Isolation and molecular identification of Rhizoctonia solani and Fusarium solani isolated from cucumber (Cucumis sativus L.) and their control feasibility by Pseudomonas fluorescens and Bacillus subtilis

This study was conducted to isolate and molecularly identify two pathogenic fungi namely Rhizoctonia solani and Fusarium solani implicated in root rot and seedling damping-off disease of cucumber, Cucumis sativus. Besides, the efficacy of Pseudomonas fluorescens and Bacillus subtilis as bacterial agents in controlling these two pathogens was also evaluated in vitro and in a greenhouse pot experiment. Results of polymerase chain reaction (PCR) amplification and nucleotide sequence analysis using BLAST demonstrated that R. solani isolate was genetically different from the R. solani isolates in the National Centre for Biotechnology Information (NCBI). Therefore, it was recorded in GenBank under the accession number MK105921. P. fluorescens and B. subtilis showed a complete inhibition of the mycelial growths of R. solani and F. solani in vitro. In the pot experiments, soil treatment with a suspension of P. fluorescens and B. subtilis before planting significantly reduced the damping off of cucumber seedlings caused by R. solani and F. solani. This study suggests that these bacterial antagonists could have a good potential as biological control agents to protect cucumber plants from the infection with R. solani and F. solani.


Background
Cucumber, Cucumis sativus L., is one of the most important vegetable crops worldwide but, unfortunately, is affected by many pathogens in the field causing serious yield losses (Mohammed and Hasan 2018). Rhizoctonia solani Kühn and Fusarium solani (Mart.) Sacc. are devastating pathogens that cause damping-off diseases of cucumber plants either in the greenhouses or in the fields (De Curtis et al. 2010). Both R. solani and F. solani have a wide host range and are capable to resist the extreme environmental conditions. In addition, they are capable to remain for a long period in soil and plant residues (Šišić et al. 2018).
The accurate diagnosis of plant pathogenic fungi is one of the urgent needs due to its importance in reaching rapid and efficient disease management systems to reduce or prevent damages caused by the fungal infections (Balodi et al. 2017). In previous studies, morphological characteristics are widely used in diagnosing many pathogenic and non-pathogenic fungi (Rezaee et al. 2018). Although identification of fungi based on morphological characteristics may sometimes give accurate results, many researchers do not rely on these characteristics because they require high experience in the field of classification, especially when dealing with closely related fungal groups such as Fusarium spp. In addition to its need for time and effort, morphological methods are sometimes inaccurate due to some factors such as the nature of growth, light, darkness, humidity, and some other factors that affect the sizes, shapes, and colors of spores and fungal colonies. Therefore, many researchers have shifted to other methods such as polymerase chain reaction (PCR), which is one of the molecular techniques used to target a specific region of the organism's genome that can show the genetic relationships between the fungal isolates to support the morphological identification. This technique has been found to be one of the most important, accurate, and rapid techniques in the detection and identification of many microorganisms including fungi, bacteria, and viruses (Alhussaini et al. 2016).
Different measures for controlling of plant pathogens have been developed such as the use of resistant varieties (Borrelli et al. 2018), plant extracts (Han et al. 2018), chemical control (Karim et al. 2018), and biological control (Yendyo et al. 2017). Overuse of the chemicals can cause environmental problems, negatively affecting human health and increasing the pest resistance to pathogens. Thus, the need for using new biological approaches as alternative safe control methods has been increasing. (Nicolopoulou-Stamati et al. 2016). Many microbial species such as P. fluorescens and B. subtilis have been shown to effectively control plant pathogens (David et al. 2018).
Bacillus subtilis had a great potential as biocontrol agent against Rhizoctonia solani on maize and pepper plants (Madhavi et al. 2018). Zaim et al. (2018) found that B. subtilis was effectively used to suppress 93.67% of the disease caused by F. oxysporum f. sp. ciceris of chickpea and also improved the plant growth leading to increased plant height, root length, and fresh and dry weights of shoot and root. Notz et al. (2002) reported that P. fluorescens was effectively reduced root rot disease of bean (Phaseolus vulgaris) caused by Sclerotium rolfsii and Fusarium wilt of tomato caused by F. oxysporum f. sp. lycopersici and wheat caused by F. oxysporum. The objective of the present study was to isolate and molecularly identify R. solani and F. solani, the causal agents of cucumber seedlings damping off, and to evaluate the efficacy of P. fluorescens and B. subtilis as biocontrol agents against these fungi.

