Selected rhizobacteria strains as potential growth promoters and biocontrol agents against chocolate spot disease in faba bean grown in pots

Abstract The faba bean is a grain legume used for food and feed. It suffers from several yield limiting diseases. Botrytis fabae Sard is a fungal pathogen which causes chocolate spot disease in faba bean all over the world. Searching for solutions to the problem of yield loss due to B. fabae is critical. One sustainable solution is the utilization of plant-growth-promoting rhizobacteria as biocontrol agents. This study aimed to demonstrate the effect of rhizobacteria strains on controlling chocolate spots and promoting the growth of faba beans under pot conditions. Three rhizobacteria strains that previously demonstrated promising potential were used and tested for antagonistic activity against B.fabae in dual culture. All tested rhizobacterial strains inhibited the mycelial growth of B.fabae with different potential, and Chryseobacterium strain GY04 showed a maximum percent of mycelial growth inhibition of 72.38%. Also, all rhizobacteria strains significantly reduced the incidence and severity of chocolate spot disease, with different efficacy in both faba bean varieties under pot conditions. Chryseobacterium strain GY04 showed the highest disease severity reduction on the local variety at 75 (79.62%) and 90 (73.57%) days after sowing. Rhizobacterium Pseudomonas chlororaphis GY07 resulted in the highest disease severity reduction on the Dosha variety (80.12, 43.85%) at 75 and 90 days after sowing, respectively. All tested rhizobacteria improved the main plant growth parameters in both varieties. The rhizobacterial strains examined in this study could be used as biocontrol against Chocolate spot disease and as growth promoters under pot conditions.


Introduction
The faba bean is a grain legume used as food and feed.It is a source of protein, mineral nutrients, vitamins and numerous bioactive compounds (1).It also plays a significant role in improving soil fertility through symbiotic nitrogen fixation and, as a result, improving crop production (2).
Although this crop suffers from several yield limiting diseases, over a hundred pathogens attack it (3).In Ethiopia, more than 26 diseases have limited the production of faba beans (You et al. 2021).Chocolate spot disease of the faba bean is one of the world's major diseases, caused primarily by Botrytis fabae Sard and, to a lesser extent, Botrytis cinerea Pers.It causes crop losses ranging from minor to complete yield loss through damage to the foliage, limiting photosynthesis activity, and finally reducing production (4).It is one of the most significant faba bean diseases in Ethiopia.Also, this disease is a major problem in the study area (You et al. 2021).
Various control options are applied globally to manage the chocolate spot disease in faba beans (Sahile et al. 2008;5-9).In Ethiopia, to manage Chocolate spot disease, the use of fungicides (chlorothalonil or mancozeb) and late planting were suggested (Sahile et al. 2008).Due to the high cost of fungicides and the intermittent rainfall distribution, farmers can not adopt these options fully (9).The fungicide mancozeb has a considerable deleterious impact on nutrient uptake and limits plant growth (10).Using resistant varieties is another chocolate spot management option (5).However, these options have not been realized in Ethiopia because all available varieties in the country are not well resistant to chocolate spot disease (11).
As a result, it is critical to find an alternative solution that is simple to implement, cost-effective and non-hazardous to the environment.Currently, biocontrol agents as an alternative disease management option have gained popularity for managing plant disease (12).Using rhizobacteria as biocontrol agents is an excellent option for managing plant pathogens without environmental pollution.The application of rhizobacteria as a biocontrol is mandatory to eliminate the development of resistance to fungicides (13).
Many bacterial species from different parts of the world have shown antagonistic potential against Botrytis infection in faba beans (6)(7)(8)14).Few studies on the control of the chocolate spot disease of faba beans by rhizobacteria have been conducted in Ethiopia (15)(16)(17).All these studies are limited to the well-studied rhizobacteria Pseudomonas florescence and Bacillus species.There is a need to search for extra rhizobacteria to achieve effective biocontrol agents against B. fabae.
We have recently reported that Chryseobacterium spp.and Pseudomonas chlororaphis had promising potential to control chocolate spot disease, growth and yield-related parameters of the faba bean under field conditions (27).Microbes in the rhizosphere usually live in groups and rarely as individuals.The result obtained in the previous research may be attributed to the interaction effects of different microbes with our inoculant strains.To develop a potent inoculant, evaluation of the growth-promoting ability of a single microorganism in controlled environments is critical.The aim of the present study was to demonstrate the effect of rhizobacteria strains on controlling chocolate spots and promoting the growth of faba beans under pot conditions.

Study pathogen
The virulent Botrytis fabae isolate, the causative agent of chocolate spot disease in faba beans, was obtained from the culture collection centre of the Biology Department at the University of Gondar.The Botrytis fabae isolate culture was refreshed by following Mohamadpoor et al. (2022).

