The fungus Rhizoctonia solani Kühn is an economically important pathogen involved in root rot and seedling damping-off disease of sugar beet (Beta vulgaris L.). In Morocco, this is the first detailed report of R. solani as the causative agent of sugar beet damping-off. The identification of the pathogen was based on combining the morphological characteristics and molecular characterization using the ribosomal DNA internal transcribed spacer (rDNA ITS) region. Antifungal activities of four commercial fungicides; azoxystrobin, difenoconazole, tebuconazole, and trifloxystrobin were assessed in vitro, displaying effective inhibition of the mycelial growth of the pathogen strains using PDA medium. A commercially available Bacillus subtilis Y1336 was evaluated as a potential biological control agent (BCA) against seedling damping-off caused by R. solani. The biological control activities of B. subtilis Y1336 was demonstrated by dual culture bioassays on PDA medium, in vitro seed inoculation with R. solani and B. subtilis, and by applying the BCA as seed coat under greenhouse conditions. This is the first time that the commercially available B. subtilis Y1336 has been proved as an effective BCA against the damping-off disease of sugar beet. This commercial antagonist should be incorporated into the integrated disease management startegy for sugar beet seedling mortality together with effective tested fungicides.
Research Article
Identification, pathogenicity, fungicide sensitivity and biological control of Rhizoctonia solani associated with damping-off disease of sugar beet in Morocco
https://doi.org/10.21203/rs.3.rs-1517415/v1
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Beta vulgaris
Rhizoctonia solani
antagonistic bacteria
fungicide
damping off
Sugar beet (Beta vulgaris L.) is considered one of the most important sugar crops in many countries worldwide. The sugar production from this crop is the second in the world after that from sugar cane (Abd El Lateef et al. 2019). In Morocco, sugar beet is cultivated in about 57,171 hectares with a yield of 3.7 million tonnes during 2019 (FAOSTAT. 2021). The Beni Mellal-Khenifra region in central Morocco is recognized for its vast agricultural plain, with a part is reserved for industrial crops. In this region, sugar beet contributes, respectively, 20.5% and 21.2% to the national area and the national production of sugar beets in Morocco (Redani 2015). However, In many regions of Morocco, including the Beni Mellal-Khenifra region, the appearance of numerous soil-borne pathogens and outbreaks has led to economically significant losses of sugar beet yields (Snaiki et al. 2005; Chenaoui et al. 2017; Farhaoui et al. 2022). Among them, Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris [Frank] Donk) is one of the most destructive pathogens affecting sugar beet in Morocco (Rhmiza 1991; Chenaoui et al. 2017). This pathogen causes root rot and damping-off in sugar beet (Ruppel 1972; Abada 1994; El-Tarabily 2004). According to the hyphal compatibility reaction, anastomosis groups (AGs) are used to identify and classify R. solani isolates. Moreover, Some AGs can be classified into subgroups based on criteria like biochemical attributes and host range (Carling et al. 2002; Liu et al. 2021). R. solani AG 2–2 is a pathogen that causes both post-emergent damping-off and root rot of sugar beet. Whereas R. solani AG 4 is more often involved in damping-off (Kirk et al. 2008; Liu et al. 2019). Worldwide, R. solani is one of the most aggressive soil-borne pathogens that grow in soil, generating sclerotia and does not produce asexual spores. Sclerotia produced by this pathogen can persist in the soil for many years. Moreover, R. solani has a wide host range. Thereby, it can persist in the soil for long periods, making the control of the disease using traditional means very difficult (Rush and Winter 1990; Anees et al. 2010; Gonzalez et al. 2011). The most important aspect of R. solani management in the European Union is the use of resistant cultivars (Buddemeyer & Märländer, 2005). The use of resistant cultivars is thus the most important aspect of R. solani management in the European Union (Buddemeyer and Märländer 2005). However, the quantitative resistance of the available cultivars does not result in complete disease management and is associated with a yield penalty in uninfected fields (Buddemeyer and Märländer 2005). Many fungicides have been used in the management of R. solani. Since 2000, Azoxystrobin was labeled for foliar and in-furrow application to manage R. solani in sugar beet (Kiewnick et al. 2001). Currently, azoxystrobin is considered the standard fungicide in the management of R. solani in sugar beet in several countries, including the United States (Liu and Khan 2016a). Several studies have shown the effectiveness of this fungicide in controlling sugar beet diseases caused by R. solani (Kiewnick et al. 2001; Stump et al. 2002). Other active ingredients such as prothioconazole and pyraclostrobin have shown their ability to reduce crown and root rots caused by R. solani in sugar beet, but the dose required to achieve protection similar to that achieved by Azoxystrobin is significantly higher (Liu and Khan 2016b). Besides, penthiopyrad can be used as a seed treatment in sugar beet to control seedling mortality caused by R. solani (Liu et al. 2021). No isolate resistant to azoxystrobin has been described in sugar beet. However, the emergence of azoxystrobin resistant isolates derived from other plants such as rice and potato (Olaya et al. 2012; Djébali et al. 2014), highlights the need for alternative fungicides for durable fungicidal management of R. solani in sugar beet. For this reason, an alternative approach is required that minimizes the risk of resistance development while providing a level of protection comparable to that obtained by azoxystrobin alone.
