Abstract
The identification of microorganisms in closely related groups is challenging. The present work focused on the different molecular methodology for the accurate microbial identification in the five commercially available organic agriculture materials enriched with effective microorganisms. From the tested five organic agricultural materials, a total of seven distinct bacterial colonies (A-1, B-1, C-1, D-1, E-1, E-2, and E-3) were isolated and processed for sequential identification utilizing HiCrome™ Bacillus agar, biochemical tests with API CHB50, 16S rRNA gene analysis, random amplified polymorphic DNA (RAPD), and species-specific PCR analysis. All the isolated microorganisms were Gram-positive rods and spore former belonging to Bacillus group and appeared as a differential characteristic feature on HiCrome™ Bacillus agar. All isolates showed high-percentage similarities with the different members of Bacillus species in biochemical testing and 16S rRNA gene analysis. The collective identification results revealed isolates, A-1, B-1, and C-1, close to B. velezensis. Further RAPD-PCR and species-specific PCR discriminated and provided confirmatory evidence for D-1 as B. thuringiensis and E-1, E-2, and E-3 as B. licheniformis, respectively. In addition, presence of B. thuringiensis was also confirmed by toxin crystal protein staining. In conclusion, the species-specific primers could be used as a rapid and accurate identification tool to discriminate closely related Bacillus species such as B. subtilis, B. licheniformis, and B. thuringiensis.
Highlights
In the past few decades, the frequent use of chemical fertilizers for agricultural purposes has increased drastically to meet the food demands of a rapidly increasing population
Microscopy examination and investigation of colony morphology in solid medium and appearance in liquid medium revealed the presence of single types of bacteria in products agriculture materials enriched with effective microorganisms (AMEM)-A, AMEM-B, AMEM-C, and AMEM-D, and that of three distinct types of bacteria in AMEM-E, which were named as A-1, B-1, C-1, and D-1, and E-1, E-2, and E-3, respectively
The AMEM-producing company highlighted the presence of Bacillus species in their products, so as the first step of identification, all the isolates were grown on chromogenic HiCromeTM Bacillus agar, a differential media for Bacillus species [20]
Summary
In the past few decades, the frequent use of chemical fertilizers for agricultural purposes has increased drastically to meet the food demands of a rapidly increasing population. The excessive use of chemical fertilizers has an adverse impact on the agricultural ecosystem. Research has focused on alternatives to chemical fertilizers to improve crop productivity without affecting the soil health and agricultural ecosystem. Several microorganisms have proven their potential as suitable candidates for sustainable agricultural development. Such microorganisms have been intensely introduced into soil to improve fertility and plant growth [1,2] and are broadly known as beneficial soil microorganisms [3]. Many studies have proposed microorganisms derived from organic fertilizers as safe and effective alternatives to chemical fertilizers [4]
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