Faba bean varieties
We used two faba bean varieties in this study.One local variety susceptible to chocolate spot disease was obtained from local farmers in the Gondar Zuria district.One variety with moderate resistance (Dosha variety) to chocolate spot disease was obtained from the Gondar Agricultural Research Centre, respectively (11).

Dual culture assay
The dual culture assay was performed to determine the potential of rhizobacteria against the test pathogen B. fabae (25,34).Briefly, mycelial discs of B. fabae were taken from the 4-day-old culture and inserted in a 9 cm plate containing PDA modified with 10% sucrose powder.A loopful of rhizobacteria culture (48 h old) grown on NA was streaked at the centre of the plate and 3 cm away from the mycelial disc of B. fabae.The B. fabae mycelial disc was also inserted in a 9 cm plate containing PDA modified with 10% sucrose powder without rhizobacteria which served as a control.The plates were incubated at 28 ± 2 °C and observed daily for 9 days.Mycelial growth inhibition was assessed by measuring the radial growth of B. fabae in the treated plate.The experiment was performed in a completely randomized design (CRD) with three replicates.The radial growth of B. fabae mycelium was measured on the 9th day, and the percentage inhibition of growth over the control was calculated, using the formula, where, PIRG is the percent inhibition of radial growth; R1 is the radius of B. fabae in the control, and R2 is the radial growth of B. fabae toward the bacterial isolate in the treated Petri plates.

Pot experiment treatments and experimental design
This experiment was designed to evaluate the potential of rhizobacteria species against the chocolate spot disease and improve faba beans growth under pot conditions.This experiment was carried out at the University of Gondar in 2022 in sterilized pots (20 cm in diameter) containing sterilized soil in a CRD approach, with three replications.The pots were sterilized as described previously (35) with a 5% formalin solution and then the pots were left for two weeks for formalin evaporation.The soil was sterilized at 121 °C for 30 min.The experiment included the following treatments in both varieties:

Preparation of B.fabae and soil infestation
From the purified Botrytis fabae culture, a 5-mm mycelia agar plug was taken and inoculated in a 250-mL flask containing 100 mL potato dextrose broth (PDB), and was incubated at 25 °C for 15 days.After 15 days of incubation, the suspensions were adjusted to 4.5 × 10 5 spores per milliliter using a hemocytometer (36).Soils collected from farmers' fields in the Gondar Zuria district were sterilized at 121 °C for 30 min.Pots containing the sterilized soil were infected with the adjusted spore concentration of B. fabae at a rate of 50 mL/pot and were watered for 7 days to enhance fungal growth at 25 °C and 12 h day and 12 h night.The control pots were treated with the same amount of sterilized PDB without pathogens before planting (17).

Rhizobacterial inoculum preparation and seed coating
A single colony from each strain was separately inoculated into 150 mL (nutrient broth culture media) in 250 mL Elenmeyer flasks.The flasks were incubated at 30 °C on orbital shaker at 120 rpm until the cell concentrations reach 10 8 cfu mL −1 (OD650 nm = 1.0).Healthy seeds of the same size were selected and surface-sterilized with 70% ethanol for 1 min, then rinsed with 5% sodium hypochlorite for 3 min and washed with three changes of sterile distilled water.Seeds were air dried and checked for germination on a Petri plate (37).Faba bean seeds that found germination were dipped into each rhizobacteria species nutrient broth culture (approximately 10 8 cells mL −1 ) for 5 hours.Germinated seeds dipped into sterile nutrient broth served as a control after being treated with a 4% solution of carboxymethyl cellulose (CMC) as a sticker for 15 min at a rate of 3 mL per 100 seeds.Three germinated seeds were sown per pot, and three pot replicates (i.e. 9 plants) were used for each treatment.Treated and non-treated pots were irrigated with enough water daily for 90 days.

Disease assessment and data collection
The disease developments were assessed in terms of disease incidence and severity.The disease incidence was recorded for up to 90 days after sowing (DAS).Disease incidence was expressed as a percentage of infected leaves out of the total leaves per treatment (17).Disease severity was expressed as percent of affected leaf based on symptoms, according to (38), using a rating scale of 0-5 (0 = no symptoms, 1= up to 5%, 2 = 6-10%, 3 = 11-25%, 4 = 26-50% and 5 = 51-100% of leaf area affected).The scale (1-5) was rated to infected leaves on the basis affected areas' disease strength through visual observations of symptoms.Finally, the disease severity was expressed as Percent Disease Index (PDI).