Overuse of the chemicals could cause negative impacts on human health and environmental problems. Biological control using beneficial microorganisms has also been considered an important component of plant disease management (Gardener and Fravel 2002; Mahmoud 2016a). In fields with severe infection with R. solani, Bacillus subtilis strain MSU-127 was able to reduce root rot index by 28.11% and increase significantly sucrose yield (Kiewnick et al. 2001). Numerous bacterial species such as B. subtilis and Pseudomonas fluorescens have demonstrated their ability to effectively control damping-off diseases (Kondoh et al. 2001; Pastor et al. 2010; Mahmoud 2016a; Al-Fadhal et al. 2019). Few trials have been conducted to evaluate the management of sugar beet damping-off induced by R. solani using bacterial antagonists (Nielsen et al. 1998; Moussa 2002; Jorjani et al. 2012; Afify et al. 2018). The commercially available B. subtilis strain Y1336 is a biological fungicide used to control various pathogens evolving on many crops (vegetable crops, fruit trees, and vines) while leaving no toxic residue. This bacteria was proved to be an efficient control agent for rose powdery mildew and to improve the yield and quality of rose-cut flowers (Wang et al. 2018a). In addition, this biological agent can act as antagonistic bacteria for suppression of balloon flower stem diseases caused by R. solani (Lee et al. 2012). There is a lack of research on the effectiveness of adding this beneficial bacterium to the soil to protect sugar beet against damping-off caused by R. solani.
The objective of this study was to evaluate in vitro and planta antifungal activity of the commercially available B. subtilis strain Y1336 and the antifungal effects of four commercial fungicides against damping-off diseases caused by R. solani.
Isolation and morphological identification of R. solani
Throughout 2020 growing seasons, sugar beet seedlings showing symptoms of post-emergence damping-off with brown to black lesions on the hypocotyl and the roots (Fig 1), were collected from two Moroccan towns : Souk Essebt Oulad Nemma (32°15'35.9°N, 6°48'54.4°W) of Fquih Ben Salah Province and Taghzirt ( 32°26'04.6"N 6°12'04.6"W) of Beni Mellal province, in which, the damping-off of sugar beet results in a significant reduction in yield each growing season. The causative pathogen R. solani was isolated from infected roots using Potato Dextrose Agar medium (PDA). Briefly, infected hypocotyls were cut into 5 mm pieces which were then surface sterilized by immersion in 0.5 % sodium hypochlorite solution for 3 minutes, rinsed three times with sterile distilled water (SDW), and left dried under laminar flow. Afterward, disinfected fragments were deposited on PDA medium amended with 50 mg/l of streptomycin and incubated for 4 days in darkness at 25°C. Colonies showing characteristics of R. solani were then purified by selecting a single hyphal tip. Two isolates of this pathogen were obtained. Each isolate represents a region. Both isolates were maintained by periodic subculturing and stored long-term on sorghum seeds. Morphological characteristics of the pathogen (colony color and shape of mycelium and sclerotia) were examined. Measurements of sclerotia were made using 20 days old cultures.
DNA extraction, PCR, and sequencing
Both R. solani isolates were grown in 8 cm diameter Petri dishes containing PDA and incubated for 3 days at 25 °C in the dark. Afterward, fungal DNA extraction was performed following the protocol previously described by Doyle (1990). The amplification of the Internal Transcribed Spacer (ITS) region of the extracted DNA was done using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3 ') (White et al. 1990). Amplified PCR products were visualized by agarose gel electrophoresis 1.5 % using tris borate EDTA buffer (TBE) (0.5×). The gels were then stained using ethidium bromide and visualized by a UV transilluminator to evaluate the presence of amplicons and their size. Sequencing was carried out at the STAB Vida Lda laboratory in Portugal. The primers used in the sequencing were the same as those used in the amplification reaction. Sequences obtained were edited and aligned using the DNAMAN sequence analysis software (version 6.0, Lynnon Biosoft, Canada). Afterward, sequences obtained were verified using Blast search to find homologous sequences in GenBank databases (National Center for Biotechnology Information) and then deposited in GenBank. A phylogenetic tree was generated from obtained sequences. The two isolates were rearranged to create major clusters using MEGA 5.0 software. The Sclerotium rolfsii (AY684917) sequence was chosen as an out-group. Phylogenetic relationships were estimated using the maximum likelihood (ML) method. Support for each branch in the inferred tree was assessed operating 1000 bootstrap replications.
