Euwallacea perbrevis (Coleoptera: Curculionidae: Scolytinae), a confirmed pest on Acacia crassicarpa in Riau, Indonesia, and a new fungal symbiont; Fusarium rekanum sp. nov.

Non-native Acacia plantations in Indonesia were first reported to be infested by a native ambrosia beetle species, identified as Euwallacea fornicatus in 1993. Recently the level of infestation in these plantations by ambrosia beetles has steadily increased. The recent redefinition of the taxonomic parameters of the Euwallacea fornicatus species complex has resulted in the identity of the ambrosia beetle species in these plantations becoming unclear. This is also true for their obligate fungal associates. Therefore, the aim of this study was to identify the ambrosia beetle species, as well as its corresponding fungal associate/s, infesting Acacia crassicarpa plantations in Riau, Indonesia. Morphological identification and phylogenetic analysis of the mitochondrial cytochrome oxidase c subunit I (COI) gene, revealed that the beetles are E. perbrevis, previously a synonym of E. fornicatus and commonly referred to as the Tea Shot Hole Borer A (TSHBa). Multi-locus phylogenetic analyses of the fungal associate of E. perbrevis revealed a Fusarium sp. that is among members of the Ambrosia Fusarium Clade (AFC), but that is genetically distinct from other previously identified Fusarium symbionts of Euwallacea species. This novel fungal species is described here as Fusarium rekanum sp. nov.

High-resolution melting curve analysis: A detection assay for Ceratocystis eucalypticola and C. manginecans in infected Eucalyptus.

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species.

Novel Fusarium mutualists of two Euwallacea species infesting Acacia crassicarpa in Indonesia.

Several species in the Euwallacea fornicatus complex have emerged as important pests of woody plants globally, particularly in habitats where they are invasive aliens. These beetles live in obligate symbioses with fungi in the genus Fusarium. In this study, we identified Euwallacea spp. and their fungal mutualists that have emerged as pests of planted Acacia crassicarpa in Riau, Indonesia. Morphological identification and phylogenetic analyses of the mitochondrial cytochrome oxidase c subunit I (COI) gene confirmed that E. similis and E. perbrevis are the most abundant beetles infesting these trees. Multilocus phylogenetic analyses of their fungal mutualists revealed their nonspecific association with six Fusarium species. These included F. rekanum and five novel Fusarium mutualists within the Fusarium solani species complex (FSSC), four of which reside in the Ambrosia Fusarium Clade (AFC). These new species are described here as F. akasia, F. awan, F. mekan, F. variasi, and F. warna.