Common root rot (CRR) caused by Cochliobolus sativus (Ito &amp; Kuribayashi) Drechs. ex Dastur is a serious disease of barley causing significant economic losses worldwide. An in vitro technique was developed for assessing directly the antagonistic potential of Bacillus atrophaeus isolate SY15b against CRR on barley. Seeds of the susceptible barely cv. WI 2291 were inoculated by mixing them thoroughly with C. sativus-SY15bsuspension and placed onto 1.5% water agar in sterile Petri dishes for 10 days. Our results demonstrated that B. atrophaeus had significant antagonistic capacities against the CRR by decreasing the percentage of barley infected area of sub-crown internodes (SCIs) as compared to controls after 10 days post incubation. Highly significant correlation coefficients (r = 0.85, P = 0.001) were found between in vitro and pot methods, indicating that in vitro co-culture testing procedure is reliable. The developed method is both simple and precise and could be used directly for a rapid assessment of the antagonistic activity of Bacillus on barley plants under uniform conditions for selecting suitable biocontrol agent against fungal soil-borne pathogens

Mohammed Imad Eddin Arabi

Mohammed Jawhar

Mohannad Harba

UNIVERSITY OF THE PUNJAB

Mycopath (2021) 19(1): 29-33                                                                         Research Article  Mycopath (2021) 19(1): 29-33 A simple method for assessing antagonistic activity of Bacillus atrophaeus against common root rot of barley *Mohannad Harba, Mohammed Jawhar and Mohammed Imad Eddin Arabi Department of Molecular Biology and Biotechnology, AECS, P. O. Box 6091,  Damascus, Syria *Corresponding author’s email: ascientific@aec.org.sy  Abstract Common root rot (CRR) caused by Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur is a serious disease of barley causing significant economic losses worldwide. An in vitro technique was developed for assessing directly the antagonistic potential of Bacillus atrophaeus isolate SY15b against CRR on barley. Seeds of the susceptible barely cv. WI 2291 were inoculated by mixing them thoroughly with C. sativus SY15b suspension and placed onto 1.5% water agar in sterile Petri dishes for 10 days. Our results demonstrated that B. atrophaeus had significant antagonistic capacities against the CRR by decreasing the percentage of barley infected area of sub-crown internodes (SCIs) as compared to controls after 10 days post incubation. Highly significant correlation coefficients (r = 0.85, P = 0.001) were found between in vitro and pot methods, indicating that in vitro co-culture testing procedure is reliable. The developed method is both simple and precise and could be used directly for a rapid assessment of the antagonistic activity of Bacillus on barley plants under uniform conditions for selecting suitable biocontrol agent against fungal soil-borne pathogens. Keywords: Bacillus atrophaeus, Barley, Biocontrol, Cochliobolus sativus, In vitro test.   Introduction Cochliobolus sativus is a soil-borne pathogenic fungus that causes common root rot of barley (Hordeum vulgare L.), a disease responsible for considered reduction in this crop worldwide (Van Leur et al., 1997; Kumar et al., 2002). CRR disease produces a brown to black discoloration of the sub-crown internode (SCI), which is commonly used for determining barley reaction towards this disease (Kokko et al., 1995; Mathre et al., 2003). The economic damage caused by CRR can be avoided by using large amounts of fungicides, which has become one of the major concerns in barley fields due to great dangerous to humans and environment (Bailey et al., 1997). Therefore, biocontrol has been suggested as an alternative approach to chemical control because it is highly selective method with less time consuming (Sellittoet al., 2021). Many biocontrol agents were isolated by screening a big number of soil microorganisms for antagonism against fungal pathogens in vitro or in planta (Köhl et al., 2019). Among those, Bacillus species have the benefit of being able to form endospores, which makes them very stable such as biofungicides or biofertilizers (Schisler et al., 2004), they could suppress various soil borne diseases in different environments (Cao et al., 2018). Bacillus atrophaeusis a Gram-positive bacterium used in various studies as a classical non-pathogenic surrogate of Bacillus