In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease

Abstract Main conclusion Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all b- and a-pinene rich. Cocultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode. Keywords Maritime pine ! Monoxenic culture ! Pinewood nematode ! Relative water content ! Shoots structure ! Volatiles Abbreviations BAP 6-Benzylaminopurine DAI Days after inoculation EPPO European and Mediterranean Plant Protectio

Accidental transfer of non-native soil organisms into Antarctica on construction vehicles

Antarctic terrestrial ecosystems currently include very few non-native species, due to the continent’s extreme isolation from other landmasses. However, the indigenous biota is vulnerable to human-mediated introductions of non-native species. In December 2005, four construction vehicles were imported by contractors to the British Antarctic Survey’s (BAS) Rothera Research Station (Antarctic Peninsula) from the Falkland Islands and South Georgia (South Atlantic) on board RRS James Clark Ross. The vehicles were contaminated with >132 kg of non-Antarctic soil that contained viable non-native angiosperms, bryophytes, micro-invertebrates, nematodes, fungi, bacteria, and c. 40,000 seeds and numerous moss propagules. The incident was a significant contravention of BAS operating procedures, the UK Antarctic Act (1994) and the Protocol on Environmental Protection to the Antarctic Treaty (1998), which all prohibit the introduction of non-native species to Antarctica without an appropriate permit. The introduction of this diverse range of species poses a significant threat to local biodiversity should any of the species become established, particularly as the biota of sub-Antarctic South Georgia is likely to include many species with appropriate pre-adaptations facilitating the colonisation of more extreme Antarctic environments. Once the incident was discovered, the imported soil was removed immediately from Antarctica and destroyed. Vehicle cleaning and transportation guidelines have been revised to enhance the biosecurity of BAS operations, and to minimise the risk of similar incidents occurring