pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate-minus mutants of Sclerotinia sclerotiorum.

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.

Farming of a defensive fungal mutualist by an attelabid weevil.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called 'proto-fungiculture', involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial 'weeds' and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in 'proto-fungiculture' animals.

Phylogeography and evolution of a fungal-insect association on the Tibetan Plateau.

Parasitoidism refers to a major form of interspecies interactions where parasitoids sterilize and/or kill their hosts typically before hosts reach reproductive age. However, relatively little is known about the evolutionary dynamics of parasitoidism. Here, we investigate the spatial patterns of genetic variation of Chinese cordyceps, including both the parasitoidal fungus Ophiocordyceps sinensis and its host insects. We sampled broadly from alpine regions on the Tibetan Plateau and obtained sequences on seven fungal and three insect DNA fragments from each of the 125 samples. Seven and five divergent lineages/cryptic species were identified within the fungus and host insects, respectively. Our analyses suggested that O. sinensis and host insects originated at similar geographic regions in southern Tibet/Yunnan, followed by range expansion to their current distributions. Cophylogenetic analyses revealed a complex evolutionary relationship between O. sinensis and its host insects. Significant congruence was found between host and parasite phylogenies and the time estimates of divergence were similar, raising the possibility of the occurrence of cospeciation events, but the incongruences suggested that host shifts were also prevalent. Interestingly, one fungal genotype was broadly distributed, consistent with recent gene flow. In contrast, the high-frequency insect genotypes showed limited geographic distributions. The dominant genotypes from both the fungus and the insect hosts may represent ideal materials from which to develop artificial cultivation of this important Chinese traditional medicine. Our results demonstrate that both historical and contemporary events have played important roles in the phylogeography and evolution of the O. sinensis-ghost moth parasitoidism on the Tibetan Plateau.