Host Plant Resistance (HPR) Against Nematodes

Rev 12/21/10

Sources:  

Mainly from Ferris et al (1992) Beyond Pesticides - Biological Approaches to Management in California, based on material from P.A. Roberts and V.M. Williamson. Also from subsequent additional material.

Introduction

The prospects for major contributions to protecting world food and fiber production through crop resistance and tolerance to nematodes are truly exciting. Agricultural scientists consistently identify plant resistance as the highest research priority for nematode pest management. The advantages and benefits of breeding crop plants resistant to injurious parasitic nematodes, and growing them on infested land, are many and varied.

Resistant crops provide an effective and economical method for managing nematodes in both high- and low-cash value cropping systems. In annual cropping systems, resistant crops can reduce nematode populations to levels that are non-damaging to subsequent crops, thereby enabling shortening and modification of rotations. They are environmentally compatible and do not require specialized applications, as opposed to most chemicals and, apart from preference based on agronomic or horticultural desirability, do not require an additional cost input or deficit. In less developed countries and in low-cash crop systems, plant resistance is probably the most viable solution to nematode problems. 

Understanding the genetic diversity of the nematode population will require continued effort in introducing new resistance gene sources as virulent biotypes are selected.

Definitions

 The term, "host" is used in nematology to indicate a plant species on which a plant-parasitic nematode species can feed and reproduce. The general condition, however, is that a given plant species is a non-host to most nematode species; that is, most nematode species do not use the plant as a food source. 

The term, "resistance" is used in a non-epidemiological sense in nematology to describe the effect of a plant on nematode reproduction. A highly resistant plant allows little or no nematode reproduction, whereas a susceptible plant supports abundant reproduction. Partially or moderately resistant plants permit intermediate levels of reproduction.

Current Status

The current availability and/or use of resistant cultivars and rootstocks for nematode management reflect the success to date of research efforts in identifying and evaluating resistance sources, incorporating them into commercially acceptable crop selections, and implementing them in management programs. The relatively few crop and nematode combinations where plant resistance or tolerance to nematodes are in use is probably less a reflection of the difficulty of the task and more a reflection of limited research emphasis. The potential for success in this area seems enormous in light of the essentially untapped sources of resistance to nematodes in a broad range of botanical groups, and the rapid technical advances such as in-embryo rescue, somatic hybridization, and direct gene transfer that should promote a more efficient genetic transfer across conventionally difficult biological barriers (e.g., sexual incompatibility, polyploidy, unacceptable gene linkages).

Plant resistance has been found and incorporated mainly to the highly-specialized parasitic nematodes such as Globodera, Heterodera, Meloidogyne, Rotylenchulus, Tylenchulus, and Ditylenchus; these nematodes have sedentary endoparasitic relationships with their host at least for a portion of their life cycle. 

Resistance in a given cultivar or rootstock may be conferred to nematode species of different genera, to more than one species from the same genus, to a single species, or to certain within-species variants. With few exceptions, such as Xiphinema index resistance in grapevines, resistance to ectoparasitic nematodes has not been identified, although many examples of non-hosts (immunity) exist.

These patterns of resistance may reflect the co-evolutionary forces between host and parasite, the more highly specialized relationships having resulted in specific genes for resistance and parasitism as genetic advantage was sought. The root-browsing, ectoparasitic nematodes, with less specific feeding requirements, apparently have not been a strong selection force for resistance in plant hosts, and have not received much attention in breeding programs, although general tolerance traits could be useful in such programs.

Implementation in California

California agriculture is intensive and diversified. About 200 food and fiber crops are grown in a range of soil and climatic types and where many of the important phytoparasitic nematode genera are represented by one or more species. Of the nematode-resistant annual crops available, relatively few are grown in this state (Table 1). Resistance to some species of Meloidogyne in beans (large lima), cowpeas, sweet potatoes, and tomatoes are the only cases where plant resistance is currently used as a nematode management tactic. Resistant cultivars, especially of sweet potatoes and tomatoes, have been used mostly in conjunction with a preplant fumigation treatment.