anthracis (Nakamura, 1989). It is easy to culture, has been identified for its resistance characteristics, and is being used in various applications (Sella et al., 2014). Such bacteria have been reported to control different crop pathogens (Rajaofera et al., 2017, 2020). The search for an environmental-friendly disease management approach has led to search bacterial agents with antagonistic activities. Although a wide spectrum of Bacillus antagonists has been reported during the past decades (Sharf et al., 2021), more efficient and new Bacillus species antagonists might be around waiting for detection. However, it should be clearly understood that not all Bacillus are considered effective for controlling pathogens and therefore it is crucial to find out the Bacillus species with biocontrol potential, which is possible through in vitro tests prior to field experiments. The common method for assessing the antagonistic effects of bacterial and fungal antagonists against fungal pathogens is based on measuring the radial colony growth of fungi in co-culture system without plants (Khan and Javaid, 2020, 2021; Khan et al., 2021; Sharf et al., 2021). However, this method does not take into account changes in hyphal density or specific growth rate (Bolivar-Anillo et al., 2021), and cannot give any information regarding this antagonism directly on plant for implementation as a biocontrol agent. The aim of this study was to develop a reliable in vitro laboratory assay for measuring directly the biocontrol capacity of B. atrophaeus isolate SY15b on barley plants against strains of C. sativus as a simple and rapid method.    30 Harba et al. Mycopath (2021) 19(1): 29-33 Materials and Methods   Bacterial isolate Soil samples were isolated from different locations in Syria (Ammouneh et al., 2011). The samples (15 g each) were added to 100 mL of sterile deionized water and mixed for 10 min. Serial dilutions were made and plated on nutrient agar (NA) and incubated at 37 °C for 2 days. Bacterial colonies were screened among 525 isolates on nutrient broth (NB) culture according to Wulff et al. (2002). The B. atrophaeus isolate SY15b was identified based on both physiological characteristics and on 16S rDNA analysis (Harba et al., 2020). Homology blast search was achieved against the database and deposited in GenBank (Accession No. MT159352). The B. atrophaeusis SY15b solution was diluted to 2 × 108 cell for using in the experiments.  C. sativus inoculum preparation The most virulent pathotype Pt4 of C. sativus (Arabi and Jawhar, 2002) was used in this work. The isolate was grown on Petri dishes containing potato dextrose agar (PDA) medium (DIFCO, Detroit, MI. USA) at 22 ±1 ºC for 10 days in the dark. The conidial suspension was adjusted to 5 × 105 conidia mL-1 and 40 mL of spore suspension was mixed in a plastic Petri dish with 50 g sterile neutralized peat (Bailey et al., 1997).   Seed inoculation by C. sativus and bacteria Barley seeds of susceptible cultivar WI 2291 from Australia were surface sterilized with 5% sodium hypochlorite solution for 5 min and then soaked three times in sterilized distilled water. They were inoculated by mixing thoroughly with beat-gum conidia inoculums and B. atrophaeusis SY15b (Arabi and Jawhar, 2002).  Pot experiment One part of inoculated seeds was planted in pots filled with sterilized peat moss in a depth of 6 cm for elongating the SCIs (Kokko et al., 1995). Each experimental unit consisted of 10 seedlings with three replicates. A full replicate consisted of 10 pots inoculated with Pt4 isolate.  Where control pots inoculated with C. sativus without bacteria was maintained as control. They were placed in a growth chamber at temperatures 22±1 ºC (day) and 17±1 ºC (night) with a daylength of 12 h and a relative humidity of 80–90%.   In vitro experiment Second part of inoculated seeds were placed on 1.5% water agar, and to elongate SCIs, the Petri dishes were incubated 21±1 °C for 5 days in the dark. Then they were kept under a daylength of 12 h at the same temperature. Whereas, Petri dishes containing C. sativus inoculated seeds without bacteria were maintained as control.   Evaluation of antagonism Common root rot responses based on the measurement of individual SCI discoloration (%) were assessed 7 weeks for pot assay, and 10 days for in vitro method post inoculation. The 0–5 scale (Kokko et al., 1995) was used for both methods. All experiments were repeated three times. Data were analyzed using analysis of variance (ANOVA) and means were separated by Tukey's test (P = 0.05).  