Antagonism of the Two-Needle Pine Stem Rust Fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum In Vitro and In Planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 × 107 conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4°C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known β-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Molecular and conventional detection and identification of Cladosporium tenuissimum on two-needle pine rust aeciospores

An integrated approach, based on the analysis of both molecular and morphological characters, has led to the unambiguous detection and identification of the rust hyperparasite Cladosporium tenuissimum from aeciospores of the two-needle pine rust fungi Cronartium flaccidum and Peridermium pini. Cladosporium tenuissimum was first detected from contaminated field-collected rust spores using the polymerase chain reaction (PCR) method. The similar-sized amplified DNA of the parasite was then separated from rust DNA using electrophoretic migration, reamplified separately with the nested PCR, and sequenced. Sequence comparison in the data banks enabled the hyperparasite to be recognised as a species of Cladosporium. Molecular detection was followed by conventional identification, obtained by plating rust spores on potato dextrose agar, a selective medium for rusts, since they are unable to grow on such a common substrate, and isolating the hyperparasite in pure culture. It was subsequently identified as C. tenuissimum. Traditional identification would not have been possible without guidance from the molecular data, which focused attention on the mycoparasite. Macro- and micro-scopic features of colonies are also given to help with future identification on spore sources from other geographical areas and, if this should occur, future identification on other rusts.Key words: mycoparasitism, PCR detection, traditional detection, Cladosporium tenuissimum, Cronartium flaccidum, Peridermium pini.