Inﬂuence of Substrates in the Development of Bean and in Pathogenicity of Rhizoctonia solani JG Kühn

: The number of plants produced in horticultural substrates has increased over the last decade. These substrates are to have some physicochemical and phytosanitary characteristics that allow for optimal growth. Rhizoctonia solani JG Kühn is one of the root and hypocotyl diseases that frequently develops in seedbeds causing severe economic losses worldwide. The aim of this work was to evaluate the substrates and additives used in bean plantlets and the R. solani disease severity. The use of vermiculite or peat did not a ﬀ ect bean germination percentage, but peat promoted the development of the aerial part and vermiculite improved the growth of the root system. R. solani reduced bean germination percentage and bean growth in both peat and vermiculite. The presence of additives (bentonite and / or cornmeal) in the substrates (vermiculite or peat) reduced bean germination and development, favouring the severity of R. solani. However, the addition of cornmeal improves bean dry weight of the aerial part. So, whenever it is necessary to add additives to the substrates to improve the installation and development of a biocontrol agent, such as Trichoderma harzianum , the mix of peat and cornmeal would improve the development of both bean and biocontrol agent.


Introduction
Every day, the number of plants produced in nurseries increases and they are transplanted both for small-scale production, or self-consumption, and for large-scale production. If the desire is to produce healthy plants free of phytopathogens starting from healthy and registered seeds, a substrate that reduces their propagation and development is needed. Integrated crop health management approaches, using both modern cultivars with resistance/tolerance to these organisms and technologies that stimulate root health and growth, accompanied by chemical, biological and cultural phytopathogen management strategies are needed for sustainable production.
Some growing media may have a lower risk because of the nature of their production. They may become contaminated or infested, depending on the type and composition of the substrate during the production process [1]. Knowledge about the relationships between plants, plant pathogens and antagonists, and about the physico-chemical properties of the soil, will help control diseases, reducing the use of chemical products.
Most potting substrates are a combination of organic (peat, bark, straw, etc.) and inorganic materials (vermiculite, perlite, clay, etc.). The addition of organic compounds, such as cornmeal, improves

Rhizoctonia Solani Isolate
R. solani R43 was used in this study, taken from the collection of "Pathogens and Antagonists" of the Laboratory Diagnosis of Pests and Diseases (University of León, León, Spain). It is an isolate collected from the production area of the Protected Geographical Indication (PGI), called "Alubia La Bañeza-León" (EC Reg. n.256/2010 published on 26 March 2010, OJEU L880/17), without any genetic manipulation.

Substrates and Bioassays
Bioassays were performed in climatic chambers in order to test R. solani activity. Peat (Pindstrup Peat 32, Pindstrup Mosebrug S.A.E., Sotopalacios, Burgos, Spain) and vermiculite (Verlite ® , Vermiculita y derivados, Gijón, Spain) and their combinations with the additives cornmeal (C) (Fioretto ® , Biemme, Saluzzo, Italy) and bentonite (B) (Bengel ® , Agrovin, Ciudad Real, Spain) were tested in this bioassay and their characteristics are described in detail by Mayo-Prieto et al. [36]. Theses substrates and additives were disinfected by autoclaving at 120 • C for 1 h on 2 consecutive days. Eight different treatments (combinations of substrates and additives) were evaluated using ten polypropylene pots (1 L capacity) used per treatment (Table 1), meaning 10 repetitions per treatment. Each pot was watered with 250 mL of water prior to inoculation. Each pot was inoculated with a 50 mL suspension of R. solani R43 by a surface irrigation (5 Petri dishes/water litter) [37]. For inoculation of the control, only PDA medium was used without any pathogen in all combinations of substrates and they were placed in the same climatic chamber as the substrates with the pathogen. Pots were kept in a growth chamber for 8 days at 25 • C/16 • C (day/night), 60% RH in the dark.
Bean seeds of "Canela" variety were surface sterilised (sodium hypochlorite 1% for 3 min and in distilled water for 6 min). After 8 days of the inoculation of R. solani R43, three seeds were sown in each pot (10 pots/treatment). The culture was maintained for 45 days with a photoperiod of 16 h light, 25 • C/16 • C (day/night), 60% RH and brightness of 3500 lux. Irrigations were performed every 4 days with about 250 mL tap water/pot [37]. A nutrient solution was added on the 2nd-4th week [38].
Bean germination percentage was evaluated 12, 17 and 24 days after sowing. Plants were then removed 45 days after sowing and the following parameters were evaluated: disease severity percentage in the aerial part (1: without chlorosis; 2: 1%-10% of the leaves with chlorosis; 3: 11%-25% of the leaves with chlorosis; 4: 26%-50% of the leaves with chlorosis; 5: more of 50% of the leaves with chlorosis; 6: plant of bean without leaves; 7: dead plant of bean; 8: dead plant after the germination), wet weight and dry weight (72 h in an oven, 82 • C) of the aerial part and root system.

