Several genera of soil fungi are pathogens that cause severe damage to crops around the world, resulting in large yield losses ( Abuzar, 2013 ; Naing et al. , 2014 ). One such soil-borne fungal pathogen is Rhizoctonia , which is widely distributed and causes diseases such as stem and root rot, damping-off, wilting, and leaf blight ( Cao et al. , 2004 ). In chili peppers ( Capsicum annuum L.), one of the most important spice crops in the world ( Smith, 2015 ), Rhizoctonia solani , along with Phytophthora capsici and Fusarium oxysporum , cause severe damage to the roots and stems which negatively impacts on plant growth and crop yield ( Velásquez-Valle et al. , 2001 ; Guillén-Cruz et al. , 2006 ; Lee et al. , 2008 ; Youssef et al. , 2016 ). Mexico is one of the largest exporters of chili peppers worldwide, and one of the challenges it faces is to reduce yield losses caused by root pathogens. This can be achieved through the use of different control strategies and one of them is to apply antagonistic microorganisms ( Mojica et al. , 2009 ; Abuzar, 2013 ).

In recent years, there has been increasing interest in biological control as an alternative to the use of agrochemicals that have a negative impact on the environment ( Huang et al. , 2012 ; Czaja et al. , 2015 ). The use of plant growth-promoting rhizobacteria (PGPR) can be a good option because, in addition to positively affecting crops, several genera are natural antagonists of phytopathogenic fungi ( Commare et al. , 2002 ; Saravanakumar et al. , 2007 ; Pliego et al. , 2011 ; Huang et al. , 2012 ). Biocontrol through rhizobacteria has a low cost and can substitute or complement chemical fungicides ( Jha & Anjaiah, 2007 ). In several parts of the world, both rhizobacteria and secondary bacterial metabolites have been isolated and characterized for their potential to inhibit the phytopathogenic fungi of agricultural crops ( Jha & Anjaiah, 2007 ; Jung et al. , 2014 ; Seiber et al. , 2014 ). Antagonist bacteria are characterized for their capacity to produce antibiotics, siderophores, hydrogen cyanide, chitinases, glucanases, and proteases ( Nagarajkumar et al. , 2004 ; Babalola, 2010 ). These characteristics give them the ability to inhibit phytopathogen growth ( Jung et al. , 2014 ).

In chili crops, obtaining healthy seedlings in seedbed is essential to avoid the spread of diseases when they are transplanted into the field. This can be done with the use of phytopathogen-free substrates or by applying chemical or biological fungicides during seedling growth. One of these fungicides could be rhizobacteria with antifungal activity. The objective of this study was to characterize the ability of three rhizobacterial strains to inhibit the growth of Rhizoctonia solani and to test its potential in chili seedbeds as biocontrol agents.

The sequences of the strains P61 and A46 had 99% similarity to the sequence of Pseudomonas tolaasii . In the case of strain S108, the sequence showed 99% similarity to the sequence of Rahnella aquatilis (Table 1). This strain also had similarity with the genera Ewingella, Hafnia, Serratia , and Rouxiella . According to its phenotypic characteristics, S108 fits better with the description of Rhanella ( Kämpfer, 2005 ; Brady et al. , 2014 ). The strain S108 has creamy-colored, convex colonies with full margins, is negative for indole production, grows at temperatures between 4 and 37 ^o C, and the cells are motile. The nucleotide sequences were submitted to the NCBI GenBank database, where the following accession numbers were assigned: KY933651.1 (strain A46), KY933652.1 (strain P61), and KY933653.1 (strain S108).

All three rhizobacterial strains used in this study inhibited the growth of Rhizoctonia solani in dual culture (Table 2). These strains have the potential to control diseases in agriculturally important crops. Biological control using rhizobacteria is linked to their allelochemical activity, which includes the production of antibiotics, lytic enzymes, bioacids, molecules involved in quorum sensing, virulence factors, and siderophores ( Chauhan et al. , 2014 ; Saraf et al. , 2014 ). In addition, it also involves competition for space or nutrients and the induction of systemic resistance in plants ( Sulochana et al. , 2014 ).

