Morphological identification, molecular characterization and optimization of cultivation for Acrobeloides maximus species extracted from bitter gourd (Momordica charantia) field at Gujarat, India.

Abstract

Abstract Background Free-living nematodes are very important part of agricultural soil ecosystems, especially members of the Cephalobidae family. Morphological and morphometric analysis is efficient for genus-level classification of Cephalobidae members, but to achieve species-level identification, molecular characterization is imperative. Methods and Results The nematodes extracted from the collected soil of the bitter gourd field were cultivated on NGM (nutrient growth medium) agar plates under standardized laboratory conditions. The morphological characteristics of 42 females and 6 males were captured under a digital microscope and processed using the ImageJ analysis tool for De-Man measurements and compared with previously submitted databases. DNA barcoding was carried out with the 18S small RNA subunit gene, and the resulting sequence was then processed to BLASTN after alignment and annotation. The phylogenetic tree was constructed from the processed data files and further evaluated by branch lengths and bootstrap values. Blast analysis showed 98% similarity with the A. maximus strain, which was also supported by high bootstrap values (96%). Acrobeloides maximus was identified based on morphological features described by Throne, 1925 and confirmed by molecular characterization. Conclusion The nematodes collected from the soil sample were cultivated and identified as A. maximus by a combined approach of morphological, pictorial, morphometric, and molecular analysis, which may further be used in the future identification of Acrobeloides spp. To our knowledge, this is the first recorded A. maximus from the bitter gourd field of Gujarat, India. Pictogram and measurement of eggs inside the female body is also shown for A. maximus for the first time.