Effect of temperature on mycelial growth of R. solani isolates
The effect of temperature on the mycelial growth of R. solani isolates was evaluated on a PDA medium. From actively growing 3-day-old cultures of each isolate, mycelial discs 5 mm in diameter were cut out and placed in the center of Petri dishes, 9 cm in diameter, containing the PDA medium, then the dishes were incubated at different temperatures (5 °C, 10 °C, 20°C, 25 °C, 30 °C, 35°C, and 40 °C). Each treatment was repeated 3 times. Colony size was measured every day. Colony diameters were measured every 24 h for 20 days, then radial growth of colonies, corresponding to each temperature, was calculated.
In vitro antifungal activities of chemical fungicides
Four fungicides were evaluated in the in vitro tests (Table 1): trifloxystrobin (Flint), azoxystrobin (Ortiva 25 SC), tebuconazole (Supreme Super), and difenoconazole (Score 250 EC). Their effect on the radial growth rate of both isolates of R. solani was tested using PDA media amended with a range of seven concentrations (0, 0.1, 1, 10, 50, 100, and 500 μg/ml). To evaluate radial mycelial growth, a 5 mm mycelial disc cut from the growing edge of 3 days old PDA cultures was placed upside down in the center of the Petri plate. Dishes were incubated at 25 ° C in the dark. Colony diameter was measured 72 h after inoculation. Each treatment was repeated 3 times, and the experiment was carried out twice. The rate of mycelial growth reduction for each isolate and concentration was determined according to the following formula:
Where R is the radial growth of fungi in amended media and r is the radial growth of fungi in non-amended media.
For each isolate, the EC50 (effective concentration that reduced mycelial growth by 50%) was calculated by regressing the percent of mycelial growth inhibition against the log10 transformation of the seven concentrations of fungicide with the XLSTAT PRO software (Addinsoft, www.xlstat.com).
In vitro study of the antagonistic activity of the commercially available Bacillus subtilis strain Y1336
Dual culture assay
The capacity of inhibiting the growth of R. solani by the commercially available Bacillus subtilis strain Y1336 was evaluated under in vivo conditions. A dual culture bioassay was performed using a PDA medium by confrontation with 5 replications. The antagonist bacteria were streaked in four equidistant streaks along the border of the Petri plate. Then, from the edge of a 3-day-old-colony of R. solani, a mycelial plug of 5 mm in diameter was removed and placed in the center of the Petri plate containing PDA medium between the 4 streaks of the bacterial strain. Culture media containing only the pathogen plug were used as a negative control. All plates were parafilm-sealed and incubated in the dark at 25 °C. Each treatment was repeated 3 times, and the test was performed twice over time. After 3 days of incubation, the inhibition rate of mycelial growth (IR) in each Petri plate was calculated using the following formula (Lahlali et al. 2020):
Where : IR is the inhibition rate; C: is the diameter of the pathogen colony in the control plates; T: Diameter of the pathogen colony in dual culture bioassays.
In vitro Inoculation of Seeds Using R. solani and B. subtilis Y1336
Preparation of bacterial suspension
Bacterial isolate was cultured on Luria–Bertani (LB) medium for 3 days at 25°C, then several loopfulls were transferred to test tubes containing sterile physiological water (NaCl, 9 g/l). Bacterial cell density was determined using a spectrophotometer (OD = 600nm) and then, adjusted to 1×109 CFU/ml, in sterile carboxymethyl cellulose (CMC) 1% solution (Karimi et al. 2016). The bacterial suspension can be used immediately or stored at 4°C for a short time.
In vitro biological control of damping-off
B. subtilis Y1336 was tested for its ability in reducing the sugar beet seedling damping-off caused by R. solani under laboratory conditions using the procedure described by Haque et al. (2021) with slight modifications. Seeds of sugar beet (cv. Barrosa) were initially surface-sterilized with 70% ethanol for 1 min and washed 3 times with SDW. Then, Seeds were placed on sterile filter paper and allowed to dry in a laminar flow hood for 2 h. The seeds were soaked in 50 ml of the bacterial suspension (CMC 1%), shaken at 150 rpm for three hours. Then, they are removed from the bacterial suspension, dried under a laminar flow hood for 4 hours (Karimi et al., 2016). Non-bacterized seeds served as a negative control, while seeds treated with a fungicide (Difenoconazol at5 μg g/ml) were served as a positive control. The test was performed using 50% PDA plates. Four seeds were planted employing sterile forceps at 1 cm apart on each culture dish. The mycelial plug of R. solani was deposited 2 cm from each seed. Seven treatments were involved : (1) Non-inoculated seeds alone; (2) Mycelial plugs of R. solani Rs1 and non bacterized seeds; (3) Mycelial plugs of R. solani RsM and non-bacterized seeds; (4) Bacterized seeds alone; (5) Mycelial plugs of R. solani (Rs1) and Bacterized seeds ; (6) Mycelial plugs of R. solani (RsM) and Bacterized seeds, and (7) Mycelial plugs of R. solani Rs1 and seeds non- bacterized but treated with fungicide. Dishes were sealed with parafilm, The number of germinated seeds was recorded after 7 days of incubation at 25 °C. This trial was carried out twice over time with 4 repetitions for each treatment.
