Endophytic Bacteria Bacillus subtilis, Isolated from Zea mays, as Potential Biocontrol Agent against Botrytis cinerea

Simple Summary Plant-microorganism associations date back more than 400 million years. Plants host microorganisms that establish many different relationships with them, some negative and others very positive for both organisms. A type of this relationship is established with microorganisms that live inside them, known as endophytic microorganisms; they can include bacteria, yeasts, and fungi. In this study, we isolate endophytic bacteria from maize plants, and we characterize them in order to check their potential for being used as biocontrol agents against Botrytis cinerea, one of the most important phytopathogenic fungi in the world. The endophytic bacteria showed this antagonistic effect during in vitro assay and also during in vivo assay in Phaseolus vulgaris. At the same time, they showed the capacity for promoting growth in Zea mays plants. Plant diseases are one of the main factors responsible for food loss in the world, and 20-40% of such loss is caused by pathogenic infections. Botrytis cinerea is the most widely studied necrotrophic phytopathogenic fungus. It is responsible for incalculable economic losses due to the large number of host plants affected. Today, B. cinerea is controlled mainly by synthetic fungicides whose frequent application increases risk of resistance, thus making them unsustainable in terms of the environment and human health. In the search for new alternatives for the biocontrol of this pathogen, the use of endophytic microorganisms and their metabolites has gained momentum in recent years. In this work, we isolated endophytic bacteria from Zea mays cultivated in Colombia. Several strains of Bacillus subtilis, isolated and characterized in this work, exhibited growth inhibition against B. cinerea of more than 40% in in vitro cultures. These strains were characterized by studying several of their biochemical properties, such as production of lipopeptides, potassium solubilization, proteolytic and amylolytic capacity, production of siderophores, biofilm assays, and so on. We also analyzed: (i) its capacity to promote maize growth (Zea mays) in vivo, and (ii) its capacity to biocontrol B. cinerea during in vivo infection in plants (Phaseolus vulgaris).This research was supported by grants from MINECO-FEDER RTI 2018-097356-B-C21, -B-C22, Research Project FEDER-UCA18-107713, and the University of Cadiz by the "Programa de Fomento e Impulso de la actividad de Investigacion y Transferencia de la Universidad de Cadiz"

‘Traditional Sacred Groves’, an ethnic strategy for conservation of microbial diversity

474-480‘Sacred groves’ represents a traditional effort to conserve biodiversity. They are rich patches of undisturbed forests and serve as a natural habitat for many endemic, rare, primitive and economically valuable organisms representing a micro-level biodiversity hotspots.<b style="mso-bidi-font-weight: normal"> In this study, Bacillus spp. and related genera characterized from native soils of the pristine sacred groves of Meghalaya, India revealed dominance of <i style="mso-bidi-font-style: normal">Bacillus, Paenibacillus, Lysinibacillus and <i style="mso-bidi-font-style: normal">Viridibacillus. All the isolates exhibited good plant growth promoting traits when screened for traits like phosphate solubilization, 1-aminocyclopropane-1-carboxylate deaminase and catalase activity, production of auxin, siderophores, hydrogen cyanide and ammonia. Bacteria native to the pristine niche of sacred groves showed better plant growth promoting activities as compared to isolates from disturbed forest as well as type strains implicating the importance of sacred groves and their potential role in microbial diversity conservation

Cultivable bacterial diversity along the altitudinal zonation and vegetation range of tropical Eastern Himalaya

