Application of Biotechnology For Nematode Control

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Application of Biotechnology for

Nematode Control in Crop Plants
CHAPTER in ADVANCES IN BOTANICAL RESEARCH MARCH 2015
Impact Factor: 1.25 DOI: 10.1016/bs.abr.2014.12.012
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CHAPTER FOURTEEN
Application of Biotechnology for

Nematode Control in Crop Plants
John Fosu-Nyarko*, Michael G.K. Jonesy, 1
*Nemgenix Pty Ltd, WA State Agricultural Biotechnology Centre, Murdoch University, Perth, WA,
Australia
y
School of Veterinary and Life Sciences, WA State Agricultural Biotechnology Centre, Murdoch University,
Perth, WA, Australia
1
Corresponding author: E-mail: [email protected]
Contents
1. Introduction
2. Early Selection for Plants with Nematode Resistance; Susceptibility, Resistance
and Tolerance
3. Biotechnological Approaches to Plant Parasitic Nematode Control
4. Natural Resistance Approach to Plant Parasitic Nematode Control
4.1 Transfer of Natural Resistance Genes to Different Species
5. Transgenic Approaches to Plant Parasitic Nematode Control
5.1 Disruption of Feeding Site Formation or Function
5.2 Overexpression of Host Genes with Modied Expression in Feeding Cells
5.3 RNAi-Based Nematode Resistance
5.4 Differences in Responses to RNAi in Different Nematode Species
5.5 Factors that Affect the Efcacy of RNAi Traits
5.6 Differences in Results between Model and Crop Plants
5.7 Broad Resistance to Different Plant Nematodes
6. Transgenic Technology Advances
7. From the Laboratory to the Market e Commercialization of Plant Parasitic
Nematode-Resistance Traits
7.1 Patenting
7.2 Commercialization Pathway
7.3 The Funding Gap for Early Stages of Commercialization
7.4 The Commercial Value of Nematode Resistance Traits
7.5 Specialist/Small-Scale Commercialization of Nematode Resistance Traits
8. Transgenic Nematode Resistance for Public Good
9. Regulation and Public Acceptance of GM Traits
10. Safety of RNAi-Based Traits
11. Genome-Enabled Development of Novel Chemical Nematicides
12. Ectopic Delivery of dsRNA e Nontransgenic RNAi
13. Other New Nematode Control Agents
14. Conclusions
References
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Advances in Botanical Research, Volume 73

ISSN 0065-2296
http://dx.doi.org/10.1016/bs.abr.2014.12.012
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2015 Elsevier Ltd.

All rights reserved.
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John Fosu-Nyarko and Michael G.K. Jones
Abstract
Effective control of plant parasitic nematodes in crop plants will contribute hundreds of
millions of dollars to global agriculture and help underpin future food security. Natural
nematode resistance genes present in gene pools of crop species and their relatives
have long been exploited with the aim of transferring such traits into economically
important crops where effective resistance is lacking. Biotechnology also contributes
to this process via marker-assisted selection to identify and combine the best nematode
resistance genes, and increasingly in providing new knowledge of target genes, and the
potential to exploit this knowledge using transgenic technology. Thus recent advances
now make it possible to exploit specic aspects of nematode-host plant interactions to
design control strategies that include enabling plants to prevent nematode invasion,
reducing effectiveness of nematode migration through tissues, preventing successful
establishment or reducing feeding ability or nematode fecundity. The knowledge of
what genes are vital for successful nematode parasitism can also be used to develop
new chemical control agents. These new strategies may either be available for public
use or be delivered commercially. For transgenic technologies, both modes of delivery
face the same issues in terms of deployment, such as substantial eld testing, meeting
environmental and human safety regulations, adequate funding to complete statutory
requirements, and public acceptance of GMOs when the product is to be marketed.
However, as technology develops, new strategies for nematode control are emerging,
both for transgenic approaches and in genome editing, which should be regarded by
regulators as a form of mutation rather than genetic modication. With such advances
in biotechnology, the release of commercial varieties of major crops with new forms of
nematode resistance, or new modes of delivery of control agents, is likely to become a
commercial reality. To improve durability, transgenic traits could be based on resistance
with different modes of action: for example, RNAi-based technology combined with
expression of peptides which disrupt sensory activities. Ideally such traits would be
added to existing crop genotypes with the best conventional or natural nematode resistance, to increase the effectiveness and durability of the nematode resistance trait.
Biotech trait expression could also be limited to roots to minimise expression in harvested parts, and this could improve public acceptability.
1. INTRODUCTION
The current status of molecular understanding of nematodeplant interactions is described in earlier chapters in this volume, and it is clear that
rapid advances are being made in unravelling the mechanisms which enable
plant parasitic nematodes to be such successful plant pests. The question
addressed in this chapter is how this new information can be translated to practical application, and used to reduce crop losses caused by these devastating
parasites. If this can be achieved it will be a signicant contribution to future
Application of Biotechnology for Nematode Control in Crop Plants
341
crop security and increased productivity in a sustainable manner. The requirement to nd new ways of controlling plant nematodes is all the more pressing
because some of the older chemical nematicides have been withdrawn or are
now under restricted use mandates: this has led to a renewed interest in developing new strategies to control plant parasitic nematodes based on genetic,
chemical or integrated approaches to manage nematode pests.
The academic advances in knowledge are now impressive, and it is clear
that research of excellent quality is being done to understand nematodeplant
interactions: in particular such research is leading to identifying what effectors
they secrete to be able to avoid or neutralize host plant defences, detect gradients, migrate within roots and, depending on the species, induce the formation
of long-term feeding sites. However, there is still a gap between this basic
research and its practical application to control these pests. As concluded by
McCarter (2009), the future of plant nematology as a discipline is dependent
on the value of commercial solutions delivered to growers. Such advances
are likely to come from both conventional and genetic approaches: McCarter
also emphasized that economically and environmentally sound methods to
control nematodes which contribute a commercial increase in crop yields
will result in more investment in the eld. A summary of the biotechnology-based strategies now available for nematode control, which include
both established breeding technologies and transgenic approaches, is provided
in Table 1, with brief explanations of the strategies and of their current status.
2. EARLY SELECTION FOR PLANTS WITH NEMATODE

