Biocontrol of Rhizoctonia Root Rot in Tomato and Enhancement of Plant Growth using Rhizobacteria Naturally associated to Tomato

1Higher Agronomic Institute of Chott-Mariem, Sousse University, 4042-Chott Mariem, Tunisia 2UR13AGR09-Integrated Horticultural Production in the Tunisian Centre-East, Regional Centre of Research on Horticulture and Organic Agriculture, University of Sousse, 4042, Chott-Mariem, Tunisia 3INRA, UMR1065 Santé et Agroécologie du Vignoble (SAVE), ISVV, F-33140 Villenave d’Ornon, France 4Université de Bordeaux, Bordeaux Sciences Agro, ISVV, UMR1065 SAVE, F-33140 Villenave d’Ornon, France 5BIOVITIS, 15400 Saint Etienne de Chomeil, France 6USC Oenologie-INRA, Université Bordeaux Segalen, Bordeaux Sciences Agro, ISVV, Villenave d’Ornon, France


Introduction
Tomato (Solanum lycopersicum L.) is the second most important vegetable crop worldwide after potato based on grown areas [1]. In Tunisia, it is a strategic and an economically relevant crop. However, this crop is still threatened by serious wilting and root-rotting pathogens both in greenhouse and open-field growing systems [2,3]. The most widely grown tomato cultivars were susceptible to soilborne infections and especially to Rhizoctonia Root Rot disease caused by Rhizoctonia solani (Kühn). This pathogen is mostly known as a damping-off agent but is also responsible for collar and root rots and eventual death of severely diseased plants leading to significant crop yield loss [4][5][6].
Efficient disease control is difficult due to the various host range of the causative agent, the persistence of its resting structure (sclerotia) in soil, the lack of genetic resistance and to the limited efficacy of chemical fungicides [7]. Such issues have focalized research efforts on development of environmentally safe, long lasting and effective alternatives such as biological control [8].
Among the group of BCAs, plant growth promoting rhizobacteria (or PGPR) have been widely used for the bio-suppression of various soilborne diseases [17]. In fact, PGPR strains can display disease-suppressive effects against various crown, root and foliar diseases through direct inhibition of target pathogens or indirectly via the induction of systemic resistance (ISR) which is active throughout the entire plant [18][19][20]. PGPR-treated plants showed enhanced emergence potential and increased vegetative and root growth [17,21,22].

Tomato cultivar and growth conditions
Tomato plants cv. Rio Grande seedlings were used for all in vivo bioassays. Seeds were disinfected with 5% sodium hypochlorite during 2 min, rinsed thrice with sterile distilled water (SDW) and air-dried. They were sown in disinfected alveolus plates and maintained under greenhouse conditions (30 ± 4°C; 13/11 h light/dark photoperiod). Seedlings were regularly watered to avoid water stress.

Pathogen origin and growth conditions
R. solani isolate used in the present study was originally isolated from tomato plants exhibiting severe Rhizoctonia Root Rot infection. Pathogen cultures were gratefully provided by the Laboratory of Plant Pathology at the Regional Centre of Research on Horticulture and Organic Agriculture of Chott-Mariem, Tunisia. Pathogen was grown onto Potato Dextrose Agar (PDA) medium amended with streptomycin sulfate (300 mg/L w/v) and incubated at 25°C for 5 days before use.

Rhizobacterial collection tested and growth conditions
The 25 bacterial isolates used in the current study were originally recovered from the rhizospheric soils of apparently healthy and vigorous tomato plants grown in various infested tomato fields. They were identified using morphological, biochemical and molecular tools. They were also characterized for antibiotic producing ability (Bacillomycin D and fengycin A) and PGPR traits such as IAA detection, siderophore production, phosphate solubilization. Their main traits were previously detailed [23].
Rhizobacterial stock cultures were stored at -20°C in Luria Bertani (LB) broth amended with 15% glycerol. Bacterial cultures used for the different tests were previously grown for 48 h onto Nutrient Agar (NA) and incubated at 28°C.
Suspensions of bacterial cells used for plant challenge were prepared as previously described and adjusted to approximately 10 8 cells/mL using an haemocytometer [24].

