Specific detection of Tirmania pinoyi in roots of artificially infected several perennial Cistaceae by Nested-PCR

Truffle farming does not exist in Iran, and no formal studies have been conducted on its culture. For commercial production of Tirmania pinoyi , it is necessary to accurate detection of it in the roots of the host plants. In this study, for the first time, mycorrhizal association of T. pinoyi with several perennial Cistaceae including Helianthemum lippii , H. almeriense , Cistus albidus , and Cistua ladanifer were investigated. In all plant species inoculated with Tirmania pinoyi , an ectendomycorrhizal association with varying degrees of sheath development was observed. Our results shows that H. lippii is suitable plant host for Tirmania pinoyi , and mycorrhization rate and the relative mycorrhizal dependency (RMD) in this native plant was about 90% and 57.85%, respectively, and higher than the other three plant species. The effect of the mycorrhizal fungus on host growth (root weight, root height, shoot weight, shoot height, plant height, and plant weight) was statistically significant compared to non-inoculated plants. To detect the roots colonized by T. pinoyi , a PCR based diagnostic method was developed with the species-specific primer pairs FTiPi and RTiPi designed from the sequence of the internal transcribed space regions (ITS1 and ITS2). The specificity of the primers was verified by PCR analysis of DNA from T. pinoyi specimens and other desert truffles. The method detected T. pinoyi in artificialy infected root plants, and no cross-reactions were observed with any other tested desert truffles. This species-specific PCR method is suitable for quick, simple, and reliable detection of T. pinoyi mycorrhizas.