In vitro evaluation of the antibacterial activity of fungicides against B. subtilis Y1336
To evaluate the effect of the 4 fungicides on B. subtilis Y1336 growth, different doses of the tested pesticides (azoxystrobin, difenoconazol, tebuconazol, and trifloxystrobin) were added to LB media. 0.2 ml of a bacterial suspension of the biocontrol agent (BCA) (1×104 CFU/ml) was spread onto the culture media. The number of bacterial colonies was counted after incubation at 27 °C for 48 hours in darkness. By counting the number of colonies, bacterial growth on LB amended with fungicides was compared to media without any pesticides. Each treatment contained 3 replications, and the trial has been conducted twice.
In planta experiments
Preparation of R. solani inoculum and pathogenicity test
Pathogenicity of the two R. solani isolates was evaluated according to the technique of Grisham and Anderson (Grisham and Anderson 1983). A quantity of 200 g of corn seeds was soaked in distilled water for 12 hours, autoclaved twice at 121 °C for 20 minutes in two successive days. The corn seeds were inoculated with ten plugs of 5 mm diameter of a 3-day old culture of R. solani grown on a PDA medium. The substrate was incubated at 25 °C for 6 weeks in darkness with daily shaking to ensure uniformity of fungus growth. Colonized corn seeds obtained were used as inoculum of the pathogen. Corn seeds not inoculated and prepared in the same way were served as control.
A soil mixture consisting of peat moss, sand, and soil (1: 1: 1) was autoclaved for one hour at 121 °C on two consecutive days. The soil was infected with the pathogen by adding 8 g of infested corn seeds for each kg of soil. Obtained soil was put into plastic pots of 20 cm in diameter at a rate of 3 kg of soil per pot. After 10 days of soil infection, twenty surfaces disinfected sugar beet seeds (cv. Barrosa) were planted in each pot. The pots were then placed in a greenhouse maintained at 25°C. Each treatment contained five replications in a randomized complete block design. The entire experiment has been conducted twice. The effectiveness of the BCA on pre-and post-emergence damping-off diseases of sugar beet seedlings was assessed by counting the number of seedlings that survived after 15 days (pre-emergence diseases) and 45 days (post-emergence disease).
Biological control activity of B subtilis Y1336 against R. solani
The Biological control potential of B. subtilis Y1336 against the highly aggressive isolates of R. solani (RsM) was examined under greenhouse conditions. The soil used for this test was inoculated with the pathogen as mentioned above. Seeds treated with fungicide difenoconazole (400 g/100 kg seed) were served as a positive control. After 7 days of soil inoculation, twenty sugar beet seeds (cv. Barrosa) were sown in each pot. In total there were 5 different treatments : (1) Non-bacterized seeds + soil not inoculated with R. solani ; (2) Non-bacterized seeds + soil inoculated (negative control) ; (3) Seeds treated with fungicide + inoculated soil (positive control) ; (4) Seeds bacterized with B. subtilis Y1336 + uninoculated soil ; (5) Seeds bacterized + inoculated soil. Pots were arranged in a complete randomized design with 5 replications, and the experiment was conducted twice over time. The number of healthy seedlings was recorded after 14 and 45 days after sowing. Then, the percentage of pre and post-emergence damping-off was calculated.
Statistical Analysis
Statistical analysis was executed with SPSS statistical software (version 20, IBM SPSS Statistics 20). Data were subjected to analysis of variance (ANOVA), and the least significant difference (LSD) at 5% level was used to compare mean values between treatments.
Morphological characteristic of R. solani
Both isolates showed rapid mycelial growth so that the mycelium reached the limits of the Petri dish (90 mm) after only 3 days of incubation at 25°C in the dark. Fungal colonies had typical morphological characteristics of R. solani. On PDA medium, the young fungal colonies have a white and cream color, with many floccose aerial hyphae (Fig 1B). Furthermore, dark brown colonies with dark brown sclerotia were formed after 15 days of incubation. The majority of macrosclerotia (1,1 – 2 mm) are blackish-brown and appear as a very small sphere with a diameter ≤ 1.1 mm. While microsclerotia emerge as a thin crust on the culture medium surface. Microscopic examination showed that the mycelium of the pathogen exhibits right-angled hyphal branching with a constriction and a septum near the ramification, and is composed of multinucleate hyphae (4 to 9 nuclei per cell), 7.1-8.0 μm wide (mean 7.5 μm) (Fig 1).
