Volatile Emissions and Relative Attraction of the Fungal Symbionts of Tea Shot Hole Borer (Coleoptera: Curculionidae)

Euwallacea perbrevis is an ambrosia beetle that vectors fungal pathogens causing Fusarium dieback in Florida avocado trees. Current monitoring lures contain quercivorol, a fungus-produced volatile, but the exact attractant is unknown since lures contain a mixture of p-menth-2-en-1-ol isomers and both α- and β-phellandrene. This study used pure cultures of six symbiotic fungi isolated from E. perbrevis to document volatile emissions and determine the relative attraction of symbionts in binary choice assays. In a comparative test, headspace solid-phase microextraction followed by gas chromatography–mass spectroscopy was used to identify and quantify emissions from 3-week-old cultures. In a temporal study, Super-Q collection followed by gas chromatography–flame ionization detection was used to measure cis- and trans-p-menth-2-en-1-ol emissions for three months. A total of 15 compounds were detected, with monoterpene hydrocarbons and oxygenated monoterpenoids predominating. Only trans-p-menth-2-en-1-ol was common to all six symbionts. Peak levels of both isomers were observed at day 7, then gradually declined over a 90 day period. In choice tests, avocado sawdust disks inoculated with Fusarium sp. nov. were the most attractive. This symbiont produced only two volatiles, trans-p-menth-2-en-1-ol and limonene. The combined results indicate that trans-p-menth-2-en-1-ol is the primary female attractant emitted from symbiotic fungi, but limonene may be a secondary attractant of E. perbrevis.

Characterization of the Exo-Metabolome of the Emergent Phytopathogen Fusarium kuroshium sp. nov., a Causal Agent of Fusarium Dieback

Fusarium kuroshium is the fungal symbiont associated with the ambrosia beetle Euwallacea kuroshio, a plague complex that attacks avocado, among other hosts, causing a disease named Fusarium dieback (FD). However, the contribution of F. kuroshium to the establishment of this disease remains unknown. To advance the understanding of F. kuroshium pathogenicity, we profiled its exo-metabolome through metabolomics tools based on accurate mass spectrometry. We found that F. kuroshium can produce several key metabolites with phytotoxicity properties and other compounds with unknown functions. Among the metabolites identified in the fungal exo-metabolome, fusaric acid (FA) was further studied due to its phytotoxicity and relevance as a virulence factor. We tested both FA and organic extracts from F. kuroshium at various dilutions in avocado foliar tissue and found that they caused necrosis and chlorosis, resembling symptoms similar to those observed in FD. This study reports for first-time insights regarding F. kuroshium associated with its virulence, which could lead to the potential development of diagnostic and management tools of FD disease and provides a basis for understanding the interaction of F. kuroshium with its host plants.

Nanoencapsulation of Permethrin in Polylactic Acid to Enhance Insecticide Persistence for Scolytinae Pest Control

Nanotechnology can be used to protect plants against Fusarium Dieback and the Laurel Wilt, that are new and lethal insect-vectored diseases that can host over 300 tree species, including avocado trees. The vectors of these diseases are beetles members of the Scolytinae subfamily, notoriously difficult to control because they spend most of their lives hidden within galleries. Nevertheless, when tested on avocado bolts, some insecticides (including permethrin) provided a reduction in the number of entrance holes or beetle emergence, but the persistence of pesticide residues might have been influenced by factors like rainfall and sunlight. The present study aims to encapsulate permethrin in polylactic acid nanospheres, conferring protection against losses by physic and chemical factors, ultimately increasing its persistence. The particle size, zeta potential, and encapsulation efficiency obtained were 393nm, -32mV, and 27%, respectively. After 96 h of exposure to UV-A light, the insecticidal activity of unencapsulated permethrin was severely diminished, having a reduction in mortality in scolytinae beetles from 80% to 40%, while the nanoencapsulated permethrin retained a 70%. The study has concluded the potential advantage of formulating permethrin into nanometric biodegradable spheres, enhancing the persistence of the insecticide while removing the use of toxic organic solvents as vehicle for the active ingredient, reducing the environmental impact.)

