Physiological Effects of Microbial Biocontrol Agents in the Maize Phyllosphere.

In a world with constant population growth, and in the context of climate change, the need to supply the demand of safe crops has stimulated an interest in ecological products that can increase agricultural productivity. This implies the use of beneficial organisms and natural products to improve crop performance and control pests and diseases, replacing chemical compounds that can affect the environment and human health. Microbial biological control agents (MBCAs) interact with pathogens directly or by inducing a physiological state of resistance in the plant. This involves several mechanisms, like interference with phytohormone pathways and priming defensive compounds. In Argentina, one of the world's main maize exporters, yield is restricted by several limitations, including foliar diseases such as common rust and northern corn leaf blight (NCLB). Here, we discuss the impact of pathogen infection on important food crops and MBCA interactions with the plant's immune system, and its biochemical indicators such as phytohormones, reactive oxygen species, phenolic compounds and lytic enzymes, focused mainly on the maize-NCLB pathosystem. MBCA could be integrated into disease management as a mechanism to improve the plant's inducible defences against foliar diseases. However, there is still much to elucidate regarding plant responses when exposed to hemibiotrophic pathogens.

In vitro studies of biofilm-forming Bacillus strains, biocontrol agents isolated from the maize phyllosphere.

We aimed to assess how biofilm formation by three Bacillus isolates was affected by changes in temperature, water potential, growth media, time, and the combinations between these factors. The strains had been selected as potential biological control agents (BCAs) in earlier studies, and they were identified as B. subtilis and B. velezensis spp. through 16 rRNA sequencing and MALDI-TOF MS. Maize leaves (ML) were used as one of the growth media, since they made it possible to simulate the nutrient content in the maize phyllosphere, from which the bacteria were originally isolated. The strains were able to form biofilm both in ML and biofilm-inducing MSgg after 24, 48, and 72 h. Biofilm development in the form of pellicles and architecturally complex colonies varied morphologically from one strain to another and depended on the conditions mentioned above. In all cases, colonies and pellicles were less complex when both temperature and water potential were lower. Scanning electron microscopy (SEM) revealed that changing levels of complexity in pellicles were correlated with those in colonies. Statistical analyses found that the quantification of biofilm produced by the isolates was influenced by all the conditions tested. In terms of motility (which may contribute to biofilm formation), swimming and swarming were possible for all strains in 0.3 and 0.7% agar, respectively. A more in-depth understanding of how abiotic factors influence biofilm formation can contribute to a more effective use of these biocontrol strains against pathogens in the maize phyllosphere.

Efficacy of epiphytic bacteria to prevent northern leaf blight caused by Exserohilum turcicum in maize / Eficacia de bacterias epifíticas en la prevención del tizón foliar del maíz causado por Exserohilum turcicum

Eight potential biological control agents (BCAs) were evaluated in planta in order to assess their effectiveness in reducing disease severity of northern leaf blight caused by Exserohilum turcicum. The assay was carried out in greenhouse. Twenty-six-day-old plants, V4 phenological stage, were inoculated with antagonists by foliar spray. Only one biocontrol agent was used per treatment. Ten days after this procedure, all treatments were inoculated with E. turcicum by foliar application. Treatments performed were: C-Et: control of E. turcicum; T1: isolate 1 (Enterococcus genus) + E. turcicum; T2: isolate 2 (Corynebacterium genus) + E. turcicum; T3: isolate 3 (Pantoea genus) + E. turcicum; T4: isolate 4 (Corynebacterium genus) + E. turcicum; T5: isolate 5 (Pantoea genus) + E. turcicum; T6: isolate 6 (Bacillus genus) + E. turcicum; T7: isolate 7 (Bacillus genus) + E. turcicum; T8: isolate 8 (Bacillus genus) + E. turcicum. Monitoring of antagonists on the phyllosphere was performed at different times. Furthermore, the percentage of infected leaves and, plant and leaf incidence were determined. Foliar application of different bacteria significantly reduced the leaf blight between 30-78% and 39-56% at 20 and 39 days respectively. It was observed that in the V10 stage of maize plants, isolate 8 (Bacillus spp.) caused the greatest effect on reducing the severity of northern leaf blight. Moreover, isolate 8 was the potential BCA that showed more stability in the phyllosphere. At 39 days, all potential biocontrol agents had a significant effect on controlling the disease caused by E. turcicum.