Molecular identification of R. solani
Both R. solani isolates were confirmed by molecular sequencing of the ITS rDNA region, R. solani MZ854089 (Rs1) and R. solani OM955658 (RsM). Based on the sequence analysis, a phylogenetic tree was obtained by the neighbor-joining method. Results clearly showed that both isolates were closely related to R. solani strains (Fig. 2). Collectively, soil-borne pathogens Rs1 and RsM should be identified as members of R. solani.
Effect of temperature on growth rates of R. solani
The effect of temperature on the mycelia growth of two strains of R. solani is illustrated in figures 3 and 4. In the darkness, both R. solani isolates showed maximum growth at 25°C on PDA medium. At this temperature, both strains of R. solani tested showed similar growth. The RsM strain also recorded its optimal growth at 30°C. A large difference in growth between the two strains of R. solani was recorded at 35°C, at this temperature the RsM isolate showed a radial growth equal to 5.79 mm/day. In general, both R. solani isolates showed better growth at 20°C compared to 35°C. The two tested strains of R. solani showed no growth at 5°C after 20 days of in vitro culture. Only the RsM strain showed mycelial growth at 40°C with a low rate (0.08 mm/day). This strain also showed a mycelial growth higher than that of Rs1 in the temperatures 10 °C, 15 °C, and 20 °C. To check if the temperatures of 5°C and 40°C were lethal or not for the two tested strains, their culture plates were transferred to 25°C. After 3 days of incubation in darkness, the two isolates showed mycelial regrowth.
Effect of fungicides on R. solani mycelia growth
Four fungicides: azoxystrobin, difenoconazole, tebuconazole, and trifloxystrobin were used to examine their antifungal activities against the two strains of R. solani isolated from two different regions of Morocco. The results are presented in figure 5. All fungicides revealed significant antifungal activities against both strains of R. solani tested in a dose-dependent manner, shown by reduced radial growth of the pathogen. The fungicides difenoconazol and tebuconazol showed total inhibition of mycelial growth of the strains tested at 500 μg/ml. whereas azoxystrobin completely inhibited the mycelial growth of the RsM strain from 50 μg/ml, but the Rs1 strain continues to grow even at 500 μg/ml of this fungicide. In this study, trifloxystrobin appeared to be the least effective fungicide in inhibiting the mycelial growth of both tested isolates. The Inhibition rate of mycelial growth does not exceed 78%, even at 500 μg/ml of trifloxystrobin. In parallel, this fungicide recorded the highest EC50 values, 1.96 μg/ml and 4.08 μg/ml, for Rs1 and RsM, respectively (Table 2), while difenoconazole showed the lowest value of EC50 (0.07 μg/ml for RsM).
In vitro evaluation of the antagonistic activity of B. subtilis strain Y1336
In vitro antagonistic activity and effect of the antagonist bacteria on the mycelial structure of R. solani
The commercially available B. subtilis Y1336 was tested for its antagonistic effect against R. solani employing dual culture trial. Inhibition rates were evaluated after 3 days of incubation. Obtained results emphasized that the evaluated strain exhibited an important antifungal effect against the two tested strains of R. solani. Mycelial growth of both pathogens strains Rs1 and RsM were inhibited, with inhibition rates of 74.43 and 73.27%, respectively. Microscopic observations of the zone of inhibition showed several forms of cytological alteration and damaged morphology occurred in R. solani hyphal compared to the untreated control (Fig. 6A). Generally, the alterations correspond to loss of structural integrity of the hypha and many varieties of deformations as swelling of parts of the hypha and thinning of others. Sometimes hyphal alteration was associated with the formation of conglobated clumps along the mycelia and vacuolation of the head's structure as a result of interaction with the antagonist. In addition, perforations and fragmentations of the hyphae were shown.
In vitro seed inoculation with B. subtilis
To evaluate the ability of B. subtilis Y1336 in controlling damping-off caused by R. solaniin in vitro. Co-cultivation of sugar beet (cv. Barrosa) seeds treated with the antagonist was performed in 50% PDA inoculated with the pathogen. After 7 days of incubation, the results showed that the two strains of R. solani resulted in total damping-off in the absence of the antagonist (Fig. 8). While, seedling emergence of 75.43 and 73.27% was observed in the combined treatment of seeds treated with B. subtilis Y1336 and the pathogen Rs1 and RsM, respectively (Fig. 9). No incidence of damping-off was shown in the controls not inoculated with the pathogen, and also, in the plates containing seeds treated only with the antagonist In the confrontation plates. B. subtilis limits the pathogen proliferation and causes a change in the color of the fungal mycelium in the zone of inhibition.
Impact of fungicides on the growth of B. subtilis Y1336.
To investigate the possibilities of biological damping-off control in sugar beet in combination with chemical fungicides, we examined the viability of the commercially available B. subtilis Y1336 in response to four active components having antifungal activities against the pathogen R. solani.. Among the 4 tested fungicides, difenoconazole and tebuconazole did not reduce the growth of the bacterial antagonist in the range of 0.1-500 µg / ml assayed (Fig. 4). However, 500 µg / ml of trifloxystrobin significantly decreased bacterial growth in vitro. Whereas, azoxystrobin significantly reduced the colonies of the antagonist bacteria from the concentration 100 µg / ml.