Sampling and fungal isolation
Root samples were collected from some diseased cucumber plants showing typical symptoms of damping off, growing in some plastic tunnels located in the desert farms in Najaf province, Iraq. The samples were transferred to the laboratory of plant diseases at the Faculty of Agriculture, Kufa University, Iraq, for isolation of fungal pathogens. Collected roots were washed by tap water, cut into small pieces, sterilized with NaOCl (1%) solution for 2 min, and washed with sterile distilled water to remove any residues of NaOCl. Root pieces were then dried using filter papers to remove any excess water and transferred to Petri dishes containing potato dextrose agar (PDA) media, supplemented with chloramphenicol antibiotic at a concentration of 200 mg/L. All Petri dishes were incubated at a temperature of 25 ± 2°C for about 4 days.

Morphological identification
The appeared fungi were purified and maintained on the same medium (PDA) and were used for morphological identification by microscopic examination.

Molecular characterization
The isolated fungi were molecularly identified using PCR technique and determining the nucleotide sequences as follows:

DNA extraction
From each fungal isolate, 50-100 mg of fresh 5-day-old colonies were taken by a sterile scalpel and transferred into an Eppendorf tube for DNA extraction using a specific extraction kit (Zymo Research, Cat. No. D6005), following the manufacturer's instructions. The quality and quantity of DNA extracted from each isolate were measured by a UV spectrophotometer (Thermo Scientific, Germany). DNA was then stored at − 20 until use.

PCR amplification and DNA sequencing of rDNA-ITS region
The internal transcribed spacer (ITS) region of R. solani isolates were amplified, using the universal primers ITS1 (TCCGTTGGTGAACCAGCGG) and ITS4 (TCCT CCGC TTATGATATGC) (White et al. 1990) using Taq DNA polymerase (Roche, Cat. No. 11146 173 001). The final volume of each PCR reaction mixture (sample) was 20 μl containing; 2 μ1 10 × PCR buffer, 1 μl of each primer (10 pmol), 2 μl dNTPs (2 mM), 3 μl template DNA (30 ng/μ1), 1 unit Taq polymerase, then completed to 20 μl by adding nuclease-free sterile distilled water. PCR amplification was performed using the following conditions: initial denaturation at 94°C for 1 min followed by 35 cycles each consisting of final denaturation at 94°C for 30 s, annealing temperature at 55°C for 30 s, initial extension for 1 min, and final extension at 72°C for 5 min (White et al. 1990. PCR-amplified products were electrophoretically separated on a 1% agarose gel for 140 min at 80 V and 400 mA and visualized with ethidium bromide under UV illumination, and images were captured using Vilber Lourmat, Taiwan, gel documentation system. For DNA sequencing, the PCR-amplified products were gel-purified using the FavorPrep PCR Purification Kit (Cat. No. FAGCK 001, Favorgen, Taiwan) and sent along with the primer pair (ITS1 and ITS4) to the Macrogen DNA sequencing service in Korea. PCR products were directly sequenced in both directions using the respective forward and reverse primers. The obtained nucleotide sequences were aligned and compared with the sequences belonged to the R. solani isolates in the NCBI database using the Basic Local Alignment Search Tool (BLAST) (Zhang et al. 2012). Using the MEGA6 software, multiple alignments of the nucleotide sequences and construction of phylogenetic trees were performed using the neighborjoining method (Tamura et al. 2013).

Pathogenicity of F. solani and R. solani to cucumber
Sterilized soil (1 kg/pot) was distributed in 14-cm diameter pots and F. solani and R. solani isolates separately grown on millet grains were added into the potting soil at 1% (W:W). The pots were watered and kept for 4 days before sowing. Seeds of cucumber were surface sterilized in 1% sodium hypochlorite solution for 2 min, then were rinsed in sterile distilled water, and sown in the pots (5 seeds/pot). Seeds were also sown in non-infested soil to serve as a control. Four replicates (pots) were established for each treatment, and the pots were randomly distributed in the greenhouse, where they were watered and fertilized as needed. The percentages of pre-and post-emergence damping off were determined after 20 days of sowing. The percentages of seed germination were calculated according to the following formula: seed germination (%) = (number of seeds germinated/total number of seeds) × 100.
Efficacy of P. fluorescens and B. subtilis as bio-control agents against R. solani and F. solani on cucumber

Preparation of fungal inoculums
Clean 250-ml flasks were filled with millet grains and autoclaved at 121°C for 1 h for two successive days. Five-millimeter diameter discs from the margins of the fungal colonies (R. solani or F. solani) were added to the flasks. Flasks were incubated at 25 ± 2°C for 2 weeks and were shaken every 2 days to ensure uniform colonization of the fungus.