Screening of the antifungal potential of tomato-associated rhizobacteria against Rhizoctonia solani
The antifungal activity of the 25 rhizobacterial isolates against R. solani was screened in vitro using dual culture and distance culture bioassays for elucidating the suppressive effects of their diffusible and volatile compounds, respectively. Dual culture assay R. solani 5 day-old cultures were used for this bioassay. Agar plugs (5 mm in diameter) were cut using a sterile cork borer and placed at one side of a Petri plate (9 cm in diameter) containing PDA medium. At the opposite side, 10 μL of a bacterial cell suspension (10 8 cells/ mL) were dropped into a well (5 mm in diameter) performed using sterile cork borer in Petri plates containing PDA. Control plates were challenged with pathogen plugs and bacterial suspension was replaced by a same volume of SDW. Plates were maintained at 25°C for 5 days. Three plates were used per each individual treatment. The diameter of pathogen colony and the inhibition zone were measured and the percentage of inhibition of pathogen growth was calculated as previously described [23].

Distance culture assay
Antifungal activity of volatile metabolites of the tomato-associated rhizobacteria against R. solani was assessed using the distance culture assay also known as the sealed plate method. For this test, 10 µL of 48 h-old bacterial culture adjusted to 10 8 cells/mL were dropped into wells (5 mm in diameter) performed using sterile cork borer in Petri plates containing NA medium. A second PDA Petri plate was challenged with pathogen plug only (5 mm in diameter). Both half plates were wrapped together with parafilm to seal in the bacterial volatile compounds. For control plates, pathogen-challenged half plate was inverted over a half one containing NA only. The paired plates were incubated at 25°C for 5 days. Three plates were used per each individual treatment. After the incubation period, the diameter of pathogen colony was measured and the percentage of growth inhibition was calculated as previously described [24].

Assessment of Rhizoctonia Root Rot-suppressive and plant growth-promoting abilities
The ability of the 25 rhizobacterial isolates to limit the in vivo expression of R. solani and to enhance plant growth was screened based on pot experiments maintained under greenhouse conditions. Rhizobacteria and pathogen cultures were prepared as described above. Tomato cv. Rio Grande seedlings (at the two-true-leaf growth stage), grown in alveolus plates, were watered at the collar level with 30 mL of a suspension of bacterial cells (adjusted to 10 8 cells/mL). Seven days post bacterial treatment, 30 mL of R. solani inoculum (mycelial fragments) were poured at the same level to each seedling. Control seedlings were watered with SDW only. One day post pathogen challenge, seedlings were transplanted into pots (16 cm in diameter) filled with peat previously infected with 40 ml of fungal inoculum. A reminder bacterial treatment was performed 24 h post-transplanting [24]. Overall, the bioassay included a positive control (pathogenfree and rhizobacteria-free seedlings), a negative control (R. solaniinoculated and untreated seedlings) and 25 treatments consisting of tomato seedlings pathogen-challenged and individually treated with the tested 25 rhizobacterial isolates.
Two months after transplanting, tomato plants were uprooted and washed for eliminating the adhering peat. Three growth parameters (plant height and aerial parts and roots fresh weights) were recorded. Disease severity on collars and roots was estimated based on a 0-5 scale depending on root browning extent on the whole root system where: 0 = no symptom, 1 = 0-25% of root browning, 2 = 26-50% of root browning, 3 = 51-75% of root browning, 4 = 76-100% of root browning, and 5= plant death [24]. Disease incidence was also calculated for each individual treatment by dividing the number of symptomatic plants over the total number of plants.

Statistical analysis
The results were subjected to one-way analysis of variance and means separations were carried out using the Duncan's Multiple Range test at P ≤ 0.05. ANOVA was performed using SPSS version 16.0. Experiments were conducted according to a completely randomized design both for the in vitro (26 individual treatments, 3 replications) and the in vivo trials (27 individual treatments, 5 replications). Correlation analyses between Rhizoctonia Root Rot severity and plant growth parameters were carried out using Pearson's correlation analysis at P ≤ 0.05.

Results
Antifungal activity of diffusible metabolites from tomatoassociated rhizobacteria toward R. solani ANOVA  noted after 5 days of incubation at 25°C, depended significantly (at P ≤ 0.05) upon bacterial treatments tested. In fact, data given in Table  1 showed that all the 25 rhizobacterial isolates had significantly (at P ≤ 0.05) lowered pathogen mycelial growth over the control. The percentage of growth reduction, versus the untreated control, varied between 34.44 and 59.26% depending on isolates and exceeded 40% using 18 isolates out of the 25 tested. Figure 1) and B. subtilis B6 (KT921427) isolates were found to be the most active in inhibiting R. solani radial growth by 48.89-59.26%.
This assay also showed that some tested rhizobacterial isolates led to the formation of inhibition zones when dual cultured with R. solani.
Dimension of this zone ranged between 3 and 10.3 mm depending on isolates and was more than 5 mm when R. solani was dual-cultured with 13 out of the 25 isolates tested ( Table 1)