The Northeastern part of India sprawls over an area of 262 379km2 in the Eastern Himalayan range. This constitutes a biodiversity hotspot with high levels of biodiversity and endemism; unfortunately, is also a poorly known area, especially on its microbial diversity. In this study, we assessed cultivable soil bacterial diversity and distribution from lowlands to highlands (34 to 3 990m.a.s.l.). Soil physico-chemical parameters and forest types across the different altitudes were characterized and correlated with bacterial distribution and diversity. Microbes from the soil samples were grown in Nutrient, Muller Hinton and Luria-Bertani agar plates and were initially characterized using biochemical methods. Parameters like dehydrogenase and urease activities, temperature, moisture content, pH, carbon content, bulk density of the sampled soil were measured for each site. Representative isolates were also subjected to 16S rDNA sequence analysis. A total of 155 cultivable bacterial isolates were characterized which were analyzed for richness, evenness and diversity indices. The tropical and sub-tropical forests supported higher bacterial diversity compared to temperate pine, temperate conifer, and sub-alpine rhododendron forests. The 16S rRNA phylogenetic analysis revealed that Firmicutes was the most common group followed by Proteobacteria and Bacteroidetes. Species belonging to the genera Bacillus and Pseudomonas were the most abundant. Bacterial CFU showed positive but insignificant correlation with soil parameters like pH (r=0.208), soil temperature (r=0.303), ambient temperature (r=0.443), soil carbon content (r=0.525), soil bulk density (r=0.268), soil urease (r=0.549) and soil dehydrogenase (r=0.492). Altitude (r=- 0.561) and soil moisture content (r=-0.051) showed negative correlation. Altitudinal gradient along with the vegetation and soil physico-chemical parameters were found to influence bacterial diversity and distribution. This study points out that this is a biome with a vast reservoir of bacteria which decrease with increasing altitudes, and highlights the microbiological importance of the poorly studied Eastern Himalayan range, justifying efforts to explore the prevalence of novel species in the biome.La parte noreste de la India se extiende sobre una superficie de 262 379km2 en la cordillera oriental del Himalaya. Es un punto de acceso con altos niveles de biodiversidad y endemismo; desafortunadamente, también es una zona poco conocida, sobre todo su diversidad microbiana. En este estudio se evaluó la diversidad de bacterias cultivables del suelo, su diversidad y distribución de las tierras bajas a las altas (34 a 3 990m.s.n.m). Se caracterizaron los parámetros físico-químicos del suelo y tipos de bosques a lo largo del gradiente altitudinal y se correlacionaron con la distribución y diversidad bacteriana. Los microbios del suelo se cultivaron en placas de agar enriquecido Muller Hinton y Luria-Bertani, e inicialmente se caracterizaron mediante métodos bioquímicos. Parámetros tales como actividad de la deshidrogenasa y ureasa, temperatura, contenido de humedad y de carbono, pH y densidad aparente del suelo se midieron en cada sitio. Aislamientos representativos también se sometieron al análisis secuencial de 16S rADN. Un total de 155 aislamientos bacterianos cultivables se caracterizaron para estimar los índices de riqueza, equidad y diversidad. Los bosques tropicales y subtropicales albergan una mayor diversidad bacteriana en comparación con los bosques templados de pino y coníferas, y los bosques subalpinos de rododendro. El análisis filogenético de 16S rARN reveló que Firmicutes fue el grupo más común, seguido de Proteobacteria y Bacteroidetes. Especies pertenecientes a los géneros Bacillus y Pseudomonas fueron las más abundantes. Las UFC bacterianas mostraron una positiva pero insignificante correlación con los parámetros del suelo, tales como pH (r=0.208), temperatura (r=0.303), temperatura ambiente (r=0.443), contenido de carbón (r=0.525), densidad aparente (r=0.268), ureasa (r=0.549) y deshidrogenasa (r=0.492). La altitud (r=-0.561) y el contenido de humedad del suelo (r=-0.051) mostraron una correlación negativa. Se encontró que el gradiente altitudinal, junto con la vegetación y los parámetros físico-químicos influyeron en la diversidad bacteriana y la distribución. Este estudio señala que este es un bioma con un vasto reservorio de bacterias que disminuyen con la altitud y pone en relieve la importancia microbiológica de la pobremente estudiada zona del este del Himalaya, lo que justifica los esfuerzos para explorar la prevalencia de nuevas especies en el bioma

Cultivable bacterial diversity along the altitudinal zonation and vegetation range of tropical Eastern Himalaya