Diseasescale plant number inthatclass
Highest scale tota / l lnumber of plants The biological control efficacy (BCE) of the rhizobacteria over the control was calculated according to (39): BCE%= (Dc-Dt/Dc) ×100%, where, Dc is disease severity/incidence of control pots, Dt is disease severity/incidence of the treatment pots).
After 90 days of planting, the plants were uprooted to measure the growth parameters including shoot length (SL), shoot fresh weight (SFW), shoot dry weight (SDW), root length (RL), root fresh weight (RFW), and root dry weight (RDW) (40).

Data analysis
Data are presented as mean values with standard error of the means.The statistical analysis was performed by two-way analysis of variance (ANOVA) using SPSS version 25.The comparisons among means were done by using Tukey HSD analysis at α = 0.05.Differences were considered significant at the p < .05level (17).

Plant growth promoting properties of the rhizobacteria species
All the tested rhizobacteria strains showed multiple plant growth-promoting traits (27).

In-vitro antagonistic effects of rhizobacteria species against the test pathogen (B. fabae)
At day 9 of incubation, B. fabae mycelial growth was measured in both treated and control plates.The results revealed that all of the rhizobacteria species significantly inhibited the mycelial growth of the test pathogen.The tested rhizobacteria, Chryseobacterium strain GY04, Chryseobacterium proteolyticum GY05 and Pseudomonas chlororaphis GY07, reduced mycelia growth of B. fabae with (72.38%), (63.62%) and (61.94%) inhibition, respectively, vs. the control treatment (B.fabae solely) (Table 1).

Biological control potential of rhizobacteria species against faba bean chocolate spot disease under pot conditions
All the tested rhizobacterial strains caused reductions in chocolate spot disease incidence and severity in both varieties (Tables 2 and 3).In the pots with the faba bean (local) variety, all of the tested rhizobacteria treatments resulted in a significantly (p < .05)lower incidence percent of chocolate spot disease (19-33%) compared to control (75%) at 75 days after sowing (DAS).Also, the disease severity on faba bean (local) variety treated with all of the tested rhizobacterial species significantly decreased: it was 7.92-25.32%compared with the control (38.88%) at 75 DAS (Table 2).
Treatment of the faba bean seeds (local) variety with any of the rhizobacterial strains resulted in lower incidence of chocolate spot disease (27-35.5%)when compared to the control (83%) at 90 DAS (Table 2).The maximum disease incidence reduction percent (BCE %) at 90 DAS was recorded on the pot treated with Chryseobacterium strain GY04 (67.46%), whereas the minimum value was on the pot treated with Chryseobacterium proteolyticum GY05 (57.12%).Regarding the disease severity reduction percent (BCE %) at 90 days after sowing, the maximum reduction percent was observed on the pots treated with Chryseobacterium strain GY04.
At 75 DAS, all of the tested rhizobacteria treatments significantly reduced the incidence percent of the chocolate spot disease (7.69-12.31%)compared to the control (44.33%) in the Dosha variety (Table 3).Furthermore, the results showed that the disease severity decreased significantly (2.98%-4.44%)compared to the control (14.98%) at 75 DAS.At 75 DAS, the maximum reduction in the disease severity percent (BCE %) was observed on pots treated with Pseudomonas chlororaphis GY07 (80.12%), and the minimum value was recorded on pots treated with Chryseobacterium proteolyticum GY05.Note.%Di: percent disease incidence, Bce: biocontrol efficacy, %DS: percent of disease severity Values in each column followed by the same letter are not significantly different at p < .05according to tukey hSD analysis of two Way anoVa.
At 90 DAS, all of the tested rhizobacterial species significantly reduced the incidence of chocolate spot disease (23.52-26.34%)compared to the control (46.2%).The disease severity also decreased significantly (11.05-12.77%)compared with control treatment (19.4%) at 90 DAS with all tested rhizobacteria.The maximum disease incidence reduction percent at 90 DAS was recorded on the pot treated with Chryseobacterium strain GY04 (49.09%), whereas the minimum value was recorded on the pot treated with Pseudomonas chlororaphis GY07 (42.98%).Regarding the disease severity reduction percent at 90 DAS, the maximum reduction percent observed on pots treated with Pseudomonas chlororaphis GY07 was 43.85%, whereas the minimum value recorded on pots treated with Chryseobacterium strain GY04 was 34.17% (Table 3).The interaction effect of host varieties and treatment on the chocolate spot disease severity was found significantly (p < .05)different (Table 3.1).