Antifungal activity of volatile metabolites from tomatoassociated rhizobacteria toward R. solani
Data analysis revealed that the diameter of pathogen colony, recorded after 5 days of incubation at 25°C, varied significantly (P ≤ 0.05) upon bacterial treatments tested. In fact, as shown in Table 1, R. solani growth was lowered by 18.52 to 45.37% over control due to the inhibitory effects of volatile metabolites released by the rhizobacterial

Suppression of Rhizoctonia Root Rot disease using tomatoassociated rhizobacteria
Disease incidence, calculated 60 days post-transplanting and estimated based on the presence of typical root browning symptoms whatever their levels of extent, varied from 0 to 100% depending on bacterial isolates used for seedling treatments ( Table 2). Higher disease incidence (100%) records seemed to be more associated to root browning indexes ranging between 1 and 2.4.
Rhizoctonia Root Rot severity depended significantly (P ≤ 0.05) upon tested bacterial treatments. As shown in Table 2, this parameter ranged from 0 to 2.4 (using 0-5 scale) for all rhizobacteria-based treatments and these disease severity scores were significantly (P ≤ 0.05) lower than that recorded on pathogen-challenged and untreated control plants (disease index 4.6).
It should be highlighted that disease index values did not exceed 1 on tomato plants treated with 19 rhizobacterial isolates and were less than 0.5 with 13 out of the 25 tested indicating approximately total suppression of disease development. Moreover, compared to R. solaniinoculated and untreated control, 21 out of the 25 tested isolates led to more than 70% decrease in disease severity and this percent ranged between 47 and 53% for the four remaining ones (

Improvement of growth of R. solani-inoculated tomato plants using tomato-associated rhizobacteria
The 25 bacterial isolates, naturally associated to tomato, were screened for their plant growth-promoting effects based on various growth parameters and their data were compared to those of the untreated control plants (R. solani-inoculated or disease-free controls). ANOVA analysis indicated that the plant height and the aerial parts and roots fresh weights depended significantly (P ≤ 0.05) upon tested bacterial treatments. Their comparative abilities to promote growth of above-and below-ground plant parts were detailed below.

Plant height increase
All the rhizobacterial isolates had significantly (P ≤ 0.05) augmented the plant height of R. solani-inoculated and treated plants over the inoculated and untreated ones (

Aerial parts fresh weight increase
Data given in Table 2 showed that plant treatment with the majority of tested isolates, excluding two isolates of B. amyloliquefaciens namely B18 (KT923052) and B22 (KT923053) and one isolate of B. subtilis B20 (KT921431), led to significant (P ≤ 0.05) increase in the aerial parts fresh weight relative to R. solani-inoculated and untreated control. Enhancements recorded in the aerial parts fresh weight ranged between 34.72 and 67.25% depending on tested bacterial treatments and exceeded 50 and 60% using 11 and 3 isolates out of the 25 tested, respectively.
Based on their capacity to increase the aerial part growth of tomato plants already infected with R. solani, E. cloacae B16 (KT921429), B. subtilis B10 (KT921327), and B. thuringiensis B2 (KU158884) were found to be the most promising PGPR candidates generating 67.25, 63.84, and 62.99% increase in this parameter over the inoculated control, respectively. Moreover, increments in the fresh weight of the aerial parts allowed by these isolates were significantly higher than that recorded on pathogen-free and untreated control plants.

Roots fresh weight increase
Data presented in Table 2 revealed that the majority of the tested isolates, excepting B. subtilis B25 (KU161091), had significantly (P ≤ 0.05) improved root development. In fact, root fresh weight increment, compared to pathogen-inoculated and untreated control plants, ranged from 53.33 to 86.27% depending on tested bacterial treatments and reached up to 70% using 18 isolates.
The greatest root growth-promoting effects, expressed by more than 84% increase in the root fresh weight, were obtained using B. thuringiensis B2 (KU158884), E. cloacae B16 (KT921429), B. megaterium B1 (KU168423), and B. subtilis B10 (KT921327) ( Figure  2). Furthermore, plants infected with R. solani and treated with these four isolates exhibited 68-71% significantly higher root growth relative to pathogen-free and untreated control ones. This indicates that these tomato-associated bacterial isolates have additionally bio-fertilizing benefits.