TABLE 1.  Major annual and perennial crops in which resistance to nematodes is available and used for nematode pest management (Roberts, 1982).
Crop Nematode
Beans  Meloidogyne incognita*, M. javanica,
Pratylenchus scribneri
Carrot Meloidogyne incognita*, M. javanica
Cowpea  M. incognita*
Cotton  M. incognita
Potato  Globodera pallida, G.  rostochiensis
Soybean   M. incognita, M. javanica, Heterodera
glycines
, Rotylenchulus reniformis
Sweet potato M. incognita*, M. javanica*, M. arenaria*
Small grains 
(wheat, barley, oats)
Heterodera avenae, Ditylenchus dipsaci
Tobacco M. incognita, M. arenaria, Globodera spp.
Tomato M. incognita*, M. javanica*, M. arenaria*
Alfalfa M. incognita*, D. dipsaci*
Apricot Meloidogyne spp.*
Citrus Tylenchulus semipenetrans*
Grape Meloidogyne spp.*, Xiphinema index
Walnut Meloidogyne spp.*
Prunus rootstock-Nemaguard (almond, nectarine, peach, plum) M. incognita*, M. javanica*, M. arenaria*
*Nematode-crop combinations in which resistance is used in California.

 Of approximately 30 perennial crops grown in California, resistance has been implemented extensively in some crops and not at all in others.  Citrus rootstocks (e.g., Poncirus trifoliata and its citrange hybrids) resistant to citrus nematode (Tylenchulus semipenetrans) have been used extensively and successfully for some 25 years, although certain T. semipenetrans pathotypes are now known to circumvent this resistance and reduce yield.

Meloidogyne-resistant grapevine rootstocks are available,
but are not preferred due to a tendency to promote vegetative growth.  However, there are some vineyards established on the resistant "Harmony"  and "Freedom" rootstocks. Partial resistance and tolerance in some grape cultivars such as "Thompson Seedless" are used in sandy soils in the interior San Joaquin and Coachella Valleys, where problems are likely from Meloidogyne spp. 

Probably the most successful plant resistance implementation program in California has involved the use of Meloidogyne-resistant "Nemaguard" rootstock (derived from Prunus davidiana) for Prunus crops. Approximately 75% of the almond, nectarine, peach, and plum plantings-about 600,000 acres-are on Nemaguard rootstock. After 30 years of use, there is no evidence of selection of Meloidogyne populations that can circumvent resistance in Nemaguard; however, problems with some other nematode species occur (e.g., Criconemoides xenoplax).

Alfalfa cultivars resistant to Ditylenchus dipsaci and to species of Meloidogyne, other than M. hapla, are used in California.

Meloidogyne resistance in walnut is achieved through the use of Black Walnut rootstocks and "Paradox" hybrids. These rootstocks and hybrids are used in 95% of the walnut acreage. 

Thus, apart from Prunus, citrus, and walnut rootstocks, the implementation of nematode-resistant cultivars and rootstock plantings of California crops is limited. 

Phil Simon, USDA/ARS carrot breeder based at the University of Wisconsin, Madison, in collaboration with Phil Roberts, UCR Nematology).has conducted field and laboratory tests with derivatives of the Brasilia carrot cultivar. The cultivar  has good resistance to Meloidogyne javanica.   In 1990, Simon isolated a single gene from Brasilia that provides that resistance.  He has since incorporated resistance  to M. incognita. The goal is to develop carrots that can be grown without nematicides (Bryant, 2005).

Utilization Strategies For Nematode Host Plant Resistance  

Resistance can be important for annual crops in two major respects:  

Rotational aspects are of less concern in most perennial crops.
Most important is tolerance to injury associated with resistance, and traits such as limited tissue damage (i.e., root galling or lesions) that will reduce potential for root rots and other diseases resulting from secondary infection of nematode-disrupted tissue.

An important consideration for the utility and longevity of HPR is knowledge of the range and nature of variability of genotypes in the nematode population. How rapidly can gene frequencies in the nematode population be expected to change so that the population is dominated by virulent, resistance-breaking biotypes? How should the gene frequencies in the population be selected and managed to preserve the utility of HPR?

