Ultrastructural Examination of the Fungus‐To‐Fungus Interactions of <i>Lecanicillium uredinophilum</i> and <i>Phakopsora pachyrhizi</i>

Asian soybean rust (ASR) is caused by the biotrophic fungus <i>Phakopsora pachyrhizi</i> Syd. & P. Syd., and is one of the most important diseases of soybean [<i>Glycine max</i> (L.) Merr.], with recorded yield losses of up to 100%. The fungus <i>Lecanicillium uredinophilum</i> isolate PP2018-001, originally isolated from wild strawberry rust pustules, revealed hyperparasitic abilities on the rust urediniospores of soybean and frangipani (<i>Plumeria</i> spp.). This study examined the fungus-to-fungus interactions of the hyperparasite (<i>L. uredinophilum</i>) and <i>P. pachyrhizi</i> using confocal laser-scanning microscopy (CLSM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to reveal whether <i>L. uredinophilum</i> isolate PP2018-001 employed mycoparasitism as its mechanism of action. The CLSM used an <i>Agrobacterium tumefaciens-</i>mediated transformation (AGTM) of <i>L. uredinophilum</i> isolate PP2018-001 with the green fluorescence protein (GFP) gene to track the hyperparasite infection process. The SEM and TEM investigations used both in vivo and in vitro co-inoculations to examine the extent and type of damage caused by <i>L. uredinophilum</i> on the urediniospores of <i>P. pachyrhizi</i>. Confocal microscopy revealed the ability of <i>L. uredinophilum</i> to penetrate and intensely colonize urediniospores within 36 h. In SEM studies, <i>L. uredinophilum</i> hyphae extensively coiled around urediniospores after both the in vivo and in vitro co-inoculations, with clear penetration and damage of outer urediniospore walls, which, with time, produced visible perforations and loss of cell integrity. TEM revealed the infection of urediniospores by <i>L. uredinophilum,</i> penetrating hyphae, germ tube pores, and the collapse of urediniospores. This study captured the parasitic nature of <i>L. uredinophilum</i> on <i>P. pachyrhizi</i> fungus-fungus interactions. It showed that mycoparasitism occurred, and possibly enzymatic activity occurred, resulting in the degradation of urediniospore outer walls and germ tubes. The results are important for the potential registration of isolates of <i>L. uredinophilum</i> for the biological control of soybean rust.