Pathogenicity test of R. solani and in planta antagonism of B. subtilis Y1336 against R. solani
Both strains of R. solani isolated in this study from infected sugar beet seedlings were found to be pathogenic and could induce pre-and post-emergence damping-off diseases of sugar beet seedlings (Fig. 11). Pots inoculated with R. solani show that the pathogen inhibits germination and causes lesions in the roots and hypocotyls whereas controls remained healthy. 'Koch's postulate was then verified by pathogen re-isolation and morphological identification. Based on the in vitro trials, commercially available B. subtilis Y1336 was subjected to greenhouse bioassay to verify its antifungal activity and its ability in minimizing the sugar beet damping-off disease caused by R. solani. In agreement with the in vitro results and as illustrated in table 3, the BCA B. subtilis Y1336 reduced the incidence of pre and post-inoculation damping-off incidence and significantly increased the number of emerged seedlings (compared to the inoculated control) under greenhouse conditions. The treatment of the seeds with B. subtilis Y1336 significantly controlled damping-off disease, caused by R. solaniRs1, by 58.75%. In soil not inoculated by R. solani, seed treatment with B. subtilis Y1336 did not show pre-or post-emergence damping-off diseases of sugar beet seedlings.
Damping-off is primarily recognized as one of the serious threats in sugar beet cultivation in Morocco. To the best of our knowledge, this is the first detailed report of R. solani that causes damping-off of sugar beet in Morocco. Trials were performed in the laboratory and under greenhouse conditions to assess the ability of a commercially available B. subtilis Y1336 and 4 fungicides to manage seedling mortality in sugar beet. As a warmth-dependent fungus, R. solani is widely spread in the soil of Morocco. R. solanI isolated in this study from infected sugar beet seedlings were found to be pathogenic and could cause pre and post-emergence damping-off in sugar beet. These results are in accord with previous ones reported that R. solani is a highly pathogenic fungus causing damping-off in many crops including sugar beet (Buttner et al. 2004; Yangui et al. 2008; Mahmoud 2016b; Inokuti et al. 2019; Avan et al. 2021). This pathogen can cause seedling pre-and post-emergence mortality as well as root and crown rot in sugar beet (Liu et al. 2019). The two strains tested in this study cause damping-off and root rot in sugar beet (data not shown). So, the AG attributed to these two isolates could be R. solani AG 2–2. The mycelial growth of the two studied R. solani isolates was strongly influenced by temperature. From 10 to 25°C, the growth rate increased with increasing temperature up to 25°C for both isolates. Mycelial growth on PDA of RsM was faster than Rs1 over the range 10 to 20°C. Maximal growth of Rs1 on PDA was at 25°C and 30°C, while optimal growth of RsM was recorded at 25°C. The temperature effect observed in this study is in agreement with results reported by (Harikrishnan and Yang 2004). Whereas Kumar et al. (1999) showed that the most favorable temperature for the studied R. solani isolates was 25°C, and growth decreased significantly at 30°C.
The application of fungicides is the most used strategy to control R. solani on sugar beet. In this study, four fungicides were assessed for their effectiveness against two strains of R. solani obtained from two different sugar beet-growing regions in Morocco. According to the classification adopted by Martin et al. (1984), In vitro sensitivity of the pathogen to fungicides exhibited that the two isolates were extremely sensitive to azoxystrobin, difenoconazole, and tebuconazole, but they were moderately sensitive to trifloxystrobin. The maximum azoxystrobin concentration used (500 µg/ml) did not completely inhibit the mycelial growth of Rs1. Arabiat & Khan, (2016) noted, after 13 years of field treatment with fungicides, a dramatic increase in mean EC50 values for azoxystrobin and trifloxystrobin in tested R. solani strains that become tolerant to these two fungicides. In addition, forms of resistance to fungicides have been observed in crop fields due to the frequent application of azoxystrobin, which has led to a decrease in the sensitivity of other pathogens that are Alternaria solani, Podosphaera fusca, and Pseudoperonospora cubensis (Ishii et al. 2001; Rosenzweig et al. 2008). However, azoxystrobin has been the most widely used active ingredient to control R. solani on sugar beet since 1997, which is the year of its registration in the USA (Khan et al. 2017). Difenoconazole and tebuconazole were effective against both strains of R. solani tested in vitro trials. This is in accord with other studies that showed the efficacy of these two fungicides against R. solani (Shan-lin 2013; Datta et al. 2016). In addition, our results show that the cultivation of sugar beet seeds in PDA medium containing difenoconazole caused no damage to seedlings. In planta, damping-off protection by difenoconazole against R. solani was around 80%. Moreover, soil treatment with this fungicide did not lead to any damage to sugar beet seedlings in our experiments.