Single Gene Resistance. 

Single gene resistance has received the most attention so far in HPR research and application. Single, dominant genes for resistance are often most convenient to select and to use in
breeding programs. For example, tomato lines which contain the Mi gene exhibit effective resistance to root-knot nematodes in the field and produce normal yields even in fields severely infested by these nematodes. 

Single genes are also desirable for molecular engineering
studies because they are easier to identify and manipulate than multiple genes with additive effects and quantitative expression. Thus, major advances in gene cloning, and formation of transgenic plants, will most likely involve single gene systems in the forseeable future. However, a negative aspect of single gene HPR is vulnerability to circumvention by aggressive isolates of nematodes arising through directional selection during frequent exposure to resistant host plants.

A well-characterized example of single-gene resistance is provided by cultivars of tomato that are resistant to root-knot nematodes (e.g., Meloidogyne spp.). These nematodes penetrate the host near root tips and cause many changes in root morphology and physiology. Changes include formation of galls and development of specialized "giant cells" for support of nematode development and egg production (Hussey, 1985).

Mi, a single, dominant locus conferring resistance to three root-
knot nematode species (M. incognita, M. javanica, and M. arenaria), but not to M. hapla, is present in many modern tomato cultivars (cf Roberts and Thomason, 1986). Mi is one of the NBS/LRR class of resistance genes.  Genes in this class gave nucleotide binding sites (NBS) and leucine rich repeats (LRR) as part of the protein architecture. The mechanism of resistance to nematodes conferred by Mi appears to involve a hypersensitive response on the part of the host. Necrotic plant cells are visible around the head of the invading nematode within six hours of inoculation of roots with nematode juveniles (Dropkin et al., 1969; Dropkin, 1969). Necrosis of host cells precludes larval growth, and the larvae die within 96 hours (Riggs and Winstead, 1959; Dropkin et al., 1969). 

The resistance due to Mi can be reversed by exogenous factors such as addition of cytokinins or elevated temperatures (Dropkin et al., 1969; Dropkin, 1969).

A hypersensitive response consisting of localized cell necrosis at the infection site is characteristic of single gene resistance to many plant pathogens including viruses, bacteria, nematodes, and fungi (Keen, 1982). These resistance genes appear to involve specific recognition by the plant of some feature of the invading parasite. Often recognition is limited to particular strains or isolates of the pathogen and is believed to be mediated by single genes called avirulence genes in the pathogen, giving rise of gene-for-gene complementarity between host and pathogen (Flor, 1955; Ellingboe, 1982). 

While avirulence genes have been cloned from bacterial pathogens (Staskawicz et al., 1984; 1987), less progress has been made in the identification of plant resistance genes. Reasons for this include the high complexity of plant genomes as well as lack of any knowledge about resistance gene products. No definitive information on the tissue localization and level of expression of resistance gene products is available. Whether resistance gene products are induced by pathogens or are constitutively expressed remains an open question. Also, it is not known if the resistance gene products act directly or indirectly in pathogen recognition or in triggering the hypersensitive response.

Identification and selection of genetic sources of HPR to nematodes will require cooperative efforts between nematologists and plant breeders. In addition to collaboration, research needs in this area should focus on the search for gene sources in wild plant gene pools and the selection of traits through classical breeding approaches.  Traditional breeding involves the introgression of selected traits into breeding lines and further selecting through recurrent backcrossing programs to develop resistant cultivars or rootstocks.

Research efforts must be expanded in the isolation and cloning of desirable traits of HPR and tolerance to facilitate understanding of mechanisms of resistance and host-parasite recognition. Cloned genes will enable direct transfer of resistance into crop plants that are unrelated to the gene donor plant. Success in transfer will depend upon adequate, non-disruptive expression of the desired HPR trait in the transgenic plants. The use of novel transfer techniques, including tissue culture, cell and protoplast fusions, embryo cultures, and embryo cloning techniques may help to overcome incompatible plant barriers. 