Design of a diagnostic system based on molecular markers derived from the ascomycetes pan-genome analysis: The case of Fusarium dieback disease

A key factor to take actions against phytosanitary problems is the accurate and rapid detection of the causal agent. Here, we develop a molecular diagnostics system based on comparative genomics to easily identify fusariosis and specific pathogenic species as the Fusarium kuroshium, the symbiont of the ambrosia beetle Euwallaceae kuroshio Gomez and Hulcr which is responsible for Fusarium dieback disease in San Diego CA, USA. We performed a pan-genome analysis using sixty-three ascomycetes fungi species including phytopathogens and fungi associated with the ambrosia beetles. Pan-genome analysis revealed that 2,631 orthologue genes are only shared by Fusarium spp., and on average 3,941 (SD ± 1,418.6) are species-specific genes. These genes were used for PCR primer design and tested on DNA isolated from i) different strains of ascomycete species, ii) artificially infected avocado stems and iii) plant tissue of field-collected samples presumably infected. Our results let us propose a useful set of primers to either identify any species from Fusarium genus or, in a specific manner, species such as F. kuroshium, F. oxysporum, and F. graminearum. The results suggest that the molecular strategy employed in this study can be expanded to design primers against different types of pathogens responsible for provoking critical plant diseases.

Screening for susceptibility of Macadamia to E. fornicatus and its fungal symbiont Fusarium euwallaceae.

The polyphagous shothole borer (Euwallacea fornicatus, PSHB), an ambrosia beetle, with its fungal symbiont, Fusarium euwallaceae, is responsible for Fusarium Dieback (FD) in a wide range of woody hosts. In 2019, the first suspected case of E. fornicatus was reported in macadamia in South Africa. The aims of this study were to confirm the E. fornicatus report and thereafter to assess the susceptibility of commercially planted macadamia cultivars to FD caused by F. euwallaceae. The identities of the beetle and associated fungal symbionts were confirmed by means of DNA sequence analysis of the 28S ribosomal large subunit gene for beetles and the internal transcribed spacer region for fungi. Isolates identified as Fusarium species were further characterised by phylogenetic analysis of the translation elongation factor 1α and the β-tubulin gene regions. Thereafter, Koch’s postulates regarding F. euwallaceae were fulfilled on a mature Macadamia integrifolia tree planted at the experimental farm of the University of Pretoria. In order to determine susceptibility against FD, additional cultivar screening was conducted on nine commercially planted cultivars by means of pathogenicity trials using sterilized or inoculated toothpicks inserted into detached branches. Detached branch inoculations showed no significant lesion development six weeks post inoculation, except for cultivar 816. The restricted growth of F. euwallaceae observed in macadamia tissues therefore suggests that macadamia may not be a suitable host for F. euwallaceae and that the threat of FD in macadamia in the event of E. fornicatus infestation is less than for other E. fornicatus hosts. Future work on beetle attraction to macadamia is recommended for a more comprehensive understanding of the interaction between E. fornicatus and its fungal symbionts and macadamia.

Pathogenicity and Distribution of Fusarium solani Isolates Associated with Erythrina Decline in Japan

Erythrina spp. trees have been declining since the 2000s worldwide, and fungi belonging to Fusarium solani species complex (FSSC) have been suggested to be a causal factor of decline and mortality of Erythrina variegata trees in Okinawa Island, Japan. In addition to the FSSC isolate grouped as “it-1” based on ITS sequence data (previously called strain A), we conducted an inoculation experiment with two isolates grouped as “it-2” (previously strain B), which is genetically close to it-1. Two it-2 isolates originating from two islands showed pathogenicity to E. variegata with the same symptoms as those caused by it-1 isolate. We also found the isolates of it-1 and it-2 were widely distributed, including on Ishigaki Island, ∼400 km south of Okinawa Island across the ocean. All isolates of it-1 and it-2 belong to the ambrosia Fusarium clade of the FSSC, a group of symbionts of ambrosia beetles, including the pathogens of Fusarium dieback in avocados and teas. The detection of ambrosia beetles Euwallacea spp. from our specimens provided information on the vectors of the pathogens. Our present results suggest the fungi of the FSSC could be responsible for the Erythrina decline in other areas with damage.

Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.