Identification and characterization of bacteria associated with decline of ironwood (Casuarina equisetifolia) in Guam

Ironwood (Casuarina equisetifolia subsp. equisetifolia) is a nitrogen-fixing tree of considerable social, economic and environmental importance that commonly occurs in tropical/subtropical zones of Asia, the Pacific, Africa, and Central America. Ironwood decline was first noticed on Guam in 2002 and is now affecting thousands of trees and impacting the ecosystem. In 2012, a survey showed that Ralstonia solanacearum and Klebsiella spp. were associated with wetwood symptoms of declining trees. R. solanacearum strains isolated from diseased ironwood in Guam were similar to R. solanacearum strain GMI1000, having similar BOX-PCR profiles and belonging to phylotype I and biovar 3. Two Klebsiella species (K. variicola and K. oxytoca) were recovered, with K. variicola being the more prevalent species. Pathogenicity tests revealed that R. solanacearum caused wilt in tomato and ironwood seedlings, whereas neither Klebsiella spp. produced symptoms. There were no differences in virulence between Guam R. solanacearum and control strains following inoculation into tomato and ironwood from Hawaii. Additionally, no observable differences in ironwood susceptibility to Ralstonia strains from Guam or Hawaii, were observed, suggesting that the association of Guam R. solanacearum with Guam ironwood is not specific. Co-inoculation studies with both R. solanacearum and Klebsiella variicola and K. oxytoca revealed that Klebsiella sp. did not affect symptoms produced by R. solanacearum alone. In planta studies were feasible only on seedlings and young trees in Hawaii; thus, possible interactions between R. solanacearum and Klebsiealla sp. in adult trees remain to be investigated. A new in-field survey of declining ironwood is needed to better understand the role of Klebsiella and Ralstonia in ironwood tree decline in Guam.