In this study, several experiments were conducted to determine the ability of rhizobacterial strains to inhibit the growth of R. solani ; their antagonistic activity is not caused by the release of volatile organic compounds. This conclusion was supported by the divided plate experiment (in which there was a barrier in the middle of the plate), in which the phytopathogenic fungus grew, crossing the barrier on the fourth day, and once it passed the division, the inhibition zone was observed (data not shown). Other studies have demonstrated that rhizobacteria can control phytopathogens via the production of volatile compounds, primarily hydrogen cyanide. For example, Pseudomonas and Bacillus inhibit fungal development via HCN production ( Zhang et al. , 2015 ).

The production of siderophores is another biocontrol mechanism that rhizobacteria have to suppress the growth of fungi that cause diseases in crops ( Chernin et al. , 1995 ; Kielak et al. , 2013 ; Brzezinska et al. , 2014 ). The three rhizobacteria considered in this study produced siderophores (Table 2). These compounds chelate iron, are quite diverse, and can only be synthesized by plants and microorganisms like bacteria and fungi ( Milagres et al. , 1999 ; Shin et al. , 2001 ; Pérez et al. , 2007 ; Louden et al. , 2011 ). It has been observed that when rhizobacteria take up the iron in the medium, they make it unavailable for other microorganisms, including phytopathogens, suppressing their growth. In this case, the production of siderophores was not the mechanism that was involved in the antifungal activity of the strains against R. solani because even in the PDA supplemented with iron, these strains inhibited the growth of the phytopathogenic fungi in the same manner as they did in the medium without iron (data not shown). In other studies, the production of chitinases by rhizobacterial strains of Pseudomonas, Serratia , and Bacillus has been reported and related to the antifungal activity of these microorganisms ( Brzezinska et al. , 2014 ; Akocak et al. , 2015 ). In this work, none of the three strains produced chitinases.

The bacterial filtrates at a 1:9 ratio (filtrate/PDA) did not have antifungal activity on R. solani , probably because the amount of antifungals was low. By increasing the bacterial filtrates to a 3:7 ratio, it was found that the S108 filtrate showed activity against the fungus, this was with the filtrate obtained from bacterial culture without the fungus or co-culture with the fungus, which suggests that strain S108 releases compounds with stronger antifungal activity. The same was true with the filtrate A46 obtained from co-culture (Fig. 2).

The production of compounds of microbial origin is influenced by several factors including temperature, culture medium (nutrients), pH, the growth phase of the microbial colony, stress conditions, and the presence of or stimuli from other microorganisms ( Berendsen et al. , 2012 ; Zha et al. , 2013 ). In particular, strain A46 only released metabolites with antifungal activity when it was co-cultured with R. solani in PDB (Figs. 1 and 2). Contrastingly, strain S108 did not require the presence of the fungus to produce compounds with strong antifungal activity. Strain P61 without the fungi or in co-culture produced metabolites with weak antifungal activity.

Pseudomonas strains reportedly produce different antimicrobials like phenazine, 2,4-diacetylphloroglu­cinol, pyrrolnitrin, hydrogen cyanide, and pyoverdine, among others ( Coraiola et al. , 2006 ; Rokni et al. , 2012 ). In the particular case of Pseudomonas tolaasii , this species causes rot in edible fungi, and the extracted toxin, which is called tolaasin, has been associated with the disease caused by the bacterium ( Cho & Kim, 2003 ; Sunhee et al. , 2011 ). In this study, the two P. tolaasii strains significantly inhibited R. solani growth in dual culture. Their antagonistic activity may be due to the production of tolaasin, even when the filtrates had very low antifungal activity. Other studies also report that bacterial filtrates obtained from strains with antagonistic activity against fungi in dual culture do not always show antifungal activity ( Li et al. , 2014 ).