Bacterial isolation
Soil samples were collected from the rhizosphere of cucumber non-symptomized plants growing adjacent to the plants that are showing damping-off or wilt symptoms. One gram of each collected sample was suspended in 9 ml of sterile distilled water and serially diluted until getting the dilution of 10 −7 . One milliliter of each sample was spread on a Petri dish plate containing nutrient agar medium, and all plates were incubated at 37°C for 24 h. Individual bacterial colonies were picked up using a sterilized loop, transferred to nutrient agar plates, and incubated at 37°C for 24 h. The developed single colonies were transferred to nutrient agar medium slants and pure cultures were stored in a refrigerator at 4°C. The isolated bacterial isolates were identified, using morphological (staining and motility), cultural (Nutrient agar, Cetrimide agar), and biochemical tests (IMIC test, triple sugar iron test, nitrate reduction test, catalase test, casein hydrolysis, oxidase test, starch hydrolysis, lipid hydrolysis, gelatin liquefaction, and carbohydrate tests).
In vitro evaluation of antifungal activity of the isolated bacterial isolates The antagonistic effects of P. fluorescens and B. subtilis against R. solani and F. solani were evaluated in vitro. A streak of either P. fluorescens or B. subtilis was placed on PDA plates at 28°C for 1 day; then a mycelial disc (0.5 cm) of either R. solani or F. solani was placed onto the center of each PDA plate. All plates were incubated at 28°C until the fungal growth of the control plates reached the edge of the plate. The reduction of the fungal mycelial growths was calculated according to Fokemma (1973). Radial growth of R. solani and F. solani was recorded and inhibition percent of the growth was calculated according to the following formula:   Effect of B. subtilis and P. fluorescens on cucumber seed germination and damping-off disease in pots A pot experiment was designed using small pots containing reasonable weight (300 g.) of sterilized soil (sandy loam). R. solani and F. solani grown on millet grains were mixed with the potting soil at 1% W:W. After 3 days, bacterial suspensions containing 1 × 10 9 CFU/ml of the tested bacterial isolates were also added, and the infested pots were irrigated for 5 days before sowing. Ten cucumber seeds were sown in each pot with 3 replicates (pots) for each treatment in completely randomized design (CRD). The experiment included 10 treatments namely non-infested soil (control), soil treated with R. solani only, soil treated with F. solani only, soil treated with B. subtilis only, soil treated with P. fluorescens only, soil treated with R. solani + B. subtilis, soil treated with R. solani + P. fluorescens, soil treated with F. solani + B. subtilis, and soil treated with F. solani + P. fluorescens. Pots were kept under greenhouse conditions till the end of the experiment (3 weeks of sowing). The percentage of seed germination and damping off of cucumber seedlings were determined at the end of the experiment.

