Do exchangeable hydrogens affect the evaluation of partial mycoheterotrophy in orchids? Insights from δH analysis in bulk, α-cellulose, and cellulose nitrate samples.

To evaluate the nutritional modes of orchids associated with 'rhizoctonia' fungi, analyses of hydrogen (δH), carbon (δC), and nitrogen (δN) stable isotope ratios are usually adopted. However, previous studies have not fully accounted for exchangeable hydrogens, which could affect these evaluations. Here, we performed standard δC, δN, and δH analyses on bulk samples. Additionally, we conducted δH analysis on α-cellulose and cellulose nitrate samples to investigate whether the heterogeneity of exchangeable hydrogens among plant species influences the assessment of nutritional modes. The δH of orchids were consistently higher than those of surrounding autotrophic plants, irrespective of the three pretreatments. Although the rhizoctonia-associated orchid exhibited lower δC, its δH was higher than those of the autotrophs. Notably, among all response variables, δN and δH exhibited high abilities for discriminating the nutritional modes of rhizoctonia-associated orchids. These results indicate that a time-efficient bulk sample analysis is an effective method for evaluating plant nutritional modes, as the heterogeneity of exchangeable hydrogens does not significantly impact the estimation. Using δN and δH benefits the assessment of partial mycoheterotrophy among rhizoctonia-associated orchids.