J.Mycopathol.Res. 62(1) :55-65, 2024, ISSN : 0971-3719 (Print), 2583-6315 (Online) © Indian Mycological Society, Department of Botany, University of Calcutta, Kolkata 700 019, India doi.10.57023/JMycR.62.2.2024.55 A Morpho-biochemical and functional comparison of Arbuscular Mycorrhizal Fungal spores cultured in two distinct conditions MAUNATA GHORUI 1,2 , SHOUVIK CHOWDHURY 1,2 , PRAKASH BALU 2 , SUMIT SUMAN 1 , KESHAB DAS 3 , AND KIRAN SUNAR 3* 1 Symbiotic Sciences Pvt Ltd., Plot no. 575, Udyog Vihar Industrial Area Phase VI, Sector 37, Gurugram, Haryana – 122004. 2 Department of Biotechnology, School of Life Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai - 600 117. 3 Department of Botany, Balurghat Mahila Mahavidyalaya, Balurghat, Dakshin Dinajpur, West Bengal – 733101. Received : 24.11.2023 Accepted : 25.01.2024 Published : 25.03.2024 The present investigation was conducted to compare between Arbuscular Mycorrhizal Fungal spores generated in vitro through Root Organ Culture and those obtained in vivo through conventional pot culture method. Our study conducts a comprehensive analysis of four AMF species viz., Rhizophagus irregularis, Rhizophagus clarus, Entrophospora etunicata, and Funneliformis mosseae cultured both in in vitro and in vivo conditions regarding their morphology, Fatty Acid Methyl Ester (FAME) profiles and infectivity potential. Morphological analysis showed that in vitro-produced spores tend to be smaller in size with smooth surface in comparison to their in vivo counterparts. The observed spore surface irregularities can be attributed to the sloughing off of the outer layer, the adhesion of Rhizobia, Pseudomonads, and the adherence of soil particles due to the release of Glomalin. FAME profiles showed the presence of marker fatty acids indicative of Glomalean nature in all AMF species. Notably, variations in the fatty acid profiles, especially the presence of Neutral Lipid Fatty Acid (NFLA) C16:ù5c, differentiate in vitro and in vivo spores, indicating the influence of growth conditions on the biochemical composition of these AMF species. It is also crucial to identify the disparities in colonization percentages between in vivo and in vitro spores when extrapolating research findings from AMF grown in controlled conditions ( in vitro) to those cultured in association with plants ( in vivo). In vivo cultivated spores of R. irregularis, R. clarus, and F. mosseae exhibited higher infectivity potential while E. etunicata showed higher infectivity potential among all the in vitro cultivated AMF species. These findings suggest interesting opportunities for further exploration for optimizing their use as bioinoculants. Keywords: AMF, comparative analysis, FAME, infectivity potential in vitro, in vivo INTRODUCTION Arbuscular Mycorrhizal Fungi (AMF) form symbiotic relationships with approximately 72% of vascular plants, playing crucial roles in both natural and agricultural ecosystems (Brundrett and Tedersoo, 2018). This symbiosis is increasingly important given global challenges such as a growing population, limited arable land, and excessive chemical fertilizer use (Huang et al. 2020; Smith and Read, 2008). Among numerous cultivation techniques, the primary approaches for cultivating these *correspondence: kiran.sunar@gmail.com microorganisms include in vivo cultivation using pot cultures and in vitro cultivation through Root organ cultures. In vitro cultivation offers controlled conditions but lacks ecological relevance, reducing host diversity and potentially impacting experimental results (Calvet et al . 2013). Morphological plasticity, where AMF spores exhibit variability in response to environmental factors, further complicates species identification (Walker et al. 2021). Studies suggest that the choice of AMF production system influences plant growth and experimental outcomes (Calvet et al. 2013). In vitro cultivation in stable conditions optimizes sporulation but neglects the fluctuating abiotic factors in natural environments (Heinemeyer and Fitter, 2004; Meyer et al. 2017). Additionally, in vitro lacks biotic interactions, essential for AMF’s