RESISTANCE; SUSCEPTIBILITY, RESISTANCE AND
TOLERANCE
The earliest reports of selection for plant resistance to nematodes date
back to the late nineteenth century, and were based on phenotypic selection
for plants which had fewer galls on roots when infected with root knot nematodes. From these selections, varieties of cowpea, sugar beet, cotton and
coffee were reported with improved resistance to root knot nematodes
(Ware, 1936; Webber & Orton, 1902; Wilfarth, 1900).
With a better understanding of nematodeplant interactions, plant nematologists now describe host interactions as compatible when a plant supports
reproduction of the parasite, in which case the host is either susceptible or
tolerant to infestation, and incompatible when the host is resistant to nematodes, and cannot be invaded successfully or only supports very limited or
no growth and reproduction by the parasite. Plants susceptible or resistant to
The introgression and combination of natural

resistance genes, for example from related or wild
species, has been the mainstay of resistance
breeding strategies
Disruption of sensory functions
Wall-degrading enzymes may be required for

migration, e.g. Endoparasites
Positional gradients in roots detected for migration
to the required site in the root
Effectors that enable nematodes to evade or
neutralize host defences
Effectors enable sedentary endoparasites to induce
giant cells and syncytia.
Disrupt feeding site formation, triggered by
nematode-responsive promoter(s)
Major or minor natural resistance

genes
Nematode migration in the

rhizosphere and root entry
Migration in the root
Avoiding host defences
Disruption of feeding site formation

or function
Table 1 Biotechnology-Based Strategies for Nematode Control

Target for Control
Considerations
Marker-assisted breeding for

nematode resistance has become
routine in many breeding
programs, although effective
resistance genes are not available
for all crops
Peptide(s) that inhibit reception of
gradients by amphids
RNAi disruption of amphid
proteins/function
RNAi downregulation of nematode
expression of cell wall-degrading
enzymes
Inhibition of sensing gradients in
roots
RNAi downregulation of expression
of effectors involved in avoiding
host defences
of key effector(s) required for
feeding site formation
Nematode responsive promoter(s)
linked to cell death gene, e.g.
barnase
Status/Example
342
New approaches for genome editing now available
Make use of basic work on nematode effectors and

genes vital for their survival: these can dene new
targets for control
There is a need to develop new more
environmentally friendly forms of chemical
control and delivery, and new forms of biological
control
Delivery of toxic compounds to the

nematodes
Develop new nematicides and

modes of delivery; new biological
control agents
Disrupt expression of genes vital for the nematode

life cycle
Many genes in nematode feeding cells are up-or
downregulated
Modify genes for host plant

susceptibility to nematodes
Overexpression of host genes with

modied expression in feeding
cells
Disrupting vital genes

of vital nematode genes
Overexpression of some host genes
with altered expression in
nematode feeding sites reduce
nematode parasitism
New technologies not necessarily
regarded as genetic modication,
more acceptable in some
jurisdictions
Use bioinformatics lters to identify
new targets for chemical control.
Design new nematicides to these
targets
A series of new nematicides are now
available commercially, based on
biological and chemical control,
separately or in combination, e.g.
using delivery by drip irrigation or
seed coating
343
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nematodes can also exhibit varying degrees of tolerance to infestation, when

they can support a level of nematode infestation without showing severe
symptoms. When applying nematode control strategies, the aim is to reduce
nematode reproduction and thereby the level of infestation, resulting in a
decrease in symptoms of root damage and associated susceptibility to abiotic
stresses and secondary attack by soil pathogens.
3. BIOTECHNOLOGICAL APPROACHES TO PLANT

PARASITIC NEMATODE CONTROL
Research on biotechnological approaches to nematode control
aims either to exploit natural resistance present in gene pools of crop species and their relatives or to employ synthetic forms of resistance, such as
those based on disruption of feeding cells, expression of specic proteins
or peptides, on gene silencing (RNAi) or on delivery of toxic compounds to the invading nematode (Table 1). To exploit natural variation
for resistance, large-scale screening of germplasm is often employed,
together with molecular markers and/or positional cloning to identify
resistance (R) genes or metabolites that confer resistance to particular
nematodes in a wide range of germplasm of crop plants and their wild relatives. Identied sources of resistance are then introgressed into the desired
germplasm. In contrast, transgenic approaches to nematode control exploit
knowledge of nematodehost interactions and can be directed to targeting
the nematode, including disorientating the infective stages to prevent
them from nding host roots, reducing the effectiveness of migration
through host tissues, reducing successful establishment in host cells or
reducing feeding ability and fecundity of nematodes on a susceptible or
tolerant host (Table 1).
4. NATURAL RESISTANCE APPROACH TO PLANT

