Scientific service support is essential for the plant health services in any country or region, and for a phytosanitary programme to work well there needs to be good cooperation between the regulatory agencies, crop consultants, and farmers and growers, as illustrated in this chapter by the success of programmes aimed at citrus and ornamentals in Florida.
The ever-decreasing skills in identification and diagnosis are in demand as an increasing number of national and international standards for plant health services are established, not only for the production of clean propagation material, inspection and sampling procedures, but also to provide the basis for PRA and eradication and con- tainment programmes. At the same time there are increasing demands to formalize quality procedures in laboratories, leading to the production of identification protocols that provide guidance for international agreement. This section discusses some of the challenges in putting science into practice to comply with phytosanitary legislation.
12.11.1. Morphological identification and the role of collections
The identification of plant-parasitic nematodes, at the time of writing, is still largely dependent on recording morphological features and subsequent judgements by nema- tode identification specialists or taxonomists. Such judgements may, of course, differ at any one time, but are important in a sector where perhaps only one or a few speci- mens may be isolated from a sample and where an international consensus over the organism causing problems is vitally important. Original descriptions are important tools, as are authenticated reference slide collections. However, the international decline in taxonomic skills and the lack of resources for curation and conservation of collections have led to concerns over the whole basis of identification of regulated nematodes (Hockland, 2005). A recent example of the importance of such issues was the international controversy over the use of names M. mayaguensis and M. enterolobii for international phytosanitary measures. This has been resolved by morphometric and morphological comparisons by experts so that M. mayaguensis is now regarded as a junior synonym of M. enterolobii (Karssen et al., 2012).
12.11.2. Molecular tools and their role in detection
An array of different technologies has been developed over the years to help special- ists in morphological identification, including electrophoresis and PCR (see Chapter 2).
They are especially important where morphological identification is particularly dif- ficult or where only immature specimens have been intercepted. However, it is often not realized that the development of such techniques as reliable, routine methods for use as quarantine identification tools requires additional intensive research. Resulting
protocols are inevitably only developed for a restricted range of species, thus still necessitating a preliminary, provisional identification by a morphological specialist to detect an unusual finding.
Analytical methods examining the genetic make-up of organisms are being con- tinually refined and adapted to develop new phylogenetic models that are becoming an integral part of nematode systematics (De Ley and Blaxter, 2002), and the associ- ated technological equipment, though expensive, is becoming a familiar part of most diagnostic laboratories. Despite great advances in the use of molecular methods for the identification of diseases, especially viruses, the pace of development of reliable, accredited diagnostic protocols in nematology remains slow for species listed in leg- islation. Recently, EPPO has taken a leading role in both development and accredita- tion practices, and collaborated with the European Co-operation for Accreditation (EA) to achieve greater cooperation in raising standards (http://www.eppo.int/
EPPOStandards/diagnostics). The emergence of a range of Meloidogyne species with the potential to cause economic damage (M. ethiopica, M. floridensis, M. graminicola, M. enterolobii and M. minor) has prompted research in biochemical and molecular tools, but personnel in plant health services should have a full understanding of the limits of molecular technology; their real value for the future probably lies in the provision of screening tools, which would indicate any requirement to check identi- ties further. This is because most current protocols for distinguishing regulated spe- cies may not include unregulated, native species of the genera that occur in the countries where interceptions or outbreaks occur, or new species that might be imported from elsewhere. Thus, plant health identification and detection services need to encompass a range of scientific skills if unnecessary statutory action is to be avoided, e.g. for other unregulated species of Bursaphelenchus, Globodera or Meloidogyne. Furthermore, research into the use of molecular tools for detection of plant-parasitic nematodes in soil needs to be developed with care and the implications fully understood, if the status of infestations is to be truly represented. Thus, the integrated role of experienced diagnosticians, taxonomists and molecular scientists in nematode identification and detection for phytosanitary services remains a vital one.
12.11.3. Science versus legislation
Phytosanitary legislation requires clarity and consistency to avoid misinterpretation.
The names of regulated plant-parasitic nematodes need to be established; however, as with other plant pests, this invariably poses a problem in taxonomy where the taxo- nomic details of some nematodes are frequently changing in line with new species concepts. Consequently, this demands an awareness that some species might be sub- ject to many taxonomic changes and there may exist many synonyms in the legisla- tion of some countries; this needs to be recognized if confusion is to be avoided and if correct phytosanitary action, including control, is to be taken.