Se evaluó a 8 potenciales agentes de control biológico (ACB) en un ensayo in planta, con el objetivo de probar su efectividad en la reducción del daño provocado por Exserohilum turcicum, agente causal del tizón foliar del maíz. El ensayo se llevó a cabo en invernadero. Plantas de maíz de 26 días, en estadio fenológico V4, se inocularon con los potenciales antagonistas por aplicación foliar como espray. Solo un agente de biocontrol fue usado por tratamiento y todos los tratamientos se inocularon con E. turcicum 10 días después, también por aplicación foliar. Los tratamientos desarrollados fueron los siguientes: C-Et: control de E. turcicum; T1: aislamiento 1 (género Enterococcus) + E. turcicum; T2: aislamiento 2 (género Corynebacterium) + E. turcicum; T3: aislamiento 3 (género Pantoea) + E. turcicum; T4: aislamiento 4 (género Corynebacterium) + E. turcicum; T5: aislamiento 5 (género Pantoea) + E. turcicum; T6: aislamiento 6 (género Bacillus) + E. turcicum; T7: aislamiento 7 (género Bacillus) + E. turcicum; T8: aislamiento 8 (género Bacillus) + E. turcicum. La monitorización en la filosfera de los antagonistas se llevó a cabo a diferentes tiempos. Además, se determinó el porcentaje de hojas infectadas y la incidencia en plantas y hojas. La aplicación foliar de diferentes bacterias redujo significativamente la gravedad del tizón del maíz: entre el 30 y el 78% a los 20 días y entre el 39 y el 56% a los 39 días. En el estadio V10 de las plantas de maíz se observó que el aislamiento 8 (Bacillus spp.) causó el mayor efecto de reducción del tizón foliar. Además, dicho aislamiento fue el potencial agente de biocontrol que mostró mayor estabilidad en la filosfera. A los 39 días, todos los potenciales agentes de biocontrol demostraban un efecto significativo sobre el control de la enfermedad causada por E. turcicum.

Efficacy of epiphytic bacteria to prevent northern leaf blight caused by Exserohilum turcicum in maize.

Eight potential biological control agents (BCAs) were evaluated in planta in order to assess their effectiveness in reducing disease severity of northern leaf blight caused by Exserohilum turcicum. The assay was carried out in greenhouse. Twenty-six-day-old plants, V4 phenological stage, were inoculated with antagonists by foliar spray. Only one biocontrol agent was used per treatment. Ten days after this procedure, all treatments were inoculated with E. turcicum by foliar application. Treatments performed were: C-Et: control of E. turcicum; T1: isolate 1 (Enterococcus genus)+E. turcicum; T2: isolate 2 (Corynebacterium genus)+E. turcicum; T3: isolate 3 (Pantoea genus)+E. turcicum; T4: isolate 4 (Corynebacterium genus)+E. turcicum; T5: isolate 5 (Pantoea genus)+E. turcicum; T6: isolate 6 (Bacillus genus)+E. turcicum; T7: isolate 7 (Bacillus genus)+E. turcicum; T8: isolate 8 (Bacillus genus)+E. turcicum. Monitoring of antagonists on the phyllosphere was performed at different times. Furthermore, the percentage of infected leaves and, plant and leaf incidence were determined. Foliar application of different bacteria significantly reduced the leaf blight between 30-78% and 39-56% at 20 and 39 days respectively. It was observed that in the V10 stage of maize plants, isolate 8 (Bacillus spp.) caused the greatest effect on reducing the severity of northern leaf blight. Moreover, isolate 8 was the potential BCA that showed more stability in the phyllosphere. At 39 days, all potential biocontrol agents had a significant effect on controlling the disease caused by E. turcicum.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize.

