CRC for Plant Biosecurity - Trogoderma granarium

DNA tool breaks this hitchhiker’s disguise

CRICHTONA — Thu, 24 Feb 2011 00:08:35 +0000

Khapra beetle (Trogoderma granarium) is one of the five highest-priority threats to the Australian grains industry. Internationally, it is considered one of the most damaging pests of stored products and is of quarantine significance. Economic analysis has estimated that the establishment of Khapra beetle in Australia could cost the grains industry at least $500 million annually in trade restrictions.

Identifying a Khapra beetle in a sample of grain can be difficult, requiring extensive training and experience. This is because the beetle will often be a little worse for wear when discovered. The grain acts like sandpaper and sloughs off the beetle’s hairy cover and most of the identifying features. This assumes that the beetle makes it at all – Khapra beetle larvae usually eat their dead adults for dinner.

It can also be difficult to identify the Khapra beetle from other, less harmful Trogoderma species and related Dermestid insects. There are more than 100 described and possibly 50 undescribed species in the Trogoderma group, which includes the Khapra beetle. There are 52 described and relatively harmless Trogoderma species endemic to Australia.

For the past three years, senior entomologists at the Department of Agriculture and Food, Western Australia, (DAFWA) have been working on the CRC for National Plant Biosecurity Trogoderma diagnostic project. There are three elements to the project: developing protocols for DNA identification of different Trogoderma species, establishing a national Trogoderma laboratory and creating an international Trogoderma reference collection.

The overall aim of the project is to protect Australia’s valuable grain export market by identifying unwanted Trogoderma visitors as quickly as possible to facilitate eradication and to distinguish them from similar native, but non-destructive, pests found in grain leaving our shores.

Working on the Trogoderma diagnostics project, DAFWA’s Mike Grimm initiated the development of a new set of protocols for molecular laboratory testing in 2007. These protocols are establishing real-time PCR (DNA) procedures that will allow species confirmation in a matter of hours.

Mr Grimm says Khapra beetles are a major threat because they are able to survive in many products and situations and will eat anything they can get their teeth into – grains, spices, herbs, dried fruit, meat, wool and many other products coming from overseas – so they can easily ‘hitch a ride’.

“Their capability to stay inactive in larval stage for years means even empty containers may have a healthy population of Khapra beetles capable of infesting anything loaded in it,” he says.

While there has only been one Khapra beetle incident in Australia (found by owners in furniture imported in a shipping container and successfully eradicated), Mr Grimm says the risk is great.

“What we are talking about is protecting the hard work by Australian farmers in producing a valuable commodity for this country,” he says. “Misidentification of exotic pest species poses just as much of a threat to our industry through the imposition of trade barriers as having Khapra beetle in this country.”

Mark Castalanelli has been working on the new DNA protocols for the CRC as part of his PhD project, through Curtin University’s Western Australia Biomedical Research Institute.

The extraction technique he has developed in Australia allows DNA to be taken without physical damage to the specimen. In the past, it had to be crushed and ground to extract DNA. By keeping the specimen intact, visual morphological identification can take place.

“We can’t have DNA identification without morphological identification,” Mr Castalanelli says. “One backs up the other and are equally important when deciding if an intercepted beetle sample is Khapra beetle or not.

“Once we are able to positively identify the Khapra beetle we need to test as many Trogoderma, Trogoderma-related and other stored grain pest species as possible to exclude the false positive results,” he says.

There is still a lot of work to be done since the protocol needs to be verified by independent laboratories; it is already being evaluated by the United States Department of Agriculture. Following its successful acceptance, the protocol may be included in the international Khapra beetle identification protocol.

In conjunction with this work, the CRC has established a national Trogoderma laboratory and international reference collection, which Mr Grimm says is the first facility of its kind for this pest in the world. “Other countries have offered their support in bringing it together,” he says.

Trogoderma laboratory project manager Dr Oonagh Byrne, a molecular entomologist with DAFWA, says the new diagnostics laboratory will be world leading through its use of a suite of tools, including DNA screening with non-destructive DNA extraction methods and imaging using photomontage and the web-based Pests and Diseases Image Library (PaDIL), and Lucid keys (a computer-based identification system).

“The diagnostic equipment purchased to date – the real-time PCR machine and the automated liquid handling system – are widely used in other DNA diagnostics, which is beneficial for other species and many people know how to use them,” she says. But the equipment is the easy part.

Also required are the new testing protocols, accrediting the laboratory and building the collection of species characteristics – through DNA profiling, the morphological validation and development of Lucid keys and an image library of species from all over the world. It is a multi-faceted approach that will take time to complete.

As part of this project, diagnostic entomologist and Trogoderma specialist Andy Szito is building the international reference collection of Trogoderma specimens by visiting collections around the world. Having travelled in Europe and the United States on his mission, this year he will visit France and Russia to view significant collections and he hopes to visit Asia and the Indian subcontinent next year.