PARASITIC NEMATODE CONTROL
Effective resistance against plant parasitic nematodes is not available in
all economically important crops. It has been argued that deploying sources
of natural resistance against pests and pathogens is the most cost-effective and
environmentally sustainable method of reducing crop losses resulting from
infection by diseases and pests. It is therefore not surprising that earlier efforts
to control nematodes focussed on using marker-assisted breeding methods
to identify sources of nematode resistance. This usually involves large-scale
345
screening for resistance in germplasm from wild ancestors or progenitors of

cultivars of particular crop plants, mapping of quantitative trait loci, positional cloning and perhaps isolation and characterization of the genes
responsible for conferring resistance. Molecular methods used for mapping
and ne mapping of populations have included RFLPs (Restriction Amplied Length Polymorphisms), AFLPs (Amplied Fragment Length Polymorphisms), RAPDs (Random Amplied Polymorphic DNA), SCAR
(Sequenced Characterised Amplied Regions)- and STS (Sequence Tagged
Site)-based methods, and more recently deep sequencing technologies.
Marker-assisted selection for nematode resistance has been a major focus
to improve crops affected by nematodes. Because cyst nematodes attack
and can cause major losses to most of the worlds important stable crops
including potato, soybean and wheat, it is not surprising that substantial
breeding efforts have been undertaken to identify stable sources of resistance
to different species and pathotypes of cyst nematodes. For example both
polygenic and monogenic genes for resistance to potato cyst nematodes
have been identied and markers closely linked to these alleles have
since been developed for use in potato resistance breeding programmes
(Table 2) (Niew
ohner, Salamini, & Gebhardt, 1995). Similarly, different
types of resistance genes have been identied, mapped and/or cloned
from host plants that confer near complete and partial resistances to Heterodera glycines, Heterodera avenae and Heterodera schachtii including the mapbased cloning of a gene encoding a serine hydroxymethyl transferase, at
the Rhg4 locus, that confers resistance to soybean cyst nematode race 4
(Table 2) (Liu et al., 2012). In Australia, where wheat and barley crops suffer
losses from infestation with the cereal cyst nematode H. avenae, characterized
nematode resistance loci Ha1 and Ha2 (allelic to Ha3) on chromosome 2,
the gene Ha4 (chromosome 5) in barley and the Cre1 locus on chromosome
2B, the Cre3 (Ccn-D1) from Triticum tauschii in wheat, and other Cre genes
have been deployed widely in cereal breeding programmes (Eastwood,
Lagudah, & Appels, 1994; Kretschmer, et al., 1997; Lagudah, Moullet, &
Appels, 1997; Williams, Fisher, & Langridge, 1996).
For root knot nematodes, ve resistant genes have been identied of
which the well-characterized Mi gene, isolated from the wild relative of
tomato, Solanum peruvianum, induces a hypersensitive response on infection
with Meloidogyne spp. (Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria) which results in the death of infective juveniles, and has
been incorporated successfully into many cultivars of tomato (Table 2).
The Mi gene is unique in that it also confers resistance to the potato aphid
Heterodera schachtii
Heterodera avenae
Globodera pallida
Heterodera glycines
Globodera rostochiensis
Cyst Nematodes
Potato
Potato
Tomato
Potato
Soybean
Soybean
Barley
Barley
Wheat
Wheat
Wheat
Sugar beet
Sugar beet
Gro1
H1
Hero
Gpa2
rhg1
Rhg4
Ha2, Ha3
Ha4
Cre1
Cre3
Cre8
Hs1pro1
Hs2
Ballvora et al. (1995), Leister et al. (1996), Kreike

et al. (1993)
Niew
ohner et al. (1995)
Ganal et al. (1995)
van der Voort et al. (1997)
Concibido, Diers, and Arelli (2004)
Webb et al. (1995)
Kretschmer et al. (1997)
Barr et al. (1998)
Eastwood et al. (1994), Williams et al. (1996)
Laguduah et al. (1997)
Lewis et al. (2009), Ogbonnaya et al. (2009)
Cai et al. (1997)
Heller, Schondelmaier, Steinr
ucken, and Jung
(1996)
Table 2 Summary of Natural Resistance Genes to Cyst and Root Knot Nematodes, and Major QTLs Associated with Resistance to
Pratylenchus spp
Crop or Source
Nematode Species
Resistance Genes
of Resistance
References
346
Mi-1
Mi-3 on chromosome 12
Mi-9
Meloidogyne incognita
Examples of QTLs on 2BS, 6DS and 6DL, 6D,

1B, 2B, 3B, 4D, 6D, 7A
Pratylenchus thornei
Thompson, Brennan, Clewett, Sheedy, and

Seymour (1999), Totkay, McIntyre, Nicol,
Ozkan, and Elekcioglu (2006), Schmidt,
McIntyre, Thompson, Seymour, and Liu (2005),
Zwart, Thompson, and Godwin (2005)
Zwart et al. (2005)
Pratylenchus neglectus
QRlnt.lrc-6D.2, QRlnt.lrc-6D.1 Wheat