Statistical Analysis
Bean germination percentage and disease severity percentage data were transformed by the formula (x + 0.5) 1/2 . Square-root transformation is appropriate for data consisting of small whole numbers, for example, data obtained in counting rare events, such as the number of infested plants in a plot and for percentage data [39,40]. Combined analysis of variance was performed using the general linear models (GLM). Orthogonal contrasts were used for additive to isolate treatment effects [41]. For the control without additive, the contrast was without additive versus cornmeal, bentonite and cornmeal-bentonite. For the additive the contrast was cornmeal-bentonite versus cornmeal and bentonite. The last contrast was cornmeal versus bentonite. All analyses were performed using SAS version 9.4 software (SAS Institute Inc., 2015, Cary, NC, USA).

Germination
Analysis of variance shows highly significant differences in pathogen and additives for germination percentage at 12, 17 and 24 days after sowing. However, there are not significant differences between substrates for germination percentage ( Table 2).
The Table 3 shows the orthogonal contrast among additives. Contrasts show highly significant differences for germination between the control and all additive combinations (control versus B + C + BC). However, the rest of the orthogonal contrasts for germination among additives [BC versus (B + C) and B versus C] are not significantly different ( Table 3).
The Figure 1 shows the germination percentage and removed seeds percentage after 24 days of sowing for substrate, pathogen and additives. The two tested substrates (vermiculite and peat) do not affect the germination percentage for either removed seeds percentage. However, the pathogen, R. solani has reduced the germination compared with control, increasing the removed seeds. The addition of cornmeal and/or bentonite to the substrates has reduced the germination percentage.
Agronomy 2020, 10, x FOR PEER REVIEW 4 of 13 in a plot and for percentage data [39,40]. Combined analysis of variance was performed using the general linear models (GLM). Orthogonal contrasts were used for additive to isolate treatment effects [41]. For the control without additive, the contrast was without additive versus cornmeal, bentonite and cornmeal-bentonite. For the additive the contrast was cornmeal-bentonite versus cornmeal and bentonite. The last contrast was cornmeal versus bentonite. All analyses were performed using SAS version 9.4 software (SAS Institute Inc., 2015, Cary, NC, USA).

Germination
Analysis of variance shows highly significant differences in pathogen and additives for germination percentage at 12, 17 and 24 days after sowing. However, there are not significant differences between substrates for germination percentage ( Table 2).
The Table 3 shows the orthogonal contrast among additives. Contrasts show highly significant differences for germination between the control and all additive combinations (control versus B + C + BC). However, the rest of the orthogonal contrasts for germination among additives [BC versus (B + C) and B versus C]are not significantly different ( Table 3).
The Figure 1 shows the germination percentage and removed seeds percentage after 24 days of sowing for substrate, pathogen and additives. The two tested substrates (vermiculite and peat) do not affect the germination percentage for either removed seeds percentage. However, the pathogen, R. solani has reduced the germination compared with control, increasing the removed seeds. The addition of cornmeal and/or bentonite to the substrates has reduced the germination percentage. Figure 1. Germination percentage and removed seeds percentage after 24 days of sowing for substrates, pathogen and additives. Blue colour represents the substrates; orange colour represents the presence or absence of R. solani; green colour represents the types of additives; solid colour represents germination and with striped colour represents removed seeds. Bars of the same colour followed by different letters (small letter for germinated seeds and capital letters for removed seeds) are significantly different (p ≤ 0.01).