In Morocco, difenoconazole fungicide has been allowed to control Sigatoka diseases in sugar beet. Although, tebuconazole and difenoconazole have not been allowed to control damping-off diseases in sugar beet. Fungicide mixtures with two different modes of action can be further explored for the apparition of damping-off in sugar beet caused by R. solani. Indeed, for a long time, the application of fungicides with different modes of action has been generally accepted to improve diseases protections synergistically, as well as to minimize the emergence of fungicide resistant strains in the fields (Gisi 1996). Azoxystrobin and difenoconazole synergism was well confirmed during the management of R. solani damping-off and root rot in sugar beet (Bartholomäus et al. 2017).
Sugar beet producers generally use seeds coated with chemical substances to avoid the damping-off phase. Nevertheless, biological control recently has become a famous alternative to conventional synthetic chemical pesticides that are harmful to the environment and human health. Several species of the genus Bacillus have shown their ability to biocontrol, have been exploited as biopesticides (Fravel 2005), and have been widely reported as agents with the ability to protect many cultures against fungal infections (Choudhary and Johri 2009; Rong et al. 2020). The commercially available B. subtilis strain Y1336 showed great potential to control rose powdery mildew (Wang et al. 2018a), and to significantly reduce the incidence of fire blight on apple and pear blossoms caused by Erwinia amylovora (Bahadou et al. 2017). B. subtilis Y1336, also, reduces significantly the incidence of Balloon flower stem rots caused by R. solani and Sclerotium rolfsii (Lee et al. 2012).
For the first time, the current study examined the effectiveness of B. subtilis Y1336 in biocontrol of the fungal pathogen known to cause damping-off. Their effect against R. solani was studied in dual culture, and tested by in vitro seed inoculation with R. solani, and by applying the BCA as seed coat under greenhouse conditions. In vitro test showed that the tested antagonistic BCA was able to reduce the growth of R. solani. Microscopic observation revealed altered mycelia morphology due to biological control activities. Additionally, in vitro inoculation of sugar beet seeds using R. solani and B. subtilis Y1336 showed that this combined treatment significantly increases the number of germinated seeds by 68.75%, compared to levels with co-cultivation of sugar beet seed and inocula of the pathogen alone. Under greenhouse conditions, coating the seeds with the BCA, significantly increased seed emergence and survival rates in addition to a significant reduction in damping-off disease severity caused by R. solani. In vitro and in vivo seed treatment with B. subtilis Y1336 did not show pre-or post-emergence damping-off diseases. Thus, this bacterium has no pathogenicity for sugar beet seedlings. As far as it is known, this is the first time that the commercially available B. subtilis Y1336 has been identified as a damping-off BCA. Pathogen management both on seeds and in the immediate environment of emerging seedlings with B. subtilis Y1336 as a seed coating or in-furrow application during seed sowing remains to be confirmed by field studies. There are several investigations on the application of B. subtilis to manage R. solani in several crops (Szczech and Shoda 2006; Ma et al. 2015; Mahmoud 2016b; Selim et al. 2017; El-Desouky et al. 2018). Various mechanisms have been suggested to elucidate the suppression of plant pathogenic fungi by B. subtilis, especially competition for space (Yu et al. 2011; Cao et al. 2012; Li et al. 2013), competition for nutrients (Moita et al. 2005; Li et al. 2013; Wang et al. 2018b), antibiosis (Landy et al. 1948; Stein 2005; Wang et al. 2018b), siderophore production (Yu et al. 2011; Singh et al. 2017; Khan et al. 2020) and induction of systemic resistance (Ongena et al. 2005, 2007; Wang et al. 2018b), or applied other biocontrol mechanisms.
Integrating fungicides with microbial antagonists can achieve protection against plant pathogenic by diminishing fungicide application. The combined application of Trichoderma asperellum SC012 and hymexazol was used to control Fusarium oxysporum causing cowpea wilt disease. Moreover, this combination could reduce the fungicide doses (Zhang et al. 2021). Fungicides with effective antifungal activity against phytopathogenic fungi must have no negative effect on the BCA growth. The BCA B. subtilis RB14-C used against R. solani was resistant to flutolanil, and the use of this bacterial antagonist reduced the quantity of flutolanil for effective management of tomato seedling mortality caused by R. solani (Kondoh et al. 2001). The control of Sclerotium sclerotium infecting witloof chicory was conducted by Coniothyrium minitans and also by a mixture of cyprodinil and fludioxonil fungicides. However, the application of this chemical mixture with C. minitans could not be suggested because 1 mg/ml of the mixture completely inhibited the mycelial growth of the antagonist (Benigni and Bompeix 2010). Our study showed that the commercial antagonist B. subtilis Y1336 was resistant to the 4 tested fungicides. These results suggest that the antagonist B. subtilis Y1336 can be integrated with a mixture of the 4 chemical fungicides to control R. solani diseases.