Examples of QTLs on chromosome 2B, 4DS, 6DS,
7AL, 3, 5, 6, 7H
QRlnn.lrc-4D.l, QRlnn.lrc-6D.l Wheat
Zwart et al. (2005)
Rlnn1 resistance locus
Wheat
Williams et al. (2002)
Pne3H-1, Pne3H-2, Pne5H,
Barley
Sharma et al. (2011)
Pne6H, Pne7H
Pratylenchus penetrans
Rlnn1 resistance locus
Wheat
Williams et al. (2002)
P. neglectus & P. penetrans QTLs on chromosome 1B, 2B Wheat
Toktay et al. (2006)
and 6D
Rlnnp6H resistance on
Barley
Galal et al. (2014)
Chromosome 6H
P. thornei & P. neglectus Xbarc 183 on chromosome 6DS Wheat
Zwart et al. (2005)
Major QTLs Identied on Chromosomes
Garcia, Stalker, Shroeder, and Kochert (1996), Chu

et al. (2011)
Solanum peruvianum Ganal and Tanksley (1996)
S. peruvianum
Yaghoobi, Kaloshian, Wen, and Williamson (1995)
S. peruvianum
Ammiraju, Veremis, Huang, Roberts, and Kaloshian
(2003)
Tomato
Klein-Lankhorst et al. (1991), Messeguer et al.
(1991), Ammiraju et al. (2003)
Pepper
Djian-Caporalino et al. (2007)
Peanut
Root Lesion Nematodes
Me3 on chromosome P9
Mi-1 and Mi-9 on chromosome 6
Mae, Mag, Rma
Meloidogyne arenaria
Root Knot Nematodes
347
348
Macrosiphum euphorbiae and the white y Bemisia tabaci (Nombela, Williamson, & Mu~
niz, 2003; Rossi et al., 1998). Although Pratylenchus species are
often regarded as being less damaging in terms of crop losses, they can be
the most economically important nematode pests in areas of low rainfall
such as the Australian wheatbelt. Not surprisingly, the most detailed
research on breeding for tolerance and resistance to Pratylenchus spp. has
been carried out in Australian cereal breeding programs (Table 2) (Jones
& Fosu-Nyarko, 2014). Genotypes with high tolerance to infestation
with Pratylenchus thornei and medium tolerance to Pratylenchus penetrans
have been identied among wheat cultivars, although they are not necessarily resistant or tolerant to other Pratylenchus species (Smiley & Nicol,
2009). Also major Quantitive Trait Loci (QTLs) for P. thornei, P. penetrans
and Pratylenchus neglectus, some of which have polygenic and additive resistance effects, have been used routinely to select for resistance for these
nematodes in Australian and CIMMYT (International Maize and Wheat
Improvement Center) wheat breeding programs (Table 2) (Williams
et al., 2002).
Study of resistance to Pratylenchus species is also important in barley
because losses caused can also be substantial. To date, ve QTL loci contributing to resistance to P. neglectus in barley germplasm have been identied on
chromosomes 3H, 5H, 6H and 7H, and these may be useful for markerassisted selection for resistance in barley (Table 2) (Sharma et al., 2011).
Although the resistance conferred by some of these genes is useful in
improving resistance to Pratylenchus species in commercial crop varieties,
there is still a need to identify new, more effective and durable sources of
natural resistance to nematodes in most major crop species.
4.1 Transfer of Natural Resistance Genes to Different Species

A major aim of identifying nematode resistance genes is to introduce them
into other susceptible crops of economic importance, to enhance crop yield
and quality and, where relevant, to reduce costs and reliance on chemical
nematicides. While there has been successful deployment of crops with a
series of nematode resistance genes (e.g. tomato cultivars with the Mi
gene, potato cultivars with the H1 gene), there have been few reports of
successful transfer of characterized R genes into new species. It appears
that the efcacy of these genes in heterologous systems is genotype and/
or species dependent and may require several elements for effective signalling in the pathways that induce a hypersensitive response, and the required
interactions with proteins may not be present in a different species. For
349
example, transfer of the Mi gene to eggplant confers resistance to M. javanica

but not to the potato aphid, M. euphorbiae. Similarly, the transfer of the tomato Hero A gene into tomato cultivars confers desirable levels of resistance
to potato cyst nematode in tomato, but not in potato (Sobczak et al., 2005).
Even in tomato cultivars carrying the Mi gene there is variation in resistance
to M. incognita attributed to their genotypic background (Jacquet et al.,
2005). A better understanding of the mechanisms of nematode resistance
offered by this group of Nucleotide Binding Site - Leucine Rich Repeat
(NBS-LRR) class of plant R genes should make their introduction into
other commercial crops more effective.
5. TRANSGENIC APPROACHES TO PLANT PARASITIC

NEMATODE CONTROL
5.1 Disruption of Feeding Site Formation or Function
Since the discovery that reproduction of sedentary endoparasitic nematodes (Heterodera spp., Gobodera spp., Meloidogyne spp., Rotylenchulus spp.,
Nacobbus spp. and Tylenchulus spp.) depends on successful formation and
function of giant cells, syncytia or similarly modied host cells (Jones,
1981), strategies which can disrupt feeding site formation have been investigated. RNAi-based methods that target the nematodes ability to induce
feeding sites are discussed below: here we consider plant processes involved
in feeding site formation and function. Success with this type of approach
very much depends on identifying plant promoters which are specically
or highly upregulated in feeding cells, and which can be linked to expression
of a cytotoxic gene which when expressed in feeding cells results in cell
death or impairment. The rst example of this approach was by Opperman,
Taylor, and Conkling (1994), who reported that the truncated (D0.3 kb)
promoter of the water channel protein TobRB7 was expressed specically
in root knot giant cells, and when linked to the cytotoxic ribonuclease barnase resulted in cell death. However, unintended or leaky expression of
such a cytotoxic gene in other cells is a serious drawback to this approach.
Even when combined with constitutive expression of the gene barstar which
can neutralize the activity of barnase (Sijmons, Atkinson, & Wyss, 1994), unless it is highly upregulated there is danger of unintended side effects on the
plant. Although a series of genes highly upregulated or downregulated in
nematode feeding cells have since been identied, such as the heat shock
promoter Hahsp17.7G4 (Escobar et al., 2003), it appears that none of these
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promoters alone is sufciently tightly expressed in the feeding cells to link