Figure 1.
Germination percentage and removed seeds percentage after 24 days of sowing for substrates, pathogen and additives. Blue colour represents the substrates; orange colour represents the presence or absence of R. solani; green colour represents the types of additives; solid colour represents germination and with striped colour represents removed seeds. Bars of the same colour followed by different letters (small letter for germinated seeds and capital letters for removed seeds) are significantly different (p ≤ 0.01).

Disease Severity
Analysis of variance shows highly significant differences in pathogen and additives for disease severity. However, there are not significant differences between substrates for disease severity ( Table 2).
The Table 3 shows the orthogonal contrast among additives. Contrasts show highly significant differences for disease severity between the control and all combinations of additives (control versus B+C+BC). However, the rest of orthogonal contrast for disease severity among additives (BC versus B+C and B versus C) are not significantly different.
Symptomatology of the aerial part has not presented significant differences between peat and vermiculite. Nevertheless, the presence of R. solani in the substrate has caused an increase in the disease severity in the aerial part being significantly different to the control plants. The addition of cornmeal and/or bentonite to the substrates has increased the bean disease severity relative to the control ( Figure 2). Agronomy 2020, 10, x FOR PEER REVIEW 1 of 13

Disease Severity
Analysis of variance shows highly significant differences in pathogen and additives for disease severity. However, there are not significant differences between substrates for disease severity ( Table 2).
The Table 3 shows the orthogonal contrast among additives. Contrasts show highly significant differences for disease severity between the control and all combinations of additives (control versus B+C+BC). However, the rest of orthogonal contrast for disease severity among additives (BC versus B+C and B versus C) are not significantly different.
Symptomatology of the aerial part has not presented significant differences between peat and vermiculite. Nevertheless, the presence of R. solani in the substrate has caused an increase in the disease severity in the aerial part being significantly different to the control plants. The addition of cornmeal and/or bentonite to the substrates has increased the bean disease severity relative to the control (Figure 2).

Figure 2.
Disease severity in aerial part at 45 days after sowing for substrates, pathogen and additives. Disease severity scale: 1, without chlorosis; 2, 1%-10% of the leaves with chlorosis; 3, 11%-25% of the leaves with chlorosis; 4, 26%-50% of the leaves with chlorosis; 5, more than 50% of the leaves with chlorosis; 6, plant of bean without leaves; 7, dead plant of bean; 8, dead plant after the germination. Blue colour represents the substrates; orange colour represents the presence or absence of R. solani; green colour represents the types of additives. Bars of the same colour followed by different letters are significantly different (p ≤ 0.01).

Dry Weight
When the dry weight of the aerial part has been analysed, bean plants in peat have been significantly larger than those in vermiculite. The presence of R. solani has caused a decrease in the weight of the bean aerial part. In regard to the additives, the greatest bean dry weight of the aerial part was found when cornmeal was in the substrate. Bean plants grown in the substrate with bentonite and cornmeal have shown the smallest bean dry weight of the aerial part ( Figure 3). Disease severity scale: 1, without chlorosis; 2, 1%-10% of the leaves with chlorosis; 3, 11%-25% of the leaves with chlorosis; 4, 26%-50% of the leaves with chlorosis; 5, more than 50% of the leaves with chlorosis; 6, plant of bean without leaves; 7, dead plant of bean; 8, dead plant after the germination. Blue colour represents the substrates; orange colour represents the presence or absence of R. solani; green colour represents the types of additives. Bars of the same colour followed by different letters are significantly different (p ≤ 0.01).

