**1. Introduction**

The common bean (*Phaseolus vulgaris* L.) is one of the most important grain legumes in many areas of the world, providing a diet rich in protein, dietary fiber, essential micronutrients and phytochemicals for more than 500 million people [1]. The global cultivated surface of *P. vulgaris* reached 33.1 million hectares in the season 2019/2020, with an annual

**Citation:** Díaz-Díaz, M.;

Bernal-Cabrera, A.; Trapero, A.; Medina-Marrero, R.; Sifontes-Rodríguez, S.; Cupull-Santana, R.D.; García-Bernal, M.; Agustí-Brisach, C. Characterization of Actinobacterial Strains as Potential Biocontrol Agents against *Macrophomina phaseolina* and *Rhizoctonia solani*, the Main Soil-Borne Pathogens of *Phaseolus vulgaris* in Cuba. *Plants* **2022**, *11*, 645. https:// doi.org/10.3390/plants11050645

Academic Editor: Shuji Yokoi

Received: 6 January 2022 Accepted: 24 February 2022 Published: 26 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

production of 28.9 million metric tons [2]. Common bean is the most important plant species for Cuba population within the group of edible legumes with an annual production of 169,900 tons. Together with rice (*Oryza sativa* L.), they form the basis of the daily diet in this geographic area [3].

In countries with a subtropical climate, the environmental conditions are favorable for the development and proliferation of a vast and heterogeneous soil microflora, including complexes of fungal species associated with root rot diseases such as *Alternaria alternata* (Fr.) Keissl., *Colletotrichum truncatum* (Schwein.) Andrus & W.D. Moore, *Fusarium oxysporum* Schltdl., *Macrophomina phaseolina* (Tassi) Goid., *Rhizoctonia solani* J.G. Kühn, and *Sclerotium rolfsii* Sacc. [4]. In addition, [5] pointed out that the incidence and severity of root rot diseases caused by these fungi depend on the climatic factors prevailing at each sowing time, as well as the characteristics of the microclimates existing in each region of the country where common beans are grown. Among them, *M. phaseolina* and *R. solani* are considered the most prevalent fungal pathogens associated with root rot diseases of common bean worldwide [6,7].

*Macrophomina phaseolina* (Ascomycota), causal agent of ashy stem blight, also affects roots and stems of host species via pycnidiospores and microsclerotia that persist in the soil, where the pathogen establishes the primary inoculum [8]. Typical symptoms in common bean include dark, irregular lesions on cotyledons, wilting, systemic chlorosis, premature defoliation, epinasty and early maturity or death in adult plants [6]. Late infections cause the appearance of grey areas on the stems, where microsclerotia and pycnidia of the fungus are produced. The occurrence of *M. phaseolina* in the seeds has major consequences since it causes the disqualification of legumes as propagation material [9]. For instance, six tons of common bean and three tons of broad bean to be used as planting material in the province of Villa Clara were disqualified between 2006–2007 because they were affected by *M. phaseolina* [10].

On the other hand, *R. solani* (Basidiomycota) is the causal agent of rhizoctonia blight, also commonly known as damping off [11]. This soil-borne pathogen can affect more than 500 plant species, including cultivated and wild plants, and causes damping off in stands, necrotic lesions in roots, seeds and stems, as well as foliar lesions with a worldwide distribution [7,12]. This fungus affects young seedlings much more than adult plant tissues. On the stem and hypocotyl of affected plants, reddish-brown cankers of various sizes appear, usually delimited by a dark border, which later become rough, dry up and destroy plant tissues [12]. It also attacks the roots causing foot rot of the plants [5]. The management of soil-borne pathogens including both *R. solani* and *M. phaseolina* is usually difficult, not only due to their intrinsic biology, but also because the lack of effective active ingredients. Thus, the use and extension of eco-friendly control methods such as biological control is required, not only to prevent plant diseases, but also contributing markedly to soil preservation and conservation [12].

Microorganisms belonging to genera *Bacillus* (bacteria) and *Trichoderma* (fungi) are the most commonly used biological control agents (BCAs) against soil-borne plant pathogenic fungi ([13,14]. Within this context, species belonging to *Trichoderma* fungal genus have been studied since 1930, and their use has been successfully applied directly to the soil or by seed treatments [15]. On the other hand, since the last century, bacteria belonging to the genus *Bacillus* have been used as BCAs due to their ability to colonize the rhizosphere of plants and inhibit the growth and development of plant pathogens. In addition, they are used as plant growth promoters. [14]. At the same time, the ability of these bacteria to form endospores gives them resistance to climatic changes, which is an important characteristic for inoculum production [13]. In addition to these well-known BCAs, research in the last decades highlights the benefits of the actinobacteria (*Streptomyces* spp. mainly) and their potential as BCAs (e.g., *Streptomyces griseoviridis*, *S. lydicus*) against soil-borne pathogens, such as species of *Rhizoctonia*, *Phytophthora*, *Fusarium*, and *Pythium* in legumes and other crops [16]. Actinobacteria, which have been traditionally known as actinomycetes, are a heterogeneous group of aerobic, filamentous and Gram-positive bacteria. Traditionally, the

main genera isolated from soil samples are *Micromonospora*, *Nocardia*, and *Streptomyces*. The genus *Streptomyces* is represented in nature by the largest number of species among the family Actinomycetaceae [17]. This genus, as a colonizer of the rhizosphere, is able to: (i) act as BCA of plant pathogenic fungi, (ii) produce siderophores, (iii) produce plant growth promoting substances, (iv) promote nodulation, (v) produce biodegradative enzymes such as chitinases, cellulases, glucanases, peroxidases, and (vi) assist *Rhizobium* bacteria in iron assimilation, or in nitrogen fixation in legumes, which indirectly contributes to the promotion of plant growth [16].

As we mentioned above, ashy stem blight and rhizoctonia blight are considered the main diseases of *P. vulgaris* in Cuba since they are associated in a complex disease of this crop that causes root rot and plant death. The control management strategies already available against this complex disease are not enough for its optimum control in the frame of the sustainable agriculture. Thus, it is necessary to explore new alternatives towards biological control of these diseases. Therefore, actinobacteria could play an important role as BCAs against the main causal agents of the disease, *M. phaseolina* and *R. solani*. However, the effect of actinobacteria as BCAs against plant pathogenic fungi is still uncertain. Consequently, no biological based compounds on actinobacteria have been developed so far. Likewise, the 'Centro de Bioactivos Químicos' Universidad Central "Marta Abreu" de Las Villas (Cuba) has a wide collection of actinobacterial strains isolated in the central region of the country, which may be explored as a new biological alternative to be included in the integrated disease management program against soil-borne plant pathogens in the common bean crop. Therefore, the main goal of this study was to evaluate 60 actinobacterial strains for their effectiveness as BCAs against *M. phaseolina* and *R. solani* by in vitro dual-cultures assays and finally to select several actinobacterial strains with high efficiency of reduction the viability of both pathogens in vitro, and the disease progress *in planta*. We expect to select several actinobacterial strains with high efficacy on reducing the viability of *M. phaseolina* and *R. solani* in vitro, and the disease progress *in planta*.

### **2. Results**