An example of this is the controversy surrounding R. citrophilus and R. similis, which are both listed in European legislation. Radopholus similis was thought to consist of different pathotypes but Huettel et al. (1984) concluded that the banana race and the citrus race were distinct species; the name R. similis was restricted to the banana race and the citrus race was described as R. citrophilus. Subsequently, Kaplan et al. (1997) synonymized R. citrophilus with R. similis; Valette et al. (1998) proposed
R. citrophilus as a junior synonym of R. similis, although in 2000 Siddiqi proposed it as a sub-species of R. similis and Elbadri et al. (2002), using molecular techniques, demonstrated marked intraspecific variation in various isolates of R. similis. This continuing taxonomic uncertainty has caused more confusion for quarantine special- ists involved in PRA work, as the host lists previously attributed to R. similis have to be used with care. Similarly, the controversy over the names M. enterolobii and M. mayaguensis illustrates that even though identification issues may have been resolved (see Section 12.11.1), studies of assessments of risk need to consider litera- ture for both species. Such difficulties require the expertise of taxonomists, whose numbers are sadly in decline but who provide the essential framework for taxonomy and identification by developing species concepts and theories for the classification and identification of organisms, and hence determine correct names, set standards for descriptions, determine key morphological characters, develop identification keys and catalogue data such as those for distribution.
The integration of morphological and molecular advances in identification can also result in scenarios that test phytosanitary legislation; it needs to remain directed at the damaging genotypes (or more strictly the absent damaging genotypes), and if these are difficult to identify or in flux then perhaps it is best that legislation contin- ues to remain cautious, with, for instance, nematodes included in X. americanum sensu lato. In 2008, Globodera spp. populations able to parasitize potato and geneti- cally distant from PCN were reported in Oregon and Idaho, USA (Skantar et al., 2011). Phytosanitary actions against these unidentified Globodera spp., which also occur in South America, are planned by USDA-APHIS. Similarly, the recent imple- mentation of a new PCN Directive in Europe has raised the issue of the identity of PCN pathotypes, or rather populations which exhibit different pathogenicity in vari- ous parts of the world, especially in their hub of diversity in Central and South America. It is essential that the potato cultivars bred and used in Europe with resist- ance only to the PCN populations that exist there are not exposed to new types of biodiversity (Anon., 2012; Hockland et al., 2012). Science has a role to play in pro- viding evidence to this effect.
12.11.4. The future of diagnostics
Whilst the highest standards of delivery have always been the aim of diagnostic labo- ratories worldwide, the development of international standards has placed increasing demands on attaining prescribed levels of quality in the delivery of services and research. International standards for phytosanitary measures and also for diagnostics are becoming increasingly important, but their adaptation in some areas, such as the identification of species, which entails the use of judgment by experts rather than the output from machines, has proved a difficult philosophy for accreditation schemes to embrace. In addition, the variability of resources available in individual laboratories means a range of protocols has to be included. Nevertheless, selected protocols are slowly achieving international status.
The combination of scarce scientific resource and the cost of providing prescribed levels and speed of delivery have led some countries to negotiate contracts for science services with those countries that still have the ability to deliver. Inevitably, this will lead to centres of expertise serving a community in a particular geographical location
or region. Whilst this may have economic advantages, it should not discourage the broad development of essential identification and diagnostic expertise that is vital for the whole basis of phytosanitary work.
At the time of writing, phytosanitary services are starting to embrace the full potential offered by advances in molecular science, computer technology and the internet. The demise of taxonomic expertise at a local level is stimulating the creation of databases and networks to take advantage of scarce skills at short notice and RPPOs like EPPO have a role to play in facilitating this (see development of the EPPO Plant Quarantine Retrieval System on the EPPO website, http://www.eppo.int/
DATABASES/pqr/pqr.htm). As the global community becomes connected and works to the new quality standards, so it is hoped that the shared experience and expertise in plant health will result in improved understanding of plant-parasitic nematodes for phytosanitary services.
Acknowledgements
The authors would like to acknowledge the valuable help in writing this chapter received from numerous colleagues working for phytosanitary services worldwide.
Thanks are also due to staff of the The Food and Environment Research Agency and the Department for Environment, Food and Rural Affairs, UK, for supporting Dr Sue Hockland in quarantine nematology. We would like to pay particular thanks to Paul Lehman and Leah Millar for their contribution to the first edition.
13.1. Introduction 384
13.2. Suppressive Soils 385
13.3. Biological Control Agents 386
13.3.1. Predators 386
13.3.2. Nematophagous fungi 387
13.3.3. Endophytic fungi 391
13.3.4. Bacteria 392
13.4. Interactions with Rhizosphere Microflora 394
13.5. Application of Biological Control Agents 395
13.5.1. Selection of biological control agents and isolates 396
13.5.2. Inoculum production and formulation 396
13.6. Integration of Biological Control with Other Control Measures 397
13.6.1. Crop rotation 398
13.6.2. Soil disinfestation 398
13.6.3. Soil amendments and green manures 398
13.7. Nematode-free Planting Material 399
13.7.1. Production 399
13.7.2. Heat treatment 400
13.7.3. Mechanical methods 401
13.8. Sanitation 401
13.9. Physical Soil Treatments 402
13.9.1. Dry heat 402
13.9.2. Steam 402
13.9.3. Solar heat 403
13.9.4. Flooding 404
13.9.5. Anaerobic soil disinfestation 404
13 Biological and Cultural Management*
NICOLE VIAENE,1** DANNY L. COYNE2