Selección de cepas de actinomicetos para el control de hongos fitopatógenos de suelo, con especial referencia a la podredumbre radical de la judía común (phaseolus vulgaris l.) (2024)

  • 1. Introducción o motivación de la tesis: The common bean (Phaseolus vulgaris L.) is the most important crop for the Cuban population within the group of edible legumes with the annual production being 136,570 tonnes and yields ranging from 1.1 to 1.4 t/ha. The environmental conditions, typical of subtropical countries, are favourable for the development and proliferation of a wide and heterogeneous soil microflora. Among them, the phytopathogenic soil-borne fungi Macrophomina phaseolina and Rhizoctonia solani, which are associated with bean root rot disease, cause important economic losses in this crop in Cuba. The low availability of active ingredients for the control of these plant pathogenic fungi, together with their intrinsic biology, typical of soil fungi, lead us to reconsider an integrated management strategy for the proper management of the disease. In this regard, the Centre for Bioactive Chemicals (CBQ) of the Central University "Marta Abreu" of Las Villas (UCLV) has a collection of 760 strains of actinomycetes, microorganisms that have not been explored in Cuba so far as biological control agents (BCAs) with agricultural applications. Within the actinomycetes, Streptomyces is the most predominant genus in the terrestrial environment and the most studied for its properties as a biocontrol agent for plant pathogens and plant growth promoter. They are characterized by the production of biologically active primary and secondary metabolites and precursors of about 45 % of all bioactive compounds. Their enzymes allow them to chemically break down cellulose, lignin, chitin and protein-rich residues, as well as to physically bind soil particles together to form aggregates and prevent erosion.

    2.Contenido de la investigación: Thus, the main objective of this PhD Thesis was to characterize 60 actinomycete strains in vitro and in vivo as ACBs against M. phaseolina and R. solani. Therefore, the first objective of this PhD Thesis was to carry out an exhaustive bibliographic review of the genus Streptomyces spp. as BCAs, with special emphasis on soil-borne pathogens. Here, aspects related to the phenotypic, biochemical and molecular characterisation of Streptomyces as well as their mechanisms of action and their application in crop protection were compiled.

    The Objective 2 aimed to evaluate the effectiveness of 60 actinomycete strains as BCAs against M. phaseolina and R. solani in vitro by dual culture assays. The most effective strains were characterized according to their cellulolytic, chitinolytic and proteolytic extracellular enzyme activity, as well as their morphological, biochemical and molecular characters. The results showed that 66.6% and 41.0% of the actinomycete strains significantly inhibited the mycelial growth of M. phaseolina and R. solani, respectively, compared with the control; and 30% of them showed a common effect against both pathogens. Significant differences were observed in their enzymatic and biochemical activity. Morphological and biochemical characters allowed us to identify all the strains as species belonging to the genus Streptomyces. Streptomyces strains CBQ-EA-2 and CBQ-B-8 showed the highest effectiveness in vitro. Finally, the effect of seed treatments with both strains (CBQ-EA-2 and CBQ-B-8), using single or mixed applications, against the disease development was evaluated on seedlings of P. vulgaris cv. Quivicán grown in pots filled with soils inoculated with M. phaseolina and R. solani. Treatments combining the two Streptomyces strains (CBQ-EA-2 + CBQ-B-8) were able to significantly reduce the disease incidence and severity for both pathogen infections compared to the nontreated and inoculated control. Furthermore, they showed a similar effect to that observed for the BCA Trichoderma harzianum A-34 and the chemical Celest® Top 312 FS (Syngenta®; Basel, Switzerland) treatments, which were included for comparative purposes.

    The Objective 3 was carried out to validate the results of the Objective 2 in three experimental fields with soils naturally infested by M. phaseolina and R. solani. In all experimental fields, significant differences in disease incidence and severity were observed between treatments compared to the nontreated controls. Overall, single treatments with Streptomyces sp. strain CBQ-EA-2 or Streptomyces sp. strain CBQ-B-8 showed significantly less effect in reducing disease incidence and severity than treatments combining the two strains (Streptomyces sp. CBQ-EA-2+ CBQ-B-8), regardless the experimental field. The three treatments with Streptomyces spp. were significantly more effective than those with Celest® Top 312 FS; in contrast, most of the Streptomyces treatments had a significantly lower effect than the BCA T. harzianum A-34. When we evaluated the components of legume yield and quality, in general, the treatment combining the two strains (Streptomyces sp. CBQ-EA-2+ CBQ-B-8), showed similar results with the chemical Celest® Top 312 FS and with the BCA T. harzianum A-34.