Effect of rhizobacterial strains inoculation on the growth parameters of faba bean under pot conditions
The pot experiment showed that treatment with all three rhizobacterial strains was associated with a significant increase (ANOVA, p < .05) in the shoot length, fresh weight, and dry weight and in the root length, fresh weight and dry weight compared to the un-inoculated control (Tables 4 and 5).
In both varieties, plants inoculated with Pseudomonas chlororaphis GY07 showed the maximum results in all growth parameters compared to the control.In this regard, a significant difference (p < .05) in shoot and root dry weight was achieved by Pseudomonas chloro raphis GY07, with 13.93 and 5.20 g in the local variety (Table 4) and 16.60 and 11.10 g in Dosha (Table 5), respectively.The shoot dry weight displayed a 64% increase over T1 (B.fabae only) in the local variety (Table 5).In the same pattern, an increase of 70% in shoot dry weight over T1 (B.fabae only) was observed in Dosha (Table 6).Chryseobacterium proteolyticum GY05 resulted in the minimum growth enhancement in all growth parameters recorded on the plants treated with it (Tables 4 and 5).The interaction effect of host varieties and rhizobacterial strains treatment on the growth parameters were found significantly (p < .05)different (Table 6).

Discussion
In north-western Ethiopia faba (6) and in other countries bean production is troubled by Chocolate spot disease initiated by Botrytis fabae (You et al. 2021).The severity and significance of the damage caused by this disease have demanded the development of effective strategies for its management.Various control options are applied globally to the chocolate spot disease of faba beans.Currently, rhizobacteria are considered an alternative option to control disease and boost faba bean productivity (41).Plant growth-promoting rhizobacteria are efficient biofertilizers, phytostimulators and biopesticides that can be used to improve crop yield, soil quality and control phytopathogens (42).
In this study, we evaluated the effect of rhizobacteria strains on the control of chocolate spot disease and enhance the growth of faba beans under pot conditions.In vitro screening for plant growth-promoting traits and lytic enzyme activities of the tested rhizobacterial species Note.%Di: percent disease incidence, Bce: biocontrol efficacy, %DS: percent of disease severity Values in each column followed by the same letter are not significantly different at p < .05according to tukey hSD analysis of two-way anoVa.Note.Shoot length (Sl), shoot fresh weight (SFW), shoot dry weight (SDW), root length (Rl), root fresh weight (RFW) root dry weight (RDW).Values are mean ± Standard error of the mean of three replications, means in each column followed by the same letter are not significantly different at p < .05according to Fisher's lSD, cV, coefficient of variance according to tukey hSD analysis of two-way anoVa.Note.Shoot length (Sl), shoot fresh weight (SFW), shoot day weight (SDW), root length (Rl), root fresh weight (RFW) Root dry weight (RDW).Values are mean ± Standard error of the mean of three replications, means in each column followed by the same letter are not significantly different at p < .05according to tukey hSD analysis of two Way anoVa.
Table 6.an interaction analysis between faba bean varieties and treatments on the growth parameter (shoot length (Sl), shoot fresh weight (SFW), shoot dry weight (SDW), root length (Rl), root fresh weight (RFW) root dry weight (RDW)) by two-way anoVa.This study showed that all tested rhizobacterial strains inhibited the mycelial growth of B. fabae in dual culture experiments (Table 1).Plant growthpromoting rhizobacteria protect plants from phytopathogens through several mechanisms such as antibiosis, competition and the production of lytic enzymes (Basu et al. 2021).All of the tested rhizobacteria strains produced the hydrolytic enzymes lipase and protease.Hydrolytic enzymes, such as glucanases, proteases, chitinases and lipases, are involved in the lysis of fungal cell walls by either digesting the enzymes or deforming the cell wall components of fungal pathogens (44,45).
The ability of the tested rhizobacteria to inhibit the mycelial growth of B. fabae may relate to production of different hydrolytic enzymes.The different efficacy of the tested rhizobacteria to inhibit radial growth may be associated with differences in hydrolytic enzyme production.(17) reported that there was variability in the percent inhibition of mycelial growth potential displayed by different PGPR isolates against B. fabae.
In our study, the maximum mycelium growth inhibition of B. fabae was recorded on the plate treated with Chryseobacterium strain GY04.(46) reported that Chryseobacterium strains had biocontrol activity against different plant pathogens.Another study by (26) showed that Chryseobacterium species produced protease and HCN with swarming activity.The effectiveness of this rhizobacterium may arise from these plant growth-promoting traits.Many studies have demonstrated that Chryseobacterium species are plant-growth-promoting rhizobacteria that suppress a wide range of plant pathogens (25).
