LAMP‐Based Assays for Detection of <i>Phlyctema vagabunda</i> and <i>Neofabraea</i> spp. Causing Bull's Eye Rot in Apple Fruits With Disease Symptoms or Latent Infection

ABSTRACT Bull's eye rot (BER) of apple and pear is caused by three fungal species belonging to the genus Neofabrae and by one species recently excluded from this genus and classified as Phlyctema vagabunda. The causal agents commonly occur in pome fruit orchards on the host plants, usually on wood cankers, and they infect apple fruits during their growth. However, BER symptoms develop on infected fruits much later, within several months of cold storage. Two sets of primers for the initiation of loop‐mediated isothermal amplification (LAMP) were designed for the fast and sensitive detection of the presence of BER‐causing fungi in asymptomatic apples and those with visible symptoms: one based on the aspartyl protease gene ( asps ) of P. vagabunda and the second based on the GTP‐encoding gene ( gtr2 ) of N. perennans , N. kienholzii and N. malicorticis . Two newly developed LAMP assays were optimised to work at one reaction temperature. These methods have high specificity for the detection of only target fungal species with high sensitivity—as low as 0.1 pg/μL of target DNA. Together with the previously developed protocol of DNA extraction from the whole apple skin (Poniatowska et al. 2024) used in P. vagabunda and Neofabraea spp. detection procedure for the first time here, the developed methods can serve as valuable tools for estimation of fruit health status for various aims and demands.