them to a cytotoxic gene without collateral damage elsewhere in the plant.
An alternative approach, based on using two nematode responsive promoters, both of which must be upregulated in nematode feeding cells for
expression of a cytotoxic gene to occur, may overcome this issue of target cell
specicity of expression (Wang, Shuie, & Jones, 2008 and unpublished data).
5.2 Overexpression of Host Genes with Modied Expression

in Feeding Cells
For a substantial time it had been predicted that there would be many
changes in the metabolism of giant cells, syncytia and other feeding cells
induced in hosts by endoparasitic nematodes (Jones, 1981). As technology
advanced it has become possible to analyze changes in patterns of expression
of genes in nematode feeding cells in ever greater detail, for example by differential display, microaspiration of feeding cell contents, laser microdissection and capture, microarrays and making use of new deep sequencing
technologies (e.g. Alkhaouf et al., 2006; Fosu-Nyarko, Jones, & Wang,
2009; Ibrahim et al., 2011; Ramsay, Wang, & Jones, 2004; Wang, Potter,
& Jones, 2003; Szakasits et al., 2009; Barcala et al., 2010; Portillo et al.,
2013). Much of the research has focused on syncytia induced in soybean
by H. glycines because of the economic importance of this nematode.
Matthews et al. (2012) selected 100 soybean genes with expression modied
in syncytia, identied using microarrays, for overexpression in a composite
hairy root soybean system. Of these, nine reduced the number of females
by 50% or more when overexpressed; conversely some enhanced the number of females. The challenge here is that the genes overexpressed would be
expected to play a role in normal plant metabolism, and so overexpression
may well confer a level of nematode resistance, but there is the risk that
in a eld situation an abnormal phenotype or reduced yield may result. It
may be possible to choose a target gene whose high expression is vital for
feeding site formation or metabolic function, but select a level of modied
expression which interferes with feeding cell formation without adversely
affecting any other parameter of plant growth.
5.3 RNAi-Based Nematode Resistance

Since the discovery of RNAi in nematodes, the potential to develop plants
which produced double-stranded RNA to nematode target genes and so to
silence expression of genes vital for their development or infection processes
has been proposed as a sustainable, environmentally friendly strategy to add
351
to current methods used for nematode control (Fire et al., 1998; Tan, Jones, &
Fosu-Nyarko, 2013; Urwin, Lilley, & Atkinson, 2002). The rst question
was how to make plant parasitic nematodes take up dsRNA from external
solutions, and this was solved by the pioneering work of Urwin et al.
(2002), who showed that upon addition of neurostimulants to the soaking
solution for H. glycines J2s they could be induced to take up sufcient dsRNA
to induce RNAi. Since that time, dsRNA feeding/soaking has been used to
assess the effects of downregulation of over 30 essential and parasitism genes
of various plant nematode species, including cyst nematodes (H. glycines, H.
schachtii, Gobodera pallida, Gobodera rostochiensis), root knot nematodes (M.
incognita, M. javanica, Meloidogyne hapla, M. arenaria and Meloidogyne artiellia),
root lesion nematodes (Pratylenchus zeae, P. thornei, Pratylenchus coffeae) and
other ectoparasitic nematodes (Radopholus similis, and Bursaphelenchus xylophilus) (Joseph, Gheysen, & Subramaniam, 2012; Li, Todd, Oakley, Lee, &
Trick, 2011; Lilley, Bakhetia, Charlton, & Urwin, 2007; Tan et al., 2013)
(Reviewed for RKNs in chapter Function of Root-Knot Nematode Effectors and Their Targets in Plant Parasitism). However, it has since been
demonstrated that neurostimulants and other chemicals are not necessarily
needed to induce RNAi using dsRNA (e.g. Fanelli, Di, Jones, & Giorgi,
2005; Kimber et al., 2007). In H. glycines and for some Pratylenchus spp, it appears that, for some genes at least, silencing resulting from soaking in dsRNA
does not always produce stable phenotypic effects, since it appears that the
effects of RNAi can wear off hours or days after the initial effect, leading
to nematode recovery or regaining of function. Nevertheless the soaking
method has been shown to be an effective method for initial screening of
gene function and for discovery of candidate target genes suitable for
plant-delivered RNAi for nematode control.
In contrast to soaking plant nematodes in solutions containing dsRNA,
host (in planta) delivery provides dsRNA continuously if expressed in host
cells from a constitutive promoter. This mode of delivery of dsRNA appears
to be an ideal and economical approach to control obligate parasites such as
plant parasitic nematodes. In planta delivery of dsRNA of two target genes
(an integrase and a pre-mRNA splicing factor) was rst demonstrated by
Yadav, Veluthambi, and Subramaniam (2006) to reduce replication of M.
incognita on transgenic tobacco plants, and this was quickly followed by
the work of Huang, Allen, Davis, Baum, and Hussey (2006) who expressed
dsRNA to an M. incognita effector protein in transgenic plants and also
showed reduced nematode reproduction. Since then a series of experimental and crop plants have been engineered to generate inverted repeats
Meloidogyne
incognita
Tomato
Grapes
Tomato
Soybean
Troponin C (tnc)
Secreted peptide (16D10)
Calreticulin (crt)
L-Lactate
Mitochondrial stress-70 protein
Soybean
Arabidopsis
dehydrogenase
Yadav et al. (2006)
>90% reduction in established