    Finally, the objective 4 was a complementary study carried out during a 6-month pre-doctoral stage at the University of Cordoba (Spain). The goal lof this work was to evaluate the effect of six of the most effective Streptomyces strains from the Objective 1 as BCAs against Verticillium Wilt of olive caused by Verticillium dahliae, one of the main olive diseases in Spain. For this purpose, trials were carried out under controlled conditions using two isolates of Verticillium dahliae (V-004 and V-323), evaluating the effect of the Streptomyces strains on mycelial growth of the pathogen by dual culture as well as their effect on the viability of conidia and microsclerotia of the pathogen in vitro. In addition, their effect on the disease development was determined in olive plants inoculated with V. dahliae isolate V-323. The six Streptomyces sp. strains and the two BCAs F. oxysporum FO12 and A. pullulans AP08 showed a significant effect on mycelial growth inhibition for both V. dahliae isolates V-004 and V-323 compared to the positive control. The reference BCA FO12 was the most effective on mycelial growth inhibition followed by AP08 for the two V. dahliae isolates; while the Streptomyces sp. strains showed a moderate effect for both V. dahliae isolates. Among the Streptomyces strains evaluated, CBQ-EA-2 was the most effective inhibiting mycelial growth of V. dahliae.

    3.Conclusión: 1. The qualitative characterization of the extracellular enzyme activities, the antagonism of the Streptomyces spp. strains, as well as the in vivo studies against M. phaseolina and R. solani under semi-controlled conditions have allowed us to characterize promising strains as BCAs, and to have a biological alternative in the framework of the integrated management of the main common bean diseases caused by soil pathogens in Cuba.

    2. A total of 62% of the Streptomyces strains revealed a high cellulolytic capacity with a halo between 80 to 90 mm in diameter, and 90% of them developed a halo with considerable extension around the colony, which denotes an important cellulolytic hydrolysis.

    3. The 66.7% of the Streptomyces strains showed chitinolytic capacity, highlighting the CBQ-EBa-5 strain, with a 35.5 mm clearance halo surrounding the colony.

    4. Based on the phenotypic and biochemical characters, all the strains were identified as Streptomyces spp. The identity of the two representative strains that showed that highest effectiveness on MGI in vitro (CBQ-EA-2, and -B-8) was con¿rmed by sequencing the 16S rRNA gene using the universal primers 27f and 1492r for eubacteria.

    5. Regarding the in vitro ef¿cacy of the 60 Streptomyces potential strains against M. phaseolina and R. solani, it varied depending on the soil-borne pathogen tested. 40 and 25 out of the 60 actinobacterial strains inhibited the mycelial growth of M. phaseolina and R. solani, respectively. Among the most effective strains, 18 of them showed a common effect against both pathogens, with the CQB-EA2, and -CD-24 being among the strains that showed greater ef cacy in inhibiting mycelial growth of the two pathogens.

    6. The treatments conducted using a mix of the two Streptomyces sp. strains (CBQ-EA-2 + -B-8) showed a signi¿cant greater effectiveness against both pathogens compared to treatments performed with the two strains alone. In addition, the effectiveness of the two combined Streptomyces strains in controlling the disease was similar to that observed for the other comparative treatments such as T. harzianum A-34 or the chemical (Celest® Top312FS).

    7. The experiments conducted in the field corroborate the results ontained under controlled controlled conditions. Therefore, treatments by coating seeds of common bean with Streptomyces sp. CBQ-EA-2 or CBQ-B-8, alone or in combination, before sowing significantly reduced the DI and DS of root rot disease associated with M. phaseolina and R. solani in the field compared with nontreated control plants. In addition, these treatments were able to improve the quality of legumes, significantly increasing the yield crop compared with the nontreated control.

    8. The six Streptomyces sp. strains and the two BCAs Fusarium oxysporum FO12 and Aureobasidium pullulans AP08 showed a significant effect on MGI of V. dahliae compared to the positive control, with the reference BCA FO12 being the most effective, followed by AP08 or the Streptomyces strains that showed a moderate effect.

    9. The six Streptomyces sp. strains and the two BCAs F012 and AP08 also showed a significant effect on MSI of V. dahliae, but in this case most of the Streptomyces strains showed similar effect than that observed for the reference BCA FO12, including the treatment combining CBQ-EA-2 and CBQ-B8. AP08 and CBQ-EBA-21 were the tretaments with least effect on MSI.

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  • Selección de cepas de actinomicetos para el control de hongos fitopatógenos de suelo, con especial referencia a la podredumbre radical de la judía común (phaseolus vulgaris l.) (2024)
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