Correlation between Rhizoctonia Root Rot severity and plant growth parameters
Pearson's correlation analysis demonstrated that plant height was significantly and negatively related to root browning index (r = -0.773; P = 4.9843 E-28) indicating that increased disease severity led to plant stunting if compared to pathogen-free control ones. Similar correlations were noted between the fresh weights of the aerial parts (r = -0.608; P = 5.5636 E-15) and roots (r = -0.675; P = 2.8624 E-19) and disease severity. This analysis indicated that the lowered Rhizoctonia Root Rot severity on tomato plants, allowed using rhizobacteria-based treatments, was linked to the registered growth promotion.

Discussion
Recently, rhizobacteria have gained more attention because of their successful ability to colonize roots and to the broad spectrum of their metabolites involved in disease biocontrol and/or growth enhancement such as antibiotics, lytic enzymes, siderophore, and phytohormones [17,[25][26][27]. In the present study, 25 rhizobacterial isolates, naturally associated to tomato plants and recovered from rhizospheric soils removed from tomato-producing sites Tunisia, were assessed against R. solani. This same rhizobacteria collection was previously tested and was shown able to inhibit S. sclerotiorum mycelial growth and myceliogenic germination of its sclerotia, to suppress Sclerotinia Stem Rot disease and to improve tomato growth [24].
Based on in vitro findings, diffusible and volatile metabolites from tested rhizobacterial isolates displayed antifungal activity against R. solani where B. thuringiensis B2, B. subtilis B6, B. subtilis B10, and E. cloacae B16 (KT921429) were found to be the most bioactive agents based on both dual and distance culture assays. According to Adesina et al. [28], the root-associated bacteria have an antagonistic potential towards Rhizoctonia spp. In fact, used as whole cell suspensions or cellfree culture filtrates, they displayed suppressive effects against root rot disease of tomato caused by R. solani. Bacillus spp. have been reported as effective biocontrol agents against R. solani in several other studies [10,[29][30][31]. These findings are also in agreement with previous studies reporting the capacity of B. subtilis and B. amyloliquefaciens to control various fungal plant pathogens including R. solani using diffusible and/ or volatile metabolites [32][33][34][35][36].
Antibiotic production by bacterial antagonists is an essential component in the biological control of fungal phytopathogens [37] and cyclic lipopeptide antibiotics, in particular, are able to suppress various phytopathogenic fungi including R. solani [38,39]. This antibiotic production ability within Bacillus spp. and their extensive uses as biocontrol agents have been reported in many reviews [40,41]. In fact, B. subtilis and B. thuringiensis synthesized at least five lipopeptide antibiotics including bacillomycin [10,42]. However, Mandal et al. [43] demonstrated that E. cloacae can produce kurstakin, iturin, surfactin and fengycin probably involved in its antifungal activity displayed toward R. solani. In fact, E. cloacae isolates tested in the current investigation were previously demonstrated able to produce fengycin A and/or bacillomycin D [23]. Chryseobacterium species were frequently encountered in soils and were effective against various soilborne pathogens [44,45]. However, their growth inhibitory effects vary depending on species and isolates. C. jejuense B11 used in the present work was previously found to be a bacillomycin D-producing agent [23]. However, in other studies [45,46], C. wanjuense KJ9C8 and Chryseobacterium species were found able to produce other antifungal compounds like hydrogen cyanide (or HCN) but not antibiotics.
The in vivo screening of the ability of the 25 rhizobacterial isolates to suppress Rhizoctonia Root Rot severity revealed that this bacterial collection contained interesting biocontrol agents. The most effective isolates allowing total disease suppression (i.e. having 0 as disease index) were B. thuringiensis B2, B. subtilis B10, E. cloacae B16, B. subtilis B8, and B. amyloliquefaciens B21. It should be highlighted that the isolates B2, B10 and B16 were previously found to be the most effective in suppressing Sclerotinia Stem Rot in tomato and in promoting plant growth [24]. Moreover, in the present investigation, B. subtilis B8 and B. amyloliquefaciens B21 isolates had totally suppressed Rhizoctonia Root Rot and, interestingly, had also enhanced by more than 70% the root fresh weight and plant height. However, in their in vitro screening, they had significantly decreased R. solani radial growth by 44-46 and 19-29%, relative to the untreated control, using dual and distance culture bioassays compared to 58-59 and 45% achieved using the three most effective isolates. These both isolates were previously shown to be fengycin A-and bacillomycin D-producing agents and also able to produce IAA and to solubilize phosphate [23]. Thus, these properties may explain their in vivo efficacy but further investigations are still required to elucidate their probable unknown features. Overall, all tested isolates were efficient in controlling Rhizoctonia Root Rot but with a varying degree. In fact, in our previous study, among the rhizobacterial isolates screened for detection of fengycin A and bacillomycin D biosynthesis genes, 20 were able to produce at least one of these antibiotics and 15 isolates were positive for both antibiotic biosynthesis genes [23]. This finding may explain the recorded disease suppression achieved using these isolates.
