Detection of Tirmania pinoyi in Roots of Inoculated Cistaceae Plant Species by Nested Polymerase Chain Reaction

Abstract

Desert truffles are edible hypogeous fungi, which grow in arid and semiarid areas of the Mediterranean region. They play an important role in the maintenance of rangelands by preventing erosion and desertification. In recent years, rangelands have experienced serious crises due to global warming and groundwater scarcity. One of the ways to improve rangelands is to use desert truffles as part of conservation or restoration efforts. For these reasons it is important to study the mycorrhizal association of these fungi with rangeland plants. For the first time, we investigated the mycorrhizal association of the desert truffle (Tirmania pinoyi) with the perennial plant species Helianthemum lippii, Helianthemum almeriense, Cistus laurifolius, and Cistus ladanifer. We inoculated all four plant species with T. pinoyi and found the presence of an ectendomycorrhizal association with varying degrees of sheath development. Our results shows that H. lippii is a suitable plant host for T. pinoyi, and the mycorrhization rate and relative mycorrhizal dependency in this native plant were 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 with noninoculated plants. To detect the roots colonized by T. pinoyi, a polymerase chain reaction (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 inoculated 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.