In conjunction with transfer of natural resistance genes, the use of novel sources of resistance (derived through induced mutations and somatic and somaclonal variations) can be explored, as well as incorporation of toxin-producing genes or inhibitors into plants.

Transgenic Tomatoes

An example of a molecular transfer candidate is, again, provided by the Mi gene of tomato. This gene was identified in the wild tomato species, Solanum peruvianum, and was introduced into cultivated tomato using embryo culture of an interspecific cross of S. lycopersicum and S. peruvianum (Smith, 1944), followed by extensive backcrossing to S. lycopersicum. Progeny of a single F1 plant are the sole source of nematode resistance in currently available fresh-market and processing tomato cultivars (Medina-Filho and Tanksley, 1983). The observation by Rick and Fobes (1974) of a tight genetic linkage between Mi and Aps-1, a gene encoding acid phosphatase-1, greatly facilitated the introduction of the resistance gene into commercial cultivars. Medina-Filho (1980) performed linkage tests to determine the genetic distance between Aps-1 and Mi. 

No recombinants were observed in 513 F2 backcross plants,
indicating that these two genes were separated by a genetic distance of less than 0.894 centimorgans (cM).

DNA markers have been mapped to loci covering much of the tomato genome through restriction fragment length polymorphisms (RFLPs) found in an interspecific cross (Bernatzky and Tanksley, 1986a,b). Two of the loci, CD67 and CD29B, have been mapped flanking Aps-1 at a distance of 7 and 11 cM away, respectively. Each of these probes has been used to analyze Southern blots of tomato lines which differ in their resistance to nematodes and in the isozyme of Aps-1 (Ma and Williamson, unpublished). Probe CD67, but not CD29B, displays RFLPs which fall into two classes depending on the Aps-1 allele present. Since RFLPs are rare within domestic tomato (Helentjaris et al., 1986), this result suggests that the region of DNA encoding CD67 was derived from S. peruvianum and was introduced into tomato along with the Mi gene. 

Four other DNA markers, tightly linked to Mi, have recently been identified (Ho and  Williamson, unpublished). The gene encoding Aps-1, itself, has also been cloned (Williamson and Colwell, in press). Understanding of the location and order of these genes will greatly facilitate isolation and cloning of Mi.

Introduction of Mi into acceptable tomato cultivars by conventional breeding has been time consuming due to the extensive backcrossing required to incorporate other desirable traits. It would be highly preferable to transfer the Mi gene as a single unit into tomato cultivars which already have other desirable traits and, thus, eliminate the need for these time-consuming backcrosses. When a clone of the resistance gene becomes available, it can be introduced rapidly into susceptible cultivars using available gene transfer methods. Also, the cloned gene can be used as a probe in a modern breeding program to monitor the segregation of Mi directly.

In future research, the cloned nematode-resistance gene from tomato will be introduced into other plant species to determine whether resistance to root-knot nematodes is conferred. The ability to introduce nematode resistance into species where no known resistance sources exist would be of great value to agriculture. In vitro modification can be carried out to change the properties of Mi, for example, altering the host range or the nematode species recognized, or increasing the temperature range for function of Mi. It is possible that Mi is homologous to other nematode resistance genes and may be useful as a probe to clone such genes. The utility of the Mi clone as a probe to identify other nematode resistance genes in wild tomato species and in other plants-many mediated by a mechanism similar to Mi-is worth exploring.

Research in this area contributes greatly to our understanding of how plants recognize and protect themselves from pathogens. Isolation of the Mi gene is not, by itself, likely to explain these mechanisms, but it will provide a key step for answering a broad range of questions that have been difficult to approach. What the gene product is, where it acts, and how it triggers the resistance response are questions of great interest. A clone of Mi will allow us to approach questions of transcriptional and translational regulation, tissue and cellular localization, and regulation of expression of this gene product.

Sequence analysis of the gene will provide information on whether it encodes a protein and provide some information on the properties of that protein. Antibody to the Mi protein product can be made by producing a fusion protein in Escherichia coli and injecting the purified fusion protein into rabbits or by synthesizing synthetic peptides and generating antibodies to these. This antibody can then be used to investigate localization and expression of the Mi gene product.