nematodes
69e83% reduction in the number of
eggs per gram root, >63% reduction
in galls and gall size
59% reduction in hatching rate of J2s
General reduction in number of eggs
per gram of hairy root
J2s recovered from silenced progeny
induces up to 84% fewer galls
57% reduction in galls per plant root,
77% reduction in RNAi nematode
diameter
diameter
Tobacco
Ibrahim et al. (2011)
Dubreuil et al. (2009)
Dubreuil et al. (2009)

Yang et al. (2013)
Huang et al. (2006)
Yadav et al. (2006)
>90% reduction in established

nematodes
Tobacco
SNF (Sucrose Non Fermentable)

chromatin remodelling
Complex component (snfc-5)
Pre-mRNA splicing factor (prp-21)
Root Knot Nematodes
Table 3 Host-Delivered RNAi for Parasitism and Essential Genes of Cyst and Root Knot Nematodes: Summary of Reduced Infectivity on
Model and Crop Plants
Nematode
Gene Silenced
Plant/Crop
Major Phenotype
References
352
Arabidopsis
Potato
Meloidogyne
hapla
Meloidogyne
chitwoodi
Arabidopsis
Arabidopsis
Arabidopsis
Meloidogyne
arenaria
Tomato
Signal peptidase complex 3

Arabidopsis
Tomato
Dual oxidase
Nematode effector protein

(NULG1a)
Soybean
Tyrosine phosphatase
Meloidogyne
javanica
Soybean
ATP synthase beta-chain

mitochondrial precursor

diameter
diameter
52% reduction in saccate nematodes,
61% reduction in total nematodes
63% reduction in saccate nematodes,
52% reduction in total nematodes
Up to 88% reduction in number of
nematodes in roots
57% and 67% reduction in egg masses
and eggs
71% and 63% reduction in egg masses
and eggs
(Continued)
Dinh et al. (2014)
Dinh et al. (2014)
Huang et al. (2006)
Huang et al. (2006)
Huang et al. (2006)
Lin et al. (2013)
Charlton et al. (2010)
Charlton et al. (2010)
353
Heterodera
schachtii
Heterodera
glycines
Up to 68% reduction in female cysts
87% reduction in female cysts
79% reduction in nematode
95% reduction in nematode
23e64% reduction in developing
females
12e47% reduction in developing
females
>50% reduction in developing females
42% reduction in developing females
Soybean
Soybean
Soybean
Soybean
Soybean
Soybean
Soybean
Soybean
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Major sperm protein

Ribosomal protein 3a (rps-3a)
Ribosomal protein 4 (rps-4)
Spliceosomal SR protein (spk-1)
Synaptobrevin (snb-1)
Beta subunit of the COPI complex
(Y25)
Pre-mRNA splicing factor (prp-17)
Uncharacterized protein (cpn-1)
Ubiquitin-like protein (4G06)
Cellulose binding protein (3B05)
SKP1-like protein (8H07)

Zinc nger protein (10A06)
Nematode secreted peptide, Hssyv46
Nematode secreted peptide, Hs5d08
Nematode secreted peptide, Hs4e02
Nematode secreted peptide, Hs4F01
30C02 effector protein
Cyst Nematodes
Sindhu et al. (2009)

Patel et al. (2008)
Patel et al. (2008)
Patel et al. (2008)
Patel et al. (2008)
Hamamouch
et al. (2012)
Li et al. (2010)
Li et al. (2010)
Steeves et al. (2006)

Klink et al. (2009)
Klink et al. (2009)
Klink et al. (2009)
Klink et al. (2009)
Li et al. (2010)
Table 3 Host-Delivered RNAi for Parasitism and Essential Genes of Cyst and Root Knot Nematodes: Summary of Reduced Infectivity on
Model and Crop Plantsdcont'd
Nematode
Gene Silenced
Plant/Crop
Major Phenotype
References
354
355
of dsRNA targeting genes expressed in pharyngeal gland cells or those

essential for development and reproduction in cyst and root knot nematodes
(Table 3). To date, reports of signicant reductions in the number of females
of soybean cyst nematode (8193%) and eggs (6895%) produced by female
cysts developing on hairy roots or composite transgenic soybean expressing
dsRNA of genes involved in RNA and protein synthesis provide a level of
condence that RNAi can be an important tool for nematode control
(Klink et al., .2009; Li, Todd, Oakley, Lee, & Trick, 2010; Li et al.,
2011; Steeves, Todd, Essig, & Trick, 2006).
5.4 Differences in Responses to RNAi in Different Nematode