Dry Weight
When the dry weight of the aerial part has been analysed, bean plants in peat have been significantly larger than those in vermiculite. The presence of R. solani has caused a decrease in the weight of the bean aerial part. In regard to the additives, the greatest bean dry weight of the aerial part was found when cornmeal was in the substrate. Bean plants grown in the substrate with bentonite and cornmeal have shown the smallest bean dry weight of the aerial part ( Figure 3).
Analysis of variance shows highly significant differences in pathogens, substrates and additives for the dry weight of the bean root system ( Table 2).
Orthogonal contrasts of the dry weight of the bean root system have shown highly significant differences between control and all additive combinations and between bentonite and cornmeal separately as well as bentonite and cornmeal combined (BC versus B + C). However, there were not significant differences found between bentonite and cornmeal (B versus C) for bean root growth (Table 3).  Analysis of variance shows highly significant differences in pathogens, substrates and additives for the dry weight of the bean root system ( Table 2).
Orthogonal contrasts of the dry weight of the bean root system have shown highly significant differences between control and all additive combinations and between bentonite and cornmeal separately as well as bentonite and cornmeal combined (BC versus B + C). However, there were not significant differences found between bentonite and cornmeal (B versus C) for bean root growth (Table 3).
Bean plants had a significantly bigger root system in vermiculite than in peat. The presence of R. solani caused a decrease in the bean root weight. In regard to the additives, bean plants in substrates with additives had a significantly lower root weight than the control. Bean plants grown in the substrates with bentonite and cornmeal showed the smallest bean root weight, as well as significant differences with the control substrate and with bentonite or cornmeal separately ( Figure  4). Bean plants had a significantly bigger root system in vermiculite than in peat. The presence of R. solani caused a decrease in the bean root weight. In regard to the additives, bean plants in substrates with additives had a significantly lower root weight than the control. Bean plants grown in the substrates with bentonite and cornmeal showed the smallest bean root weight, as well as significant differences with the control substrate and with bentonite or cornmeal separately (Figure 4).

Discussion
In horticulture, a lot of plants are produced in mineral and organic substrates. This market is directed towards the pursuit of a substrate that produces healthy plants without the need for any synthetic plant protection chemicals. For that reason, the search for combinations of substrates and

