Inoculation of alfalfa seedlings with root growth promoting microorganisms under semi-arid climate condition may improve biomass production and nutritive value. The current study aimed to investigate the effect of inoculation of alfalfa seedlings with Piriformospora indica (Pi) and co-inoculating Pi with Glomus intraradices (Gi + Pi) or Sinorhizobium meliloti (Sm + Pi) on hay yield, chemical composition, molecular structures by Fourier transformed infrared (FTIR) spectroscopy, in situ ruminal degradability and in vitro gas production. Seedlings were grown in experimental pots in a greenhouse until first cut and then transferred outside and cut a further 4 times. Biomass yield was similar across the treatments. Acid detergent fiber (ADF) concentration was higher in Pi than in control hay, and ADF decreased further with co-inoculation (P < 0.05). The ether extract (EE) concentration was lower for Pi and Gi + Pi compared with control hay, and control, Pi and Gi + Pi hays had lower EE concentration compared with Sm + Pi (P < 0.05). The FTIR spectroscopic vibration peak height ratio related to proteins (amide 1 + amide 2): total carbohydrate ratio was lower for the inoculation treatments compared with control hay (P < 0.05). In situ ruminal degradability of dry matter and organic matter were higher for hay of inoculated and co-inoculated seedlings than for control hay (P < 0.05). In conclusion, hay of alfalfa seedlings inoculated and co-inoculated with root growth promoting microorganisms had improved nutritional value compared with hay from non-treated alfalfa seedlings, and co-inoculation was the most effective, however, changes were relatively minor.