After examining thousands of specimens, Mr Szito has morphologically identified more than 100 Trogoderma species, which will back up the DNA profiles.

Locally, the first national Trogoderma trapping program is complete with pests trapped at 64 grain storage sites around Australia. The samples will be the basis of taxonomic studies and the development of DNA markers of native and non-native storage pest Dermestids, including a closely related but less serious pest, the Warehouse beetle. The trapping program will also establish the biogeographic diversity of the different species. A second trapping program will begin later in 2010.

Dr Byrne says many of the specimens held in collections are old. “While useful for morphological validation and development of Lucid keys, they are often unstable for DNA analysis – meaning DNA profiling is more likely from freshly sourced specimens from the trapping program and from sources such as the Australian Quarantine and Inspection Service.”

International Working Conference on Stored Product Protection

CRICHTONA — Wed, 02 Feb 2011 03:07:04 +0000

The 10th International Working Conference on Stored Product Protection (IWCSPP) was held in Estoril, Portugal, from 27 June to 2 July 2010. This round of IWCSPP attracted nearly 300 delegates representing countries around the globe where stored products protection is an ongoing issue. The conference included eight scientific sessions, seven workshops and 92 posters.

The delegates were welcomed with a session on overviews of stored products research across the continents, where Dr Pat Collins, the Leader of Post-harvest Integrity Program of the CRCNPB, represented Australia. This was an interesting session, which revealed that in recent years there has been significant disinvestment by governments globally in the area of stored products research.

Europe has been the worst-hit, whilst substantial funding is still available for research in countries like Australia and the USA, who are at the forefront in this research area. In the USA, currently the research is focused on pest biology, ecology, monitoring and management in small-scale storages including the flour mills; whereas in Australia the focus is on applied research including pest management in bulk storages.

Among a strong contingent of scientists from Australia, Dr Manoj Nayak represented the CRCNPB and delivered an oral presentation titled ‘Developing strategies to manage highly phosphine resistant populations of flat grain beetles in large bulk storages’. The talk was well received by the international delegates and led to further discussions with scientists from Canada, China and the USA, where this pest has either been established as a major problem or emerging as a new problem.

Papers presented at the conference covered a range of topics including biology, ecology, chemical treatments, alternatives to conventional pesticides and fumigants, non-chemical control methods, pest detection methods, quarantine and regulations in relation to export of commodities.

Dr Nayak found the fumigation session the most relevant to the research he undertakes for the CRCNPB. This session had 20 oral presentations, which included a keynote by Dr Emecki regarding fumigants for commodity protection.

Dr Nayak attended only two of the seven workshops, as some ran concurrently while others were not relevant. Both workshops he did attend focused on analysis of stored-product insect monitoring data and ecological data (movement, distribution etc.). The workshops generated excellent interaction with leading researchers from the United States Department of Agriculture (USDA), CRCNPB and Canada, where different trapping methods and the critical analysis of the trapping data and decision-making processes were discussed.

Dr Nayak also joined an international research group involving researchers from USA, Czech Republic, Croatia and China aimed at developing molecular diagnostics for identification of stored product psocids around the world. The research will commence from October 2010.

International Working Conference on Stored Product Protection

CRICHTONA — Wed, 12 Jan 2011 03:07:11 +0000

The 10th International Working Conference on Stored Product Protection was held in Estoril, Portugal from 27 June to 2 July 2010, under the auspices of the Instituto de Investigação Científica Tropical and the Instituto Superior de Agronomia in Lisbon, Portugal.

The aim of the conference was to exchange information on storage from a wide range of agricultural and economic settings, ranging from small farms to large commercial bulk storage and from tropical to temperate climates, and to present innovative solutions to storage problems that are effective, inexpensive, and environmental friendly. The organisers hoped that this conference would help to improve food availability and quality around the world.

Mr Robert Emery attended the working conference and presented on the development and use of Personal Digital Assistants (PDAs) and how they were used to support, audit and validate the eradication of the 2007 khapra beetle (Trogoderma granarium) post-border detection in Western Australia. Robert’s paper was entitled The use of hand-held computers to audit and validate eradication of a post-border detection of khapra beetle in Western Australia.

Robert’s conference paper focused on the development of pest surveillance data collection software and hardware using PDAs to provide auditing, validation, chain of evidence and increase the volume of data collected as well as its integrity through relational databases and seamless data transfer to corporate systems. The system’s first deployment was during a Trogoderma granarium eradication.