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, -1.38MPa and -4.19MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at -1.38MPa. At -4.19MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize / Selección de microorganismos epifíticos de maíz como potenciales agentes de biocontrol de Exserohilum turcicum

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, −1.38 MPa and −4.19 MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at −1.38 MPa. At −4.19 MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.(AU)

El objetivo de este estudio fue seleccionar aislamientos microbianos de la filósfera de maíz y examinar su actividad antagonista contra Exserohilum turcicum. La selección se realizó a través de la capacidad de los aislamientos de competir con el patógeno usando un índice de dominancia y también la capacidad de afectar los parámetros de crecimiento de E. turcicum. La mayoría de las poblaciones epifíticas aisladas para la selección fueron bacterias. Estos aislamientos se encontraron en el orden de 6 log de UFC por gramo de peso fresco de hoja de maíz. En base a características morfológicas y tintóreas similares, se seleccionaron 44 de 111 aislamientos obtenidos para evaluar su capacidad antagónica. A los potenciales agua, ψ, −1,38 MPa y −4,19 MPa, tres aislados del género Bacillus mostraron dominancia a distancia (5/0) y una reducción significativa de la velocidad de crecimiento del patógeno. Tres aislamientos de Bacillus disminuyeron la velocidad de crecimiento de E. turcicum a −1,38 MPa. A −4,19 MPa la velocidad de crecimiento disminuyó con tres aislamientos de Pantoea y tres de Bacillus. En este estudio se observó una correlación negativa y significante entre la velocidad de crecimiento de E. turcicum y el índice de dominancia cuando el patógeno interactuó con algunas bacterias. Esto estaría indicando que cuando disminuye la velocidad de crecimiento del patógeno se incrementa el índice de dominancia de la interacción. Se seleccionaron once posibles agentes de biocontrol contra E. turcicum.(AU)

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize / Selección de microorganismos epifíticos de maíz como potenciales agentes de biocontrol de Exserohilum turcicum

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, −1.38 MPa and −4.19 MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at −1.38 MPa. At −4.19 MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

El objetivo de este estudio fue seleccionar aislamientos microbianos de la filósfera de maíz y examinar su actividad antagonista contra Exserohilum turcicum. La selección se realizó a través de la capacidad de los aislamientos de competir con el patógeno usando un índice de dominancia y también la capacidad de afectar los parámetros de crecimiento de E. turcicum. La mayoría de las poblaciones epifíticas aisladas para la selección fueron bacterias. Estos aislamientos se encontraron en el orden de 6 log de UFC por gramo de peso fresco de hoja de maíz. En base a características morfológicas y tintóreas similares, se seleccionaron 44 de 111 aislamientos obtenidos para evaluar su capacidad antagónica. A los potenciales agua, ψ, −1,38 MPa y −4,19 MPa, tres aislados del género Bacillus mostraron dominancia a distancia (5/0) y una reducción significativa de la velocidad de crecimiento del patógeno. Tres aislamientos de Bacillus disminuyeron la velocidad de crecimiento de E. turcicum a −1,38 MPa. A −4,19 MPa la velocidad de crecimiento disminuyó con tres aislamientos de Pantoea y tres de Bacillus. En este estudio se observó una correlación negativa y significante entre la velocidad de crecimiento de E. turcicum y el índice de dominancia cuando el patógeno interactuó con algunas bacterias. Esto estaría indicando que cuando disminuye la velocidad de crecimiento del patógeno se incrementa el índice de dominancia de la interacción. Se seleccionaron once posibles agentes de biocontrol contra E. turcicum.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize. / Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize.

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ¤ê, -1.38MPa and -4.19MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at -1.38MPa. At -4.19MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

Impact of osmotic/matric stress and heat shock on environmental tolerance induction of bacterial biocontrol agents against Fusarium verticillioides.

Bacillus amyloliquefaciens and Microbacterium oleovorans reduced the Fusarium verticillioides count and significantly decreased fumonisin B(1) and B(2) levels in maize grains. The aim of this study was to determine the effect of water stress tolerance and heat shock survival upon cells of the biocontrol agents B. amyloliquefaciens and M. oleovorans. The a(w) of solid and liquid media and tryptic soy medium was modified to 0.99, 0.98, 0.97 and 0.96 by addition of ionic solute NaCl and non-ionic solutes such as glycerol and glucose. The non-ionic solute polyethylene glycol 600 (PEG 600) was used to modify matrically solid media. Bacterial incubation was at 30 °C. After 24, 48 and 72 h of incubation, samples from liquid media were spread-plate on nutrient agar medium and incubated for 24 h to determine the number of viable cells. The bacterial cells were harvested by centrifugation and heat treatment carried out in a water bath at 45 °C for 30 min. The viability of cells from different incubation times in liquid media showed statistically significant differences. Cells of B. amyloliquefaciens grown in liquid media amended with glycerol showed better tolerance at low a(w) and high survival under heat stress. These results could have important implications for optimizing and improving formulations.