Species
Depending on the target gene and the experimental procedures used (e.g.
model or crop plant species and genotype, target gene silenced, dsRNA sequences used, number of events generated and studied, the methods and
nematode genotypes used for screening, quantifying and analysis of results),
there is a wide range of reports of the efcacy of RNAi when used to reduce
nematode reproduction. As a general observation it would appear that Meloidogyne spp. are more susceptible to RNAi than Heterodera/Gobodera species,
but with more limited data from Pratylenchus species it would appear that
these are highly amenable to control by RNAi. For example, high levels
of resistance were reported in tobacco and Arabidopsis producing dsRNA
to genes of root knot nematodes, including the parasitism gene 16D10, a
gene expressed in the subventral gland cells of M. incognita, a pre-mRNA
splicing factor and an integrase gene: their expression resulted in an inability
of >90% for J2s to establish feeding sites (Yadav et al., 2006). (RNAi of the
M. incognita 16D10 gene also confers resistance to transgenic Arabidopsis
infected with four other Meloidogyne species: M. hapla, M. javanica, M. chitwoodi and M. arenaria, and RNAi of this gene has since been demonstrated
to provide a level of resistance to several important crops such as grapes and
potato (Dinh, Brown, & Elling, 2014; Huang et al., 2006; Yang et al.,
2013)).
There may also be differences in the effectiveness of this approach between species of the same genus: in some publications it seems that in planta
RNAi of H. schachtii may not be as effective as for H. glycines, because only a
1264% reduction in female nematodes (except for the 92% reported for
30C02 effector protein) was observed when genes encoding putative
secreted effector proteins, a ubiquitin-like gene, and those of a cellulose
binding protein, SKP1, and a zinc nger protein were silenced (Patel
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et al., 2008; Sindhu et al., 2009; Patel et al., 2010; Hamamouch et al., 2012).
However, silencing seven genes (elongation factor 1a, two vacuolar H
ATPases, integrase, pre-mRNA splicing factor, troponin C and tropomyosin) of H. schachtii via transgenic Arabidopsis resulted in up to 98% reduction
in adult females (Fosu-Nyarko & Jones, 2013, 2014).
The differences in effectiveness of RNAi may relate to differences in
biology of hostparasite interactions such as presence or absence of feeding
tubes: Meloidogyne feeding tubes appear to be larger and more regular in
structure than those formed by feeding cyst nematodes, and this may inuence the ability of uptake of dsRNAs, whereas Pratylenchus species do not
form feeding tubes. Alternatively there may be fundamental differences in
RNAi pathways in different nematodes, or in systemic movement of siRNAs in the nematodes. The reasons for such differences, or whether current
reports reect more differences in experimental procedures used, remain to
be demonstrated experimentally.
5.5 Factors that Affect the Efcacy of RNAi Traits

Since there are now many examples in which RNAi has been used to
confer varying degrees of resistance to root knot and cyst nematodes
(Table 3), it is important to consider what factors inuence the effectiveness
of this strategy. The rst factor is choice of target gene is it an effector vital
for successful parasitism, or a gene whose expression is vital for completing
some aspect of the nematodes life cycle? Other considerations are the
length of dsRNA used, the specic sequence chosen, whether the target
gene is a member of a multigene family, and whether there are compensating pathways for loss of a particular function. In terms of acceptability,
the target sequence chosen should preferably be unique to the nematode
specie(s) of interest, or at least not be present in mammals, benecial organisms and all non-target species for which sequence data are available. The
shorter the sequence chosen, the less chance there is of off-target effects,
and so the use of an articial miRNA vector, in which only 2024 bases
of target sequence may be used, should reduce possible off-target effects
(but the most effective sequence from the target gene should then be
used). Even when these selection criteria are applied, it seems that most
transgenic RNAi experiments give varying levels of effectiveness, with
none 100% effective. This observation may reect variability in the populations of target nematode species rather than efcacy of RNAi per se. In
any case, it is well known that, depending on the site of transgene insertion,
copy number, promoter strength and construct design, any set of transgenic
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plant events will exhibit a range of the desired property, and only the most
effective events will be chosen for progression through a commercialization
pipeline.
5.6 Differences in Results between Model and Crop Plants

Another factor that is often overlooked is the difference between using
model experimental plants such as Arabidopsis for nematode challenge experiments compared to crop plants. Model plants such as Arabidopsis have not
been selected for their resistance to plant nematodes, and so are likely to
be more susceptible, whereas in many cases crop varieties have been selected
for resistance or tolerance to nematodes, even if only partial. As a result,
promising results from model species do not always map over to crop species, since the percentage improvement in nematode resistance is that
conferred over and above the selected resistance, rather than against a highly
susceptible host.
5.7 Broad Resistance to Different Plant Nematodes

One of the attractions of developing transgenic resistance to plant nematodes
using RNAi technology is the potential to confer broader resistance to
several species in one construct, in contrast to the more specic resistance
conferred by natural resistance genes. The principle is that hairpin dsRNAs
to a number of different target genes can be made either from the same or
different species, or to target different populations of the same nematode
species. When P. thornei and P. zeae were soaked in dsRNA sequences of
two target genes from each species, there was a reduction in subsequent
reproduction on carrot discs irrespective of the target gene source (Tan
et al., 2013). However, so far there are no convincing reports from transgenic in planta experiments that two different nematode species can be
controlled with one hybrid dsRNA construct (Charlton et al., 2010).
Perhaps the RNAi mechanism can be overwhelmed if too many siRNAs
are generated, and with more subtle expression or choice of target sequence
the potential for broad resistance to different nematode species may be
achieved.
6. TRANSGENIC TECHNOLOGY ADVANCES