In the case of Rahnella aquatilis , it is a Gram negative enterobacteria that has been found in water bodies, forest soils, mosses, colonized roots, and sporomes of ectomycorrhizal fungi ( Izumi et al. , 2006 ; Opelt et al. , 2007 ; Degelmann et al. , 2009 ; Kumari et al. , 2013 ). This bacterial species has been reported as a plant growth promoting rhizobacteria ( Kandel et al. , 2017 ) and it has antagonistic activity against phytopathogenic fungi of the genera Penicillum, Botrytis, Fusarium, Rhizoctonia, Pythium , and Gaeumannomyces ( Calvo et al. , 2007 ; Opelt et al. , 2007 ; Kandel et al. , 2017 ). Other attributes that have been found in strains of this species are: the ability to produce secondary metabolites (peptides, siderophores, amino acids, organic acids, phytohormones) and solubilize phosphates ( Pintado et al. , 1999 ; Opelt et al. , 2007 ; Oulkadi et al. , 2014 ; Kandel et al. , 2017 ). In this work, the filtrates of strain R. aquatilis S108 mixed with the PDA medium at 3:7 ratio showed high antifungal activity.

Rhizosphere microorganisms establish different interactions and produce a large variety of compounds with bioactivity that depends on the microorganism, the environment and the interaction itself ( Cray et al. , 2015 ), so the use of two or more microorganisms in co-culture could be essential for the production of bioactive compounds, simulating what happens in the rhizosphere ( Bertrand et al. , 2014 ). The co-culture of two microorganisms has been shown to increase the production of metabolites that are not present in individual cultures ( Pettit, 2009 ; Bertrand et al. , 2014 ; Dashti et al. , 2014 ). For this reason, an experiment in which the bacteria were co-cultured with the pathogenic fungus was conducted in PDB, and the resulting filtrates were evaluated for their antifungal activity. Only the antifungal activity of the filtrate from the P. tolaasii A46 strain increased significantly due to the effect of co-culturing with the fungus, suggesting that the production of antifungal metabolites increased due to the presence of R. solani . This finding demonstrates that some rhizobacterial strains with the potential for the biological control of phytopathogenic fungi require fungal stimulus to trigger the production of antifungal compounds. Several factors affect the growth of microorganisms in culture medium, and in turn, these microorganisms produce several compounds ( Wang et al. , 2011 ; Zhao et al. , 2013 ; Dashti et al. , 2014 ; Djinni et al. , 2014 ). Some of these compounds are organic acids, such as lactic, phenylacetic and propionic acids, which lower the pH of the culture medium, inhibiting filamentous fungi ( Lavermicocca et al. , 2003 ; Saithong et al. , 2010 ; Li et al. , 2014 ). In our study, we observed that the pH of the medium was slightly acidified with all three rhizobacteria. Thus, to rule out this possibility as the cause of the R. solani inhibition, the pH was adjusted to 7 after the filtrates were obtained. We found that even when adjusting the pH, the filtrates maintained their antifungal activity.

The application of the strains S108 and P61 to Serrano chili seedlings decreased the mortality rate caused by R. solani , compared with the control. Strain S108 and the Captan treatment had a similar effect on the reduction of seedling mortality caused by the phytophatogen, which was maintained until the end of the experiment. This suggests that strain S108 had an effectiveness equivalent to the chemical fungicide (Fig. 3). Strains S108 and P61 have great potential as biopesticides to control R. solani in chili seedbeds and possibly other transplant vegetables.

Obtaining healthy seedlings in seedbeds is essential for the successful production of chili crops in the field. The use of inoculants prepared with rhizobacteria like the R. aquatilis S108 strain can help small farmers to control Rhizoctonia and possibly other fungi that cause root diseases in the seedbeds, being of low cost and friendly to the environment. However, it is necessary that the study continues with field tests in order to evaluate if the inoculation of the strain S108 is as effective in the control of this disease as the use of chemical fungicides.

In summary, the three strains of rhizobacteria in dual culture inhibit the growth of R. solani by between 38 and 49%. Strains produce siderophores; however, the antifungal activity was not related to these compounds. The filtrate of strain S108 had the greatest effect on R. solani with 56% inhibition when this is added to the PDA medium in a 3:7 ratio. The rhizobacterial inoculation reduced the mortality of seedlings caused by R. solani . The best treatments were the inoculation of strain S108 and the application of Captan, which had statistically the same effect. The strain of Rahnella aquatilis S108 has potential as a biopesticide to control R. solani in chili seedbeds and possibly the filtrate obtained from the bacterial culture also works to reduce the growth of the pathogen at the nursery level.