Results and Discussion   In this study, the antagonistic potential of the B. atrophaeusis SY15b was concluded and validated by reducing directly common root rot severity on barley SCI as shown in photo-plate of PDA culture plate assay comparing with controls (Fig. 1). The disease symptoms on the susceptible cv. WI 2291 were easily detected in vitro after 48 h of inoculation (Fig. 1).  Our results demonstrated that B. atrophaeus had significant antagonistic potential against the CRR by decreasing the percentage of barley infected area of SCIs as compared to controls after 10 days post incubation (Fig. 1). Inoculation with the C. sativus virulent pathotype Pt4 increased discoloration of the SCIs on cv. WI 2291 in pot  and in vitro experiments (Fig. 2). The results clearly indicate that antagonistic between B. atrophaeusis SY15b and C. sativus was similar in the two methods and that the in vitro assay was useful for bio-control agent against CRR in a short time (Fig. 2). Highly significant correlation coefficients (r = 0.85, P = 0.001) were found between in vitro and pots methods, indicating that in vitro co-culture testing procedure is reliable (Fig. 3). Our data are in accordance with those obtained by Schisler et al. (2004) and Cao et al. (2018) who reported that the antagonistic effect against plant pathogen could be attributed to the antifungal compounds production belonging to the family of iturins and subtilins that operate on the fungi's cell wall. Lyon and Muir (2003) found that the hydrophilic enzymes production to break down polysaccharides, lipids and nucleic acids and may have been also engaged. Hence the most likely explanation for reducing CRR symptoms by B. atrophaeusis SY15b is that antifungal activity is increased by in vitro co-culturing system, since B. atrophaeus has been proved to produce a wide spectrum of antimicrobial compounds, secretion metabolites preventing occurrence of systemic diseases (Rajaofera et al., 2020). Several factors have been reported to affect susceptibility of barley to common root rot disease during field experiments such as inoculum level and climate conditions (Kumar et al., 2002). In our in vitro co-culture experiments, the inoculums concentration was controlled. Therefore, precise evaluation could be made. Furthermore, the soil may contain other fungi  Antagonistic activity of Bacillus atrophaeus 31 Mycopath (2021) 19(1): 29-33 and bacteria that can cause similar SCI rotting, which can lead to misdiagnosis of disease symptoms with root rot.   Conclusion  Despite the successful and importance use of Bacillus species, information regarding B. atrophaeus application is rare. Here we developed an in vitro technique that can be considered as a rapid, simple and relatively inexpensive method for B. atrophaeus SY15b isolate antagonisms against C. sativus under uniform conditions. The technique can help to replicate infection assays, as well as to test large numbers of bacterial and fungal pathogen isolates in a short time. The SY15b isolate could be promising as a bio-control agent against this disease.  Acknowledgement We wish to sincerely thank the Director General of AECS and the Head of the Molecular Biology and Biotechnology Department for their support during this study. Thanks are also extended to Dr. H. Ammouneh and Mr. A. Albaterni for their assistance with experiments.             Fig. 1: (A): Symptoms of common root rot on barley SCI without Bacillus treatment. (B): Bacillus atrophaeus SY 15b showing antagonistic activity against C. sativus isolate Pt4 in the in vitro co-culture after 120 h of incubation. 32 Harba et al. Mycopath (2021) 19(1): 29-33       Fig. 3: Relationship between pot and in vitro co-culture for assessing the antagonistic activity of Bacillus atrophaeus against common root rot on barley susceptible cv. WI2291. 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A simple method for assessing antagonistic activity of Bacillus atrophaeus against common root rot of barley

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A simple method for assessing antagonistic activity of Bacillus atrophaeus against common root rot of barley

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