The Northeastern part of India sprawls over an area of 262 379km² in the Eastern Himalayan range. This constitutes a biodiversity hotspot with high levels of biodiversity and endemism; unfortunately, is also a poorly known area, especially on its microbial diversity. In this study, we assessed cultivable soil bacterial diversity and distribution from lowlands to highlands (34 to 3 990m.a.s.l.). Soil physico-chemical parameters and forest types across the different altitudes were characterized and correlated with bacterial distribution and diversity. Microbes from the soil samples were grown in Nutrient, Muller Hinton and Luria-Bertani agar plates and were initially characterized using biochemical methods. Parameters like dehydrogenase and urease activities, temperature, moisture content, pH, carbon content, bulk density of the sampled soil were measured for each site. Representative isolates were also subjected to 16S rDNA sequence analysis. A total of 155 cultivable bacterial isolates were characterized which were analyzed for richness, evenness and diversity indices. The tropical and sub-tropical forests supported higher bacterial diversity compared to temperate pine, temperate conifer, and sub-alpine rhododendron forests. The 16S rRNA phylogenetic analysis revealed that Firmicutes was the most common group followed by Proteobacteria and Bacteroidetes. Species belonging to the genera Bacillus and Pseudomonas were the most abundant. Bacterial CFU showed positive but insignificant correlation with soil parameters like pH (r=0.208), soil temperature (r=0.303), ambient temperature (r=0.443), soil carbon content (r=0.525), soil bulk density (r=0.268), soil urease (r=0.549) and soil dehydrogenase (r=0.492). Altitude (r=0.561) and soil moisture content (r=-0.051) showed negative correlation. Altitudinal gradient along with the vegetation and soil physico-chemical parameters were found to influence bacterial diversity and distribution. This study points out that this is a biome with a vast reservoir of bacteria which decrease with increasing altitudes, and highlights the microbiological importance of the poorly studied Eastern Himalayan range, justifying efforts to explore the prevalence of novel species in the biome.<br>La parte noreste de la India se extiende sobre una superficie de 262 379km² en la cordillera oriental del Himalaya. Es un punto de acceso con altos niveles de biodiversidad y endemismo; desafortunadamente, también es una zona poco conocida, sobre todo su diversidad microbiana. En este estudio se evaluó la diversidad de bacterias cultivables del suelo, su diversidad y distribución de las tierras bajas a las altas (34 a 3 990m.s.n.m). Se caracterizaron los parámetros físico-químicos del suelo y tipos de bosques a lo largo del gradiente altitudinal y se correlacionaron con la distribución y diversidad bacteriana. Los microbios del suelo se cultivaron en placas de agar enriquecido Muller Hinton y Luria-Bertani, e inicialmente se caracterizaron mediante métodos bioquímicos. Parámetros tales como actividad de la deshidrogenasa y ureasa, temperatura, contenido de humedad y de carbono, pH y densidad aparente del suelo se midieron en cada sitio. Aislamientos representativos también se sometieron al análisis secuencial de 16S rADN. Un total de 155 aislamientos bacterianos cultivables se caracterizaron para estimar los índices de riqueza, equidad y diversidad. Los bosques tropicales y subtropicales albergan una mayor diversidad bacteriana en comparación con los bosques templados de pino y coníferas, y los bosques subalpinos de rododendro. El análisis filogenético de 16S rARN reveló que Firmicutes fue el grupo más común, seguido de Proteobacteria y Bacteroidetes. Especies pertenecientes a los géneros Bacillus y Pseudomonas fueron las más abundantes. Las UFC bacterianas mostraron una positiva pero insignificante correlación con los parámetros del suelo, tales como pH (r=0.208), temperatura (r=0.303), temperatura ambiente (r=0.443), contenido de carbón (r=0.525), densidad aparente (r=0.268), ureasa (r=0.549) y deshidrogenasa (r=0.492). La altitud (r=-0.561) y el contenido de humedad del suelo (r=-0.051) mostraron una correlación negativa. Se encontró que el gradiente altitudinal, junto con la vegetación y los parámetros físico-químicos influyeron en la diversidad bacteriana y la distribución. Este estudio señala que este es un bioma con un vasto reservorio de bacterias que disminuyen con la altitud y pone en relieve la importancia microbiológica de la pobremente estudiada zona del este del Himalaya, lo que justifica los esfuerzos para explorar la prevalencia de nuevas especies en el bioma

Bioprospecting of Plant Growth Promoting Bacilli and Related Genera Prevalent in Soils of Pristine Sacred Groves: Biochemical and Molecular Approach.

Bacillus spp. and related genera native to soils of the pristine sacred groves from Meghalaya, India were characterized using biochemical and 16S rRNA gene analysis which revealed dominance of Bacillus, Paenibacillus, Lysinibacillus and Viridibacillus in the groves. Biochemical estimation was carried out for in vitro testing of plant growth promoting traits present in these isolates. PCR screening were performed for plant growth-promoting related genes involved in the biosynthesis of acid phosphatase (AcPho), indolepyruvate decarboxylase (ipdC), 1-aminocyclopropane-1-carboxylate deaminase (accd) and siderophore biosynthesis protein (asbA). 76% of the sacred grove isolates gave an amplified fragment for AcPho. Three of the isolates gave an amplified fragment for IpdC gene. Apart from 2 isolates, all the other isolates including the reference strains were positive for the amplification of the accd gene indicating their potential to produce ACC deaminase enzyme. 42% of the isolates gave an amplified fragment for asbA gene indicating the potential ability of these isolates to produce the catechol type siderophore, petrobactin. Overall findings indicated multiple PGP genetic traits present in these isolates which suggested that these isolates are capable of expressing multiple PGP traits. Phylogenetic and sequence analysis of accd and asbA genes from the isolates revealed that asbA genes from Paenibacillus taichungiensis SG3 and Paenibacillus tylopili SG24 indicated the occurrence of intergeneric horizontal transfer between Paenibacillus and Bacillus

PCR screening for PGP related genes from the isolates.

<p>PCR screening for PGP related genes from the isolates.</p

Comparative matches for the closest phylogenetic neighbours obtained for the isolates based on profile of 16S rRNA gene and petrobactin biosynthesis protein <i>asbA</i> gene.

<p>Comparative matches for the closest phylogenetic neighbours obtained for the isolates based on profile of 16S rRNA gene and petrobactin biosynthesis protein <i>asbA</i> gene.</p