The conference program covered the following topics:

around the world – an overview of different regions, reviews of past, present and future directions of research in different regions of the world
biology and monitoring – insects and mites biology, physiology, ecology, modeling and trapping
engineering – grain drying, handling, aeration and chilled air
fumigation – MA, SF, MB, PH3, other gases and resistance
microbiology and mycotoxins – moulds, control of moulds, mycotoxins, contamination and food safety
non- chemical control – biological control, extreme temperatures and organic pest control
residual insecticides – residual insecticides, DE, plant extracts and resistance
integrated pest management practices – methods to prevent, monitor and control pests in warehouses, processing facilities and museums, and
quarantine and regulatory – quarantine pests, control programs, MB alternatives in quarantine, wood packaging and registration of new pesticides.

As part of the conference Robert also attended a field trip visit to Companhia das Lezírias and the Orivárzea Rice Plant (Benavente), which was still partly under construction. Companhia das Lezírias is a state-run agriculture and forestry company located in the Lezíria do Tejo subregion. The company is an ecological sanctuary and peri urban farming area, near Grande Lisboa subregion - the most populated subregion of Portugal. The company produces rice, wine, cork, and livestock, as well as being a reputed horse breeder.

The International Working Conference on Stored Product Protection brought together close to 500 delegates from 27 countries, which provided an excellent opportunity to forge collaborative ties with a range of scientists, consultants, extension workers and industry, who are involved in the safe storage of the world's durable food commodities such as grains, legumes, spices, dried fruits, herbs and animal feed; and non food products such as museum artefacts and wood.

Identifying Khapra beetle

VANMEURSA — Mon, 03 May 2010 05:54:01 +0000

Khapra beetle, Trogoderma granarium is one of the most significant exotic pests of stored grain products and is capable of causing millions of dollars worth of damage to the Australian grain export industry. As such, there is increasing demand for accurate and expert identification of invasive pest species.

In the case of the Khapra beetle, curator and taxonomist with the Department of Agriculture and Food Western Australia, Mr Andras Szito is one of very few internationally accepted active specialists. Mr Szito provides the morphological identifications for the CRC supported National Dermestid Laboratory and supervises the morphological component for PhD candidate, Mr Mark Castalanelli’s Khapra beetle research.

In the last two centuries thousands of specimens belonging to the Trogoderma group have been collected and deposited in museums worldwide. To complement Mr Szito’s work with the CRC, he recently visited numerous leading natural history collections in the United States and Europe to take an inventory of the curated Trogoderma specimens.

Over the past decade greater emphasis has been placed on molecular methods to identify various pest species. Confidence in molecular methods requires diagnostic tests to be developed using specimens which have been verified using the best expert methods. Unfortunately as years go by, fewer and fewer taxonomists remain with the knowledge and skills to perform reliable morphological identifications at an internationally accepted level. Taxonomically trained diagnosticians are aging, jobs disappearing and entomological trainings at universities are shutting down. At the same time, as the pool of specialists with the required training and knowledge decreases the need for taxonomists is increasing because there are no internationally agreed standards for defining new species on molecular methods alone. Considering the economic implications of the presence or absence of Khapra beetle it is no surprise that the United States Department of Agriculture is very keen to collaborate with our Trogoderma projects.

There are over a hundred described species belonging to the Trogoderma group, making assessing the scientific literature and physically reviewing the specimens on which they are based very important. Over fifty of the described Trogoderma species are native to Australia and an estimated further fifty species are not yet described. Clearly the diagnostic molecular tests must reliably determine the pest species without false negatives or false positive results from other species. An additional major difficulty arises with identifying immature stages, for which there are much poorer morphological keys. A competent specialist can reliably separate the adults of the known pest species from the undescribed species. However identifying immature stages in the majority of cases is very difficult. Therefore, the ability to sequence the genome of the identified adult specimens and establish the association with immature specimens becomes increasingly important. Having a sequence of identified Trogoderma species identified by using the best available morphological means, will enable the researchers to identify the Khapra beetle and related pest species.

Few people appreciate the difficulties of identifying Khapra beetle using morphological methods. The descriptions of closely related species are scattered in time, geography and languages; the first being described in the early 19th century, the last in the late 20th century. There are descriptions in English, German, French, Latin, and Spanish with many of them appearing in obscure journals nearly impossible to access.

During his overseas visit, Mr Szito was able to photograph numerous references not held by Australian libraries or institutions. Due to their rarity, many institutions don’t lend these references at all, so accessing them is only possible by visiting. Purchasing them is also not possible due to their rarity - some of them have less than a dozen copies in existence, and if they are for sale, the purchase price is also prohibitive, with some of them exceeding €10,000.

Mr Szito’s visit to the US and Europe allowed him to establish collaborative working relationships with the curators of natural history collections and arrange the loan of specimens. The outcome of the project is not a complete revision of the Trogoderma species but it certainly aims to lay solid foundations for future work by establishing the identity of the common pest Trogoderma species.

Photo caption: Mr Andras Szito studying the Trogoderma collection in the Smithsonian Museum