Multiple Gene Resistance. 

Multiple gene resistance is less well understood and usually much more difficult to identify, select, and transfer through classical breeding programs.  Little or no work in nematology on multiple resistance has been attempted at the molecular level, and inheritance studies and basic genetic characterizations are limited thus far. This represents an area where more research emphasis is desirable. Generally, multiple gene HPR is considered more durable than single gene resistance and, thus, may well be of greater long-term value to agriculture. 

Because multiple gene resistance is broad-based, and may involve multiple mechanisms, resistance-breaking biotypes may not be readily selected, although studies with multigenic potato lines and the potato-cyst nematode suggest this is not necessarily always the case (Turner, 1990). Recently, the use of DNA markers has been helpful for breeding complex traits into plants, such as soluble solids into tomato.

Host-plant Tolerance. 

Tolerance of plants to nematode infection is highly desirable. It is often conferred by genes for resistance, but not always (hypersensitivity can be a problem). Tolerance can also be selected for, independent of genes for resistance, but it is very labor- intensive and programmatically expensive to select for in most cases, requiring large amounts of greenhouse and field plot facility. Research on identifying simple, linked markers for tolerance is required, which could be morphological, physiological, biochemical, or molecular in nature and that would aid in screening, selection, and breeding efforts (see Evans and Haydock, 1990). 

Microtoxin Genes. 

The expression in plants of foreign genes that produce substances toxic to nematodes, such as those being studied for insect control using B. thuringiensis (Bt), requires greater research emphasis in the future and could be a fruitful area. A major advantage of the approach is that the desirable horticultural or agronomic qualities of a cultivar are retained, and the nematode resistance is added directly into the genome. 

This should be much faster than the more traditional plant-breeding approach of: 

  1. crossing plant variety or species expressing the desired resistance with an acceptable cultivar; 
  2. screening the progeny for resistance; 
  3. repeatedly backcrossing to the acceptable cultivar to improve the desired characteristics; and 
  4. screening to ensure that resistance has been retained in each new generation of progeny.


The management potential in this area was reviewed in the Biological Control section of this chapter. In summary, the Mycogen Corporation has recently announced isolation of a Bt strain with a toxin effective against plant-parasitic nematodes. Research is needed to transgenically insert the gene responsible for toxin production in Bt into a plant genome and to determine whether the gene is expressed in the plant. 

There may be other sources of such microtoxin genes that warrant selection and isolation; for example, incorporation of a gene for collagenase which destroys the nematode cuticle.

Sources of Resistance. 

Three key potential sources of resistance are available, and with them come the obvious requisites of fundamental research to develop their potential.

(1) Wild Plant Species. 

Wild plant species represent the mostimportant source of HPR genes thus far and, no doubt, hold in their gene pools considerable additional and, as yet, unrecognized sources of resistance. However, they present a challenge in breeding work, due to problems of incompatibility, particularly among the more divergent taxa or genotypes, and the association of resistance with various undesirable traits. Embryo rescue and somatic hybridization techniques may facilitate otherwise difficult gene transfers.

(2) Induced Mutants. 

Some mutants induced by irradiation express increased levels of resistance to nematodes, e.g., in chickpea and potato, although their stability over time is questionable and, as yet, unknown.

(3) Plant Regeneration. 

The regeneration of plants from organs, tissues, and cells facilitates selection of somaclonal variants with desirable resistance traits arising from single nuclear changes. They are preferred over mutation breeding as they retain the desirable characteristics of the existing cultivar. These tissue culture-induced genetic variations, and their potential as sources of resistance to diseases and nematodes, have been little explored in research programs and warrant further investigation. Likewise, the application of in vitro tissue culture techniques, protoplast fusions, etc., toward the routine or special transfer of HPR genes across sexually incompatible boundaries between plants has not been given much research attention.

These technical areas may play a key role in our development of HPR in crop plants for nematode management in the future.