Results and discussion
Isolation and identification of F. solani and R. solani Results of the cultural and morphological characteristics showed that R. solani, isolated from diseased cucumber plants, showed slightly melanized hyphae and irregularly shaped and brownish sclerotia. Moreover, microscopic observation showed that the hyphae branch at a 90°angle and constriction of hyphae and formation of septa at a short distance from the point of the hyphal branches' origins and absence of clamp connection, conidia, and rhizomorphs (Fig. 1). Similar results concerning morphological characters of R. solani were reported by several researchers (Moni et al., 2016;Desvani et al., 2018). F. solani isolated from the infected cucumber roots produced sparse to abundant, white creamy mycelium (Fig. 1). Macroconidia are sickle-shaped with a slightly blunted apical end blunt and have 3 to 4 septa on average. Microconidia are abundant, oval to kidney-shaped, and formed in false heads on very long monophialides (Ke et al. 2016).
For confirmation of the morphological identification of F. solani and R. solani, PCR amplification of DNAs extracted from these isolates showed the possibility of amplifying PCR products with sizes ranging between 600 and 650 bp using the ITS1-ITS4 primers (Fig. 2).
The PCR-amplified ITS region (ITS1, 5.8S rDNA, and ITS4) of each F. solani and R. solani isolates were sequenced and the generated nucleotide sequences were subjected to a BLAST search. Molecular identification results confirmed the morphological identification of the tested isolates. The comparison of the whole ITS region (ITS1, 5.8S rDNA, and ITS4) of the F. solani isolate with those previously deposited in the GenBank revealed that the nearest genetic similarity (100%) of the generated ITS sequence was with F. solani from Iraq (KY662484.1 and KY662480.1) ( Table 1). As well, close phylogenetic relationships also appeared with some F. solani isolates from India (MF800959, HQ384397.1, KM017142, and KY419545.1) (Fig. 3).
Comparison of the sequence obtained from R. solani with the other R. solani isolates deposited in GenBank showed that the highest genetic similarity was 97 and Table 3 Effects of R. solani and F. solani on seed rot, and seedlings damping-off disease of cucumber on PDA The R. solani isolate identified in this study also showed genetic differences ranged 89-93% with the other R. solani isolates formerly identified and deposited in NCBI (Table 2 and Fig. 4).
Results of the nucleotide sequence analysis using BLAST demonstrated that R. solani were genetically different from the other isolates and not previously registered in GenBank; therefore, it was recorded in GenBank under the accession number MK105921. This newly identified R. solani isolate may be more dangerous and devastating for economic crops. Polymerase chain reaction (PCR) technology was used in this study to diagnose the isolates of F. solani and R. solani due to its high accuracy in the diagnosis of many organisms, including pathogenic and non-pathogenic fungi such as F. solani, R. solani, Alternaria alternata, and Aspergillus spp. Khan et al. 2018).
Pathogenicity of R. solani and F. solani R. solani and F. solani isolated in this study from infected cucumber roots were found to be pathogenic and had the ability to infect cucumber seedlings 10 days after inoculation (Table 3 and Fig. 5). The tested F. solani and R. solani isolates caused 20 and 100% seed rot and 100.0 and 0.0% seedling damping off, respectively, compared to the control treatments. These results are in agreement with previous ones reported that R. solani and F. solani are highly pathogenic fungi causing a significant reduction in seed germination of many vegetable crops including cucumber . Variability in seed rot percentages caused by R. solani and F. solani may be due to the differences in their virulence, the speed of growth, the nature of parasitism, and the sensitivity of the plant species for these pathogenic fungi (Desvani et al. 2018). R. solani produces a number of enzymes such as cutinase, cellulose, and protease, which have a significant effect on seed germination (Karima and Nadia 2012).
Antagonistic activities of P. fluorescens or B. subtillis against R. solani and F. solani In vitro investigations showed that B. subtilis and P. fluorescens showed a high reduction of the radial  growth of both R. solani and F. solani (100% growth inhibition) (Fig. 6). The potentialities of the bacterial species used in this study could be attributed to their ability to secrete hydrolytic enzymes or antifungal metabolites. As reported by Montealegre et al. (2003), B. subtilis can secrete several antifungal metabolites such as bacitracin, subtilin, bacillin, and bacillomycin which have an inhibitory effect on many fungal pathogens. Sarhan et al. (2001) also indicated that B. subtilis inhibited the mycelial growth of F. solani. It was also found that P. fluorescens is able to secrete antifungal metabolites, e.g., lipopeptide cyclic as well as several hydrolytic enzymes such as chitinase, endochitinase, β-1, 4 glucanase, β-1,3 glucanase, lipase, and protease which have an inhibitory effect on fungal pathogens (Saad 2006). In the pot experiments, it was found that B. subtilis and P. fluorescence were very effective in reducing the severity of damping-off disease of cucumber seedlings caused by F. solani or R. solani (Table 4 and Fig. 7). The production of antifungal metabolites is considered as the main mechanism of antifungal activity of P. fluorescens and B. subtilis against F. solani and R. solani (Karkachi et al. 2010). Manikandan et al. (2010) reported that P. fluorescence is an effective biocontrol agent in controlling several plant pathogens such as R. solani, F. solani, and P. fluorescens. It also has the ability to suppress fungal root diseases through different mechanisms including production of antibiotics, bio-surfactants, toxins or lytic enzymes, induction of systemic resistance, and competition for colonization sites, minerals, and nutrients (Erdogan and Benlioglu 2010). Besides, it also possesses some of the other mechanisms such as antibiotic production and spore formation and the production of siderophore (Nielsen et al., 1998). The inhibitory effect of P. fluorescens and B. subtilis against the phytopathogenic fungi might be due to the production of hydrolytic enzymes that can degrade cell walls, several cyclic lipodepsipeptides, and iron-chelating siderophores (Kim et al. 2008). Mansoori et al. (2013) reported that P. fluorescens isolates can superimposed the Bacillus isolates in reducing wilt disease of cotton. This could be due to the various antagonistic mechanisms of P. fluorescens such as antibiosis, siderophore production, hormone production, and inducing systemic resistance in host plants.

Conclusion
B. subtilis and P. fluorescens isolated in this study shown their potentiality in reduction of the damping off of cucumber seedlings. In addition, future studies may be required to determine the antagonistic activity of B. subtilis and P. fluorescens under greenhouse and field conditions as well as the antagonistic effects on other diseases of economic importance in Iraq.