This research will use the latest advancements in molecular screening to provide a better understanding of root-knot nematodes occurring in Australia from a historic perspective and provide an identification tool for these nematodes that will be much more cost effective than other methods presently available.

Background
The recent identification of a new species of highly virulent plant parasite, the Guava Root-Knot Nematode (Meloidogyne enterolobii), in Australia means that there is a great need to improve root-knot nematode identification for trade, quarantine, and pest management purposes. Critically, it is not clear if M. enterolobii is a recent arrival to Australia, or if the species has been present for some time but has only just been recognised. Historic specimens lodged in Australian collections may hold the key to answering this question, but the information is locked to traditional methods of interrogation.
Guava root knot nematode was reported in Australia for the first time in late 2022, from the Darwin area of the Northern Territory. Since this initial discovery, further detections have been made in Queensland. Guava root knot nematode is a devastating plant parasite causing significant crop losses on a global scale. This nematode has a broad host range spanning 30 plant families, including many important food crops. Notably, guava root knot nematode is a resistance-breaking species, causing severe damage to crops with genetic resistance to other root-knot nematode species.
Consequently, this nematode is a significant biosecurity risk for multiple crops in Australia and is rated in the high to extreme risk categories on the biosecurity plans for multiple industries, for example onions, potatoes, sweetpotatoes, and other vegetables. The wide host range and resistance-breaking characteristics of guava root knot nematode facilitates rapid spread, and this species is very difficult to control. Preventing establishment of this species in production areas where it was previously not present is thus critically important.

Methods
Identification of root-knot nematodes via morphological methods is time intensive, requires significant specialist expertise, and the fingerprint-like patterns present on the female body which are traditionally used to diagnose species are subject to human interpretive error. Molecular identification would be more accurate and high-throughput, however, nearly all historic material was preserved using formaldehyde-based solutions, which are generally thought to inhibit DNA extraction. This research project aims to improve root-knot nematode identification through a two-prong approach.

1. National Research Collections Australia (NRCA) is developing artificial intelligence-based image recognition for pests like Brown Marmorated Stink Bug and various weeds. This research project will develop a similar tool for rapid species-level identification of root-knot nematodes using images of perineal patterns, which are a fingerprint-like pattern of lines on the outside of adult females used for identification. The research team envision the tool as being able to provide a same-day, species-specific identification based on light-microscopy images of perineal patterns requiring minimal training to prepare. The results would thus be far quicker, cheaper, and easier than other identification methods available.

2. Despite the general assumption that DNA cannot be obtained from formalin-preserved material, a method doing just that has recently been pioneered by CSIRO. This research project will use this method to screen historic formalin preserved plant material for evidence of M. enterolobii infestation.

This project has been funded by Hort Innovation, using the research and development levies listed below and contributions from the Australian Government. Hort Innovation is the grower-owned, not-for-profit research and development corporation for Australian horticulture.

• Vegetable Fund
• Potato – Processing Fund
• Potato – Fresh Fund