During the ten last years, several workers underlined the ability of bacterial isolates belonging mainly to Pseudomonas [6,47,48] and Bacillus genera [9][10][11][12]49,50] to control R. solani but to our knowledge, few reports are available on use of C. jejuense, E. cloacae and K. pneumoniae against this pathogen. Disease suppression achieved using Chryseobacterium B12 is in accordance with Krause et al. [44] findings where C gleum was shown to be a putative biocontrol agent able to suppress Rhizoctonia damping-off on several plants.
The current study clearly demonstrated that all treatments performed using the rhizobacterial isolates had significantly increased plant growth parameters i.e. plant height by 62-73%, aerial part fresh weight by 34-67%, and root fresh weight by 53-86%. Thus, these findings showed the additional growth-promoting effects exhibited by the rhizobacterial collection when challenged to tomato plants already infected with the pathogen. According to Ahmad et al. [51], an efficient biocontrol agent is generally equipped with several tools allowing both plant growth promotion and target pathogen inhibition due to their efficient root colonization, phytohormone production ability and nutrient competition. Regarding plant growth-promoting (PGP) properties characterizing this bacterial collection, our previous study demonstrated that 20 isolates among the 25 tested were able to produce siderophore, 18 had solubilized phosphate, 19 were capable to synthesize IAA, and that interestingly 13 isolates showed positive response to at least two PGP traits [23].
Other effects lead to growth promotion during PGPR-plant interactions such as increased root permeability, enhanced ability to survive in strict competitive niche and inhibition of harmful microorganisms [52]. In the present investigation, among the tested rhizobacteria collection, the most promising strains combining both disease suppressive and growth-promoting abilities were B. subtilis B10, B. thuringiensis B2 and E. cloacae B16. Also, interestingly, C. jujuense B12 and K. pneumoniae B11 isolates had also reduced Rhizoctonia Root Rot severity and augmented root growth and plant height by more 70% compared to pathogen-inoculated control. These tomato-associated agents are able to produce antibiotic lipopeptides, IAA, and siderophore and to solubilize phosphate [23]. Bacillus and Pseudomonas species are extensively reported as phosphate solubilizing and as IAA-and siderophore-producing agents compared to the other species of rhizobacteria [53]. Additionally, Almaghrabi et al. [54] noted significant increment in shoot dry weight, plant height, and yield in tomato plants treated with B. amyloliquefaciens, B. subtilis and B. cereus and other rhizobacterial species such as Serratia marcescens, Pseudomonas putida, P. fluorescens.
In addition, in the current study, Enterobacter, Chryseobacterium and Klebsiella isolates cumulated the three PGP traits. Also, previous studies demonstrated that Chryseobacterium species represent an important bacterial group associated with plants and displaying interesting plant-growth promoting activities [55][56][57]. C. balustinum pepper-associated rhizobacteria also showed PGP properties and had improved aerial surface, aerial length and the dry weight of the aboveand underground plant parts [58]. Contrarily, reports on Klebsiella species as PGP agents are relatively rare. In fact, Ahemad and Khan [59] found that Klebsiella sp. strain PS19 solubilized the inorganic phosphate considerably, produced IAA and siderophores. K. oxytoca Rs-5, isolated from Chinese saline cotton fields are able to attenuate salt stress, to enhance plant growth and to release IAA [60]. Sachdev et al. [61] found that IAA-producing Klebsiella strains had significantly improved root length and shoot height of infected wheat seedlings relative to control.

Conclusion
The present investigation clearly demonstrated that tomato rhizospheric soils removed from tomato-growing sites of Tunisia harbor interesting biocontrol candidates belonging to Bacillus, Enterobacter, Chryseobacterium, and Klebsiella genera. These tomatoassociated rhizobacteria displayed Rhizoctonia Root Rot suppression ability and plant growth promoting capacity. Thereby, they could be developed as biofertilizing and biocontrol agents once their effectiveness demonstrated under field conditions and in different tomato-producing sites. Further studies will be focused on the assessment of their diseasesuppressive effects against Rhizoctonia Root Rot disease and their plant growth-promoting potential when used as consortia and the eventual shifts in rhizosphere microbial community occurring after their release.