Research Needs - Host Plant Resistance

Key needs in the area of genetic resistance and plant breeding
include cooperation with, and support for, classical plant breeding programs, and research dollar support in the following areas: 

  1. natural and novel gene source identification and development;
  2. classical and novel gene transfer techniques; 
  3. genetic studies on HPR, tolerance traits, nematode parasitism, and virulence factors; 
  4. bioengineering studies to facilitate molecular approaches to gene study;
  5. transfer and expression of foreign genes in plants; and 
  6. certain other potential areas such as microtoxin use in vitro, and immunization systems.

Development of a statewide computerized database is also needed to allow access to information on plant susceptibility to nematodes and on management techniques. Longer-term research needs include the development of resistant plants using germplasm from wild species, development of transgenic plants, understanding nematode genetics and its impact on stability of HPR, and understanding the mechanisms of HPR.

A considerable amount of ongoing research in molecular biology is directed toward understanding the nature of control and inheritance of HPR, but it is still unclear how the resistance traits are expressed relative to the nematode and how they are integrated into the physiology of the whole plant. 

For example, the expression of resistance conferred by the Mi gene of tomato is not thermostable. The resistance breaks down at soil temperatures above 28 C, which could be a serious constraint in warm environments or at certain times of the year.

In addition to understanding the ramifications of HPR in whole plants, it is also necessary to understand the subtleties of its expression under field conditions. Critical to this understanding is genetic variability of nematode populations, which is not readily measured and is not well documented. 

Some interesting lessons have been learned already about the impact of employing single gene resistance against nematodes in whole fields of plants. For example, single genes have been incorporated into soybeans to confer resistance to the
soybean-cyst nematode, H. glycines. Repeated culture of the resistant varieties selects for variants in the nematode population so that resistance-breaking biotypes become predominant. Consequently, the resistant cultivar is no longer effective in that field, and new sources of resistance are needed. 

Similar examples exist for the cyst-nematode parasites of potatoes and cereals in Europe. In another example, a gene for resistance to the root-knot nematode, M. incognita, has been incorporated into tobacco varieties grown in the southeastern United States. As a result, M. arenaria (another root-knot nematode more virulent than M. incognita) has now been selected for in that region.

The solution to the problem of selection for aggressive biotypes or species of a nematode pest by using resistant cultivars may lie in what Vanderplank (1984) described as "stabilizing selection." The underlying principle assumes that genes for aggressiveness to a resistant cultivar may not confer any advantage to the biotype of the nematode in the absence of the selection pressure imposed by the resistance. In other words, the original biotype or species of the pest was probably predominant because it was well adapted to that environmental situation and ecological niche, and was probably a better competitor for resources than the variants. Consequently, removal of the selection pressure, by growing a susceptible cultivar, will remove any advantage provided for the new biotype or species and allow selection for the better-adapted, original genotype. 

The superior competitive abilities of the original genotype should result in a decline of the new genotype. Obviously, this could lead to reversion to the original problem, so a delicate balance of rotating resistant and susceptible cultivars must be achieved to keep both genotypes below damaging levels.

The implementation of stabilizing selection to preserve the longevity and utility of HPR, based on single-gene sources, requires both the study and measure of a number of epidemiological and population genetics parameters: 

Clearly, in addressing these questions, the planning horizon for nematode management is extended from a single crop season to multiple growing seasons. Also, the perception of management of genotype frequencies of nematode populations in a field over time is introduced, as opposed to merely controlling the numbers of nematodes to a low level prior to planting.

A relatively untapped area for plant-parasitic nematodes is that of induced resistance. Can the plant's defensive forces, its "immune system," be activated by some stimulus so that it is better protected against nematode parasitism? Considerable research in this area, in both plant pathology and entomology, suggests that infection of a plant by a minor pathogen, or feeding by a relatively undamaging insect species, may trigger defensive responses in the plant that render it less susceptible to subsequent infection or feeding.

Several questions are suggested: 

These and other questions will provide fruitful avenues for additional research in this area.

References:

Bryant, D. 2005. Carrot research probes foreign cultivars. Western Farm Press, Apr 12, 2005.

Ferris et al (1992) Beyond Pesticides - Biological Approaches to Management in California.

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