It may be that the conditions under which the tests were carried out in the greenhouse were close to the optimal growth of B. subtilis Y1336. The effectiveness of this BCA needs to be verified and validated under field-like conditions such as cool temperatures at the start of crop establishment. Furthermore, determining optimal concentration of B. subtilis Y1336, any synergistic effects with other antagonists, and potential biocontrol effects of this bacterium against other sugar beet damping-off agents need to be studied to optimize the potential exploitation of this commercially available antagonist as a BCA of damping-off in sugar beet. In the field, the dynamics of R. solani epidemics on sugar beet is characterized by a polycyclic epidemic (Otten et al. 2003). There are two sources of inoculum that ensure R. solani epidemics: the primary resident or incoming inoculum and the secondary inoculum generated by infected seedlings and roots (Gilligan and Kleczkowski 1997). Overwintering mycelia or sclerotia of the pathogen serves as the primary inoculum for seedling mortality. Thus, measures that minimize sources of infection or suppress the spread of this pathogen can reduce damage and loss caused by R. solani. Generally, this should be conducted by removing primary and secondary inoculum. Therefore, it will be interesting to know if an application of seeds treated with B. subtilis alone, fungicides alone, or their mixture affects both the primary and secondary inocula of R. solani (Gilligan and Kleczkowski 1997). Moreover, Future studies should focus on investigating the effect of B. subtilis Y1336 on the generation, survival, and germination of Rhizoctonia sclerotia. Finally, we hope that further studies will lead us to examine the potential role of B. subtilis strain Y1336 in promoting plant growth as well as its biocontrol effect against crown and root rot caused by R. solani.
Acknowledgements
This study was financially supported by the Phytopathology Unit of the Department of Plant Protection (ENA-Meknes).
Conflict of interest: All the authors declare that they have no confict of interest.
Human and animal rights: This article does not contain any studies with human participants or animals performed by any of the authors.
Funding: No funds, grants, or other support was received.
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Table 1: Some characteristics of used fungicides
|
Active ingredient (a.i.) |
Commercial name* (manufacture) |
Chemical family |
FRAC group |
Fungicide group |
|
Azoxystrobin |
Ortiva 25 SC (Syngenta) |
Strobilurin |
11 |
QoI-fungicides (Quinone aoutside Inhibitors) |
|
Difenoconazol |
Score 250 EC (Syngenta) |
Triazol |
3 |
DMI-fungicides (DeMethylation Inhibitors) |
|
Trifloxystrobin |
Flint 50 WG (Bayer) |
Strobilurin |
11 |
QoI-fungicides (Quinone outside Inhibitors) |
|
Tebuconazol |
Supreme Super 200 EC (Upl limited) |
Triazol |
3 |
DMI-fungicides (DeMethylation Inhibitors) |
|
Bacillus subtilis Y1336 |
WG |
Biofungicide (Bacillus spp.) |
44 |
- |
* WG : Water dispersible granule, SC = suspension concentrate. EC = Emulsifiable concentrate
Table 2: Mean effective fungicide concentration that inhibited mycelial radial growth by 50% (EC50) values of azoxystrobin, difenoconazol, tebuconazol, and trifloxystrobin for two strains of R. solani isolated from sugar beet fields in the Béni Mellal-Khénifra regions.
|
Fungicide |
EC50 (μg/ml) |
|
|
Rs1 |
RsM |
|
|
Azoxystrobin |
0.32 |
0.08 |
|
Difenoconazol |
0.91 |
0.07 |
|
Tebuconazol |
0.32 |
0.20 |
|
Trifloxystrobin |
1.96 |
4.08 |
Table 3. Effect of B. subtilis Y1336 on controlling sugar beet seedling damping-off disease caused by the pathogen R. solani (Rs1) under greenhouse conditions.
|
Treatments |
Number of healthy seedlings 15 DAS |
Pre-emergence disease (%) |
Number of healthy seedlings 60 DAS |
Post-emergence disease (%) |
|
Untreated seed without R. solani |
*20 ± 0 a |
0 |
20 ± 0 a |
0 |
|
Untreated seeds + R. solani |
4 ± 1.15 d |
79.74 |
0.75 ± 0.95 d |
81.25 |
|
Seeds treated with Difenoconazol + R. solani |
17.25 ± 0.95 b |
12.65 |
16 ± 0.8 b |
7.24 |
|
Seeds treated with B. subtilis Y1336 |
20 ± 0 a |
0 |
20 ± 0 a |
0 |
|
Seeds treated with B. subtilis Y1336 + R. solani (Rs1) |
14.25 ± 0.95b c |
27.84 |
11.75 ± 2.06 c |
17.54 |
*each value represents the average of two trials over time with four repetitions. Means within the same column followed by different letters are significantly different (p ≤ 0.05) based on the least significant difference (LSD) test.