Discussion
In horticulture, a lot of plants are produced in mineral and organic substrates. This market is directed towards the pursuit of a substrate that produces healthy plants without the need for any synthetic plant protection chemicals. For that reason, the search for combinations of substrates and additives is important to achieve this goal. A problem is that different substrates can promote not only the development of the plant, but also the growth of beneficial or harmful organisms. Soil-borne plant pathogens infecting plant roots are one of the biggest problems the world of agriculture faces because of the reduction in yield and quality of the crops. An example of one of these phytopathogens is R. solani which causes an economically harmful disease on a range of agricultural crops [42]. This study has evaluated the development of bean plants in substrates with different additives and how they affect this pathogen that can decrease bean production between 5% and 60% [43,44]. Results show that R. solani caused a reduction in germination compared to the control. This is because the bean seeds and young bean plants are more susceptible to it than adult plants [35]. Adult plants suffered a reduction in the production of dry matter with the aerial part and the root system having developed less than those of control plants. In others works, bean plants inoculated with R. solani have also had a significative reduction in their growth [37,45].
In our study, the interaction between pathogen and substrate has not been significative in disease severity, indicating that neither peat nor vermiculite have modified the development of the bean plant or the symptoms produced by R. solani. In the work of Cohen et al. [46] they observed that the incidence of the disease caused by Fusarium oxysporum f. sp. melonis in melon plants was higher in plants grown in peat or peat mixed with sand than in those developed in a combination of peat and pearlite. In another study, Campelo et al. [47] studied the effect of Sclerotinia sclerotiorum and R. solani in bean plants sown in vermiculite where the first pathogen reduced the development but Rhizoctonia presented low pathogenicity both in the aerial part and in the root system.
R. solani as pathogenic fungi, are particularly sensitive to certain management practices, such as fertilization, and the C contents [48]. Harries et al. [49] studied some tobacco soil and its influence in the development of R. solani. They determined that high levels of clay, pH, organic matter content and physical stability were associated with low R. solani inoculum potential in tobacco soils. It has been observed that suppression of R. solani growth is negatively correlated to pH. A range in soil pH of 4.5-6.5 appears optimal for mycelial growth of R. solani which produces damping-off [49][50][51]. In our work, the pH of the substrates varied between 5 and 7, so this range could have promoted the development of the R. solani, increasing its pathogenicity. The extent and rate of fungal growth may also depend on the soil porosity limiting the spread of the hyphae of a phytopathogen when the space is restricted [52,53], but developing rapidly in porous sandy soil [54].
Moreover, adding cornmeal to the substrate promoted R. solani development because of it acted as a source of carbohydrates, increasing the C concentration, like it does in fields with the remains of vegetable matter. For example, when cereal or leguminous flours were incorporated to the substrate mixture, they increased the production of Penicillium conidia [55]. However, in another study, high organic matter and clay contents have also been related to soil suppression of Fusarium or Pythium [56,57].
The presence of an inoculum of a phytopathogen, a susceptible host and certain environmental conditions (soil, temperature, humidity, etc.) are all necessary for an infection [58]. One way to control of this process is practicing crop rotation, where a resistant/tolerant plant is sown, reducing the amount of inoculum. Another way to achieve this is modifying the soil or substrate in a way that favours the development of biocontrol agents. For this, biocontrol agents can be added to the substrate mixture. If a substrate is enriched with additives that provide a source of energy for the development of beneficial organisms, it will reduce the use of synthetic chemical products, therefore producing safer foods for the consumer and for the environment. In substrates with cornmeal, Trichoderma has shown higher rates of development than without this additive. Moreover, its growth has been higher whenever it has been inoculated into a mixture with peat and not vermiculite [36]. There are some studies where the use of additives has promoted the development of both the plant and Trichoderma.
Martinez-Media et al. [15] checked that the formulation with bentoniteand vermiculite and Trichoderma was effective in enhancing plant growth and reducing the incidence of Fusarium wilt in melon plants.
In another work, Bernal-Vicente et al. [16] tested some formulations with Trichoderma as liquids with spore suspension, guar gum or carboxymethylcellulose, and solids with bentonite, vermiculite and wheat bran; they verified that substrates with bentonite and vermiculite were the most effective treatments against Fusarium wilt on melon plants and resulted in the greatest plant weight.
In this study, the selection of these concentrations for bentonite (5%) and cornmeal (2%) is a result of a previous work, where the variations of these additives changed the development of bean plants. When these concentrations were increased, bean plants had a stunted development of both the aerial part and the root, promoting the development of saprophytic fungus and bacteria in the substrates and increasing their compaction (unpublished data).
The aerial part of the plants developed more in peat than in vermiculite, while the radicular system was greater in vermiculite than in peat. In this study, the incorporation of additives provided greater consistency and hardness to the substrate, reducing bean germination. Undoubtedly, the addition of additives caused significant differences in the development of the plant. The aerial part developed more with cornmeal than with bentonite or with both. This did not happen in the case of the root system, where all additives caused a significant reduction in its dry weight.
MacNish [59] found that compaction of soil had no effect on dry root weights of wheat plants grown for 21 days, but higher levels of compaction following the mixing of soils decreased the dry root weights of the plants. The reasons for this behaviour have not been explained through experimental data but it has been suggested that factors such as release of nutrients, changes in gas exchange or changes in microbiological activities are possible causes of this behaviour [54].
A non-autoclaved substrate has microbiota that counter the effects of a pathogen. Adding additives promotes the development of microorganisms, both pathogenic and not pathogenic. Biological agents restrict the development of a phytopathogen. Therefore, it is convenient to add not only additives but also biocontrol agents to the substrates in order to favour their installation and to control its pathogens. For example, bean seeds coated with Trichoderma and in a substrate with additives (bentonite and cornmeal) did not decrease their size in the presence of R. solani [37]. Nevertheless, it is important to keep in mind that additives used to promote the development of biocontrol agents may also affect the growth of some pathogens and some crops.

Conclusions
Bean germination percentage was not affected by the use of either vermiculite or peat, but peat promoted the development of the aerial part and vermiculite improved the growth of the root system. R. solani reduced bean germination percentage and bean growth in both peat and vermiculite. The presence of additives (bentonite and/or cornmeal) in the substrates (vermiculite or peat) reduced bean germination and root development, favouring the severity of R. solani. However, the addition of cornmeal improved bean dry weight of the aerial part. Therefore, whenever it is necessary to use additives in the substrates to improve the installation and development of a biocontrol agent, such as T. harzianum [36], the mix of peat and cornmeal would improve the development of both the bean and biocontrol agent. Funding: This research was funded by Junta de Castilla y León, Consejería de Educación for the project "Application of Trichoderma strains in sustainable quality bean production" (LE251P18).

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.