Pseudomonas chlororaphis GY07 was observed to exhibit reduced mycelial growth of B. fabae in vitro.Moreover, inoculation with these rhizobacteria significantly reduced the incidence and severity of chocolate spots under pot conditions in local and Dosha varieties compared to controls.These results are in agreement with previous studies which proved that Pseudomonas chlororaphis exhibits potent antagonistic activity against several phytopathogenic fungi in various plants such as Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum sp.(19), S. sclerotiorum (20) and can help control R. necatrix, which causes avocado white root rot (WRR) disease (21,23).Additionally, Pseudomonas chlororaphis YB-10 strongly inhibited the mycelium growth of Gaeumannomyces graminis var.tritici in dual cultures.Because of its ability to produce extracellular protease, cellulase, indole acetic acid (IAA) and siderophore it is able to antagonize various phytopathogens and enhance plant growth (24).P. chloro raphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the pathogenic fungus causing peanut stem rot (22).In our previous study (27), P. chlororaphis GY07 was able to produce different hydrolytic enzymes.Bacteria that can produce hydrolytic enzymes are one mechanism of suppression of phytopathogenic fungi (44).
In this study, the other rhizobacteria strain evaluated for being antagonistic to B. fabae under in vitro and pot conditions was Chryseobacterium proteolyticum GY05.This rhizobacteria strain showed inhibition of mycelial growth of Botrytis fabae (Table 1) and reduced the incidence and severity percent of chocolate spot disease in faba beans significantly in both faba bean varieties.Previous studies have demonstrated that Chryseo bacterium proteolyticum is a plant-growth-promoting rhizobacteria that suppresses a wide range of plant  pathogens (47,48).These results are in agreement with those reported by (25), who presented that Chryseobacterium proteolyticum (AS2) isolated from cocoa plants can reduce Phytophthora palmivora-caused black cocoa pods.
The data presented in Tables 4 and 5 demonstrated that three tested rhizobacterial strains significantly improved plant growth compared to the un-inoculated control.Inoculation with P. chlororaphis GY07 had pronounced potential to increase shoot and root fresh and dry weights in both varieties.This result is in line with the results of (24), who showed that inoculation with P. chlororaphis significantly promoted the growth of wheat seedlings.In our previous study, these rhizobacterial strains could produce IAA, ammonia and solubilize phosphate (27).The rhizobacterial strains that produce auxin have a pronounced effect on plant growth via nutrient uptake from the surrounding environment through enhancing root length, root branches, root laterals, and root hair (49,50).The ability of rhizosphere bacterial strains to produce ammonia is one of the mechanisms for enhancing the growth of the host plants via either increasing the root or shoot length or increasing the efficacy of the plant defence against phytopathogen attacks (51).
The utilisation of phosphate-solubilizing rhizobacteria to advance phosphorus availability to plants is important for eco-friendly, sustainable crop production (52).
In this study, the inoculation of faba bean seed with Chryseobacterium species enhanced shoot length, root length, and fresh and dry weight of shoot and root (Tables 5 and 6).The present result is in line with the results of Youseif [53], who showed that maize plants treated with Chryseobacterium sp.strain NGB-29 had increased shoot and root fresh and dry weights and showed the highest potential for IAA production.
In the present study, three rhizobacterial strains improved faba bean growth by preventing the chocolate spot disease effect under pot conditions.This indicates that these strains may be helpful as a biocontrol agent and biofertilizer for vegetables grown in pots and containers as part of the urban rooftop gardening trend, which is currently attracting interest around the globe.

Conclusions
All three tested PGPR strains improved both shoot and root growth parameters in both faba bean varieties.The tested strains also showed great potential for reducing chocolate spot disease incidence and severity.As a result, these strains may be suitable for future applications as plant growth promoters, biofertilizers and biocontrol agents for a fungal diseases in faba bean or other crops.

Table 1 .
inhibitory effect of rhizobacteria species on mycelial growth of B. fabae under in vitro tests after 9 days.Standard error of the mean of three replications, means in each column followed by the same letter are not significantly different at p < .05according to the lSD test (p ≤ 0.05).

Table 2 .
the effect of rhizobacteria species to control chocolate spot (Botrytis fabae) disease incidence and severity on local variety of faba bean using pot experiments.

Table 3 .
the effect of rhizobacteria species to control chocolate spot (Botrytis fabae) disease incidence and disease severity on Dosha variety of faba bean using pot experiments.

Table 3 .
1. an interaction analysis among the chocolate spot disease severity at 90 DaShosts varieties and treatment group by two-way anoVa.

Table 4 .
effect of rhizobacterial species inoculation on the growth parameters of faba bean local variety under pot conditions.

Table 5 .
effect of rhizobacterial species inoculation on the growth parameters of faba bean Dosha variety under pot conditions after 90 days.