 ± 2 °C for 5-7 days until a formation of the fungal growth, the growing fungi were purified by taking a small part of the edge of The fungal growths, and transferred in the center of another Petri dishes containing (PDA) average of three replicates then incubated at a temperature of 25 ± 2 °C.
Fungi isolates were diagnosed after purification of the growth of fungal colonies was completed on the PDA medium., Microscopic slides were prepared and checked using a light microscope; the fungi were diagnosed to the genus level depending on the phenotypic characteristics by using the approved taxonomic keys,21-22.
DNA of six isolates, F. nygamai (Fu1), F. nygamai (Fu2), F. solani (Fu3), F. solani (Fu4), R. solani (Rh5) and F. solani (Fu6), Scientific Progress Laboratory – Baghdad- Al-Harithiya, was extracted by using standard kit ABI OPure from the American company., The extraction was performed in the Scientific Progress Laboratory, located in Baghdad,/Iraq, and diagnosis steps were described by 23. The polymerase chain reaction (PCR) was prepared according to 24 and the primersITS1.5'-TCCGTAGGTGAACCTGCGG-3.and.ITS4 5'-TCCTCCGCTTATTGATATGC-3(Macrogen Company, Korea) were used. The lyophilized primers were dissolved in nuclease-free water to give a final concentration of 100 μl. After completing the DNA amplification and migration steps on agarose gel, the samples were sent to the Korean Macrogen Company to obtain Sequencing (Nitrogenous bases sequence).
Pathogenicity test on radish seeds
The fungus Fusarium spp and Rhizoctonia solani isolations were tested on radish seed in the Plant Protection Department, /College of Agriculture, /Anbar University, Iraq. The test of pathogenicity was carried out according to the method of 25. Data were statistically analyzed according to the completely randomized design (CRD), and the germination percentage of radish seeds average of three replicates calculated after 14 days of sowing seeds on PDA medium was calculated and compared with the control treatment them by following the equation below: Germination percentage = (number of germinated seeds)/(total seed number) × 100
Pathogenicity test on cowpea seed and seedling
Inoculum of fungal isolates was prepared according to the26 by using millet seeds, the soil was sterilized with commercial formalin, then sterilized soil was placed in sterilized plastic pots with a capacity of 2 kg, and pathogenic fungi inoculum was added at a rate of 1% (weight-weight), the process was repeated average of three replicates on all fungal isolates, and then the pot was rinsed with water and covered with polyethylene bags for 3 days after that cowpea seeds were planted at a rate of three seeds per pot, the sterilized cowpea seeds were placed without adding the pathogenic fungus inoculum as the control treatment, three replicates were used for each treatment. The test was carried out by using a CRD design, and the percentage of germination was calculated according to the following equation:
Germination percentage = (number of infected plants)/(total number of plants) x 100
The percentage of infected severity was calculated according to equation 27 as follows:
% Infected severity=
0= no injury (healthy roots )
1 light brown coloration in the secondary roots
2 light brown coloration in the secondary roots, with a small part of the main root
3 dark brown coloration in the main roots, with no rotting of the stem bases
4 dark brown coloration of the roots, with rotting of the stem bases
5 plant death
The results in Table (1) showed that cowpea root rot diseases were widespread in all studied sample collection areas, which is consistent with what was mentioned by 28-9; the condition is dangerous and widespread in most countries where the crop is grown.
The results of the phenotypic diagnosis in Table (2) and Figure 1 showed the presence of fungi (Fusarium spp., and Rhizoctonia solani), which were associated with cowpea root rot disease, which is the leading cause of the disease,29-30.
Table 1. The infected sample of cowpea plants collection areas and the collection date
Figure 1. is the morphological and microscopic check of fungus colonies in the PDA medium.
a.b Fusarium solani and c. d Rhizoctonia solani
Table 2. The associated fungi with cowpea root rot disease were isolated and diagnosed phenotypically to the genus level
The results of the molecular diagnosis by using the PCR technique in Table (3) and Figure (2), according to the arrangement of the nitrogen bases in the single DNA strand, showed that most types of fungi that cause cowpea root rot disease belong to Fusarium spp and Rhizoctonia solani. These results reinforce what was indicated by 34-35, that these fungi are the leading cause of the disease.
Figure 2. Amplification of ITS gene for unknown fungal species on agarose gel
Table 3. The associated fungi with cowpea root rot were diagnosed by using the PCR technique
Pathogenicity test on radish seeds
The results in Table (4) and Figure (3) showed that the pathogenicity test for some isolates of fungi (Fusarium nygamai (Fu1)- Khalidiya, Fusarium nygamai (Fu2)- Amriya, Fusarium solani (Fu3)- Essaouira, Fusarium solani (Fu4) Radwaniya, Rhizoctonia solani ( Rh5), Fusarium solani (Fu6), Fusarium solani (Fu7) achieved a significant reduction in the percentage of germination of radish seeds compared to the control treatment (untreated with pathogenic fungi) in which the infection rate was 0.0% 20, 36-37 or. 38-39
Table 4. Effect of some isolated fungi from cowpea roots on the germination of radish seeds on water agar medium
Figure 3. Effect of fungi isolates on the germination of radish seeds
Pathogenicity test on cowpea seed and seedling
The results of pathogenicity examination testing of cowpea seeds in Table (5) and Figure (4) showed that all the tested isolates (Fusarium solani, Fusarium nygamai, Fusarium nygamai, Fusarium solani, Fusarium solani, Rhizoctonia solani) varied in their impact on the ratio and severity of cowpea root rot disease, where, the isolate of Al-Anbar - Khalidiya Island (Fusarium nygamai) (Fu1) achieved the highest percentage and severity of infection, which amounted to 66.67% and 75%, respectively, and all tested isolates achieved a significant increase in the ratio and severity of disease compared to the control treatment (without adding pathogenic fungi), which reached 0.0% for both.
Table 5. Effect of some isolated fungi from cowpea roots on infecting the seeds and seedlings of cowpea under field conditions
The phenotypic diagnosis of F. solani was made based on the shape of the large conidia (Macrioconidia), the presence or absence of small conidia, and the presence of Chlamydiospores whose location is terminal or intertwined, 31-32—F.solani based on the shape of the crescent-shaped Macroconida,33. As for the fungus Rhizoctonia solani, the thread is branched at right angles in the area of genesis with the presence of a constriction, and it is considered one of the diagnostic features of the fungus over other fungi,29. The difference in the effect of these isolates is due to their difference in the speed of penetration of plant cells and their ability to produce toxins and enzymes that break down plant cell walls; the results of this study agree 11.
The cowpea root rot disease spread in all the governorates from which the plant samples were collected. The leading cause of cowpea root rot disease is several species belonging to the genus Fusarium and the fungus Rhizoctonia solani—the difference in the pathogenicity of fungal isolates in causing cowpea root rot disease.
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Received: May 15, 2023/ Accepted: June 10, 2023 / Published: June 15, 2023
Citation: Awad A J, Farhan T A. Isolation and diagnosis of some associated fungi with cowpea root rot disease and testing its pathogenicity. Revis Bionatura 2023;8 (2) 91. http://dx.doi.org/10.21931/RB/2023.08.02.91