There continue to be advances in the technology of genetic modication (GM) which challenge the current regulatory denitions of a
358
genetically modied organism, because it is not always clear whether the

products obtained using these techniques are subject to the prevailing GM
legislation or not (Breyer et al., 2009). Examples of new technologies or
concepts include:
Cisgenesis this involves introduction of DNA from the same or a
compatible species
RNAi downregulation of expression of existing genes
Reverse breeding
Genome editing directed mutation or precision gene editing
introduction of targeted changes to nucleotides in the genome, such
as oligonucleotide-mediated mutagenesis (e.g. Cibus), zinc nger/
designer nucleases (ZFNs) gene disruption or precise insertion of
DNA sequences (e.g. EXACT ), CRISPR-Cas systems (clustered
regularly interspaced short palindromic repeats)
Virus-delivered ZFN genome editing
Epigenetics induced differentially methylated regions
Agroinfection
Virus-induced gene silencing
Genomics-enabled technologies, e.g. ectopic delivery of dsRNA (e.g.
Biodirect Technology)
Grafting nontransgenic scions onto GM rootstocks (e.g. for vines or fruit
trees)
With advances in biotechnology, new techniques of genome editing
have emerged, that is, the ability to make tailored changes to a genome
sequence. These techniques can enable modication of expression of existing genes or introduction of targeted changes to nucleotides in the genome
(e.g. oligonucleotide-mediated mutagenesis). Such techniques began with
methods based on ZFN to dene their binding site on a DNA sequence,
linked to Fok1 endonuclease to generate double-stranded breaks in the
DNA at the dened sequence. DNA repair mechanisms are then recruited
either by nonhomologous end joining pathways or homologous repair pathways, to generate mutations or insert exogenously supplied sequences with
anking sequences homologous to the insert (Lozano-Juste & Cutler, 2014).
Tailored ZFNs are expensive to make, and other developments such as transcription activator-like effectors (TALEs; DNA-binding proteins produced
and secreted by plant pathogens into plant cells, which bind specic
DNA sequences and alter transcription on endogenous genes) are easier to
modify. TALEs have many copies of a 3335 amino acid repeat, with
DNA recognition dependent on two variable amino acids in the repeats,
359
and can be modied with addition of nuclease to make TALE nucleases

(TALENs), that can cut DNA at specic sites. The CRISPR/Cas9 system
has been developed which is technically simpler to use for genome editing
(Lozano-Juste & Cutler, 2014). Its advantage over ZFNs and TALENs is that
it uses synthetic guide-RNAs (gRNAs) rather than synthetic DNA-binding
domains, to dene the cleavage site. The CRISPR/Cas9 system is based on
a bacterial antiviral and transcriptional regulation system, modied such that
two RNA components have been combined into a single gRNA which is
transcribed from a construct containing a user-dened target sequence of 20
nucleotides complementary to the desired target sequence. Guided by the
gRNA, the Cas9 nuclease protein binds and nicks the dened sequence,
which as above, can be modied by nonhomologous end joining or homologous repair pathways, to generate mutations or insert exogenously supplied
sequences. This approach has been used to generate transgenic Arabidopsis
thaliana plants with mutations in the PDS (phytoene desaturase) locus (Nekrasov, Staskawicz, Weigel, Jones, & Kamoun, 2013), although using standard Agrobacterium transformation and selection. These developing
techniques of gene editing could be used to modify host plant genes to
confer nematode resistance, for example by disrupting or modifying expression of genes vital for feeding site formation for sedentary endoparasites.
However, there is still some uncertainty about the extent of off-target effects
for this technology.
7. FROM THE LABORATORY TO THE MARKET

COMMERCIALIZATION OF PLANT PARASITIC
NEMATODE-RESISTANCE TRAITS
7.1 Patenting
Before a nematode resistance or other trait can be commercialized,
unless the information is for public good and free use, the trait needs to
be protected by patenting. In an academic situation the approach is usually
to submit a provisional patent via the Universitys Commercialization Ofce. There is 1 year to provide additional supporting data if needed before
full patent application at which stage the costs increase substantially. In this
period the Commercialization Ofce usually looks for an industry partner
who is interested in taking on the costs of full patenting and may provide
additional funds, in return for rst use in licensing and exploiting the trait.
An alternative strategy is to establish a company to raise funds for further
development of the trait, and be responsible for patenting and licensing
360
one or more traits. In the latter case, company investors will seek a way to
recoup their investment (an exit strategy), either by trade sale to a larger
company, by public listing or by raising further investment to exploit the
trait directly. The further the product is progressed along the developmental
pathway, the higher the expected returns will be.
7.2 Commercialization Pathway

An overview of the pathway to commercialization of a biotechnology trait
conferring nematode resistance is provided in Figure 1. From an initial idea
the basic discovery research is undertaken, and if that is promising it moves
from the discovery phase to proof-of-concept, then early and advanced
stages of product development, to a prelaunch phase, and nally to commercial release to growers. The activities to be undertaken in each phase are
indicated in Figure 1, as well as indicative timescales and the probability
of success in progressing to commercial release. Basic discovery research is
more the realm of public research organizations such as universities and government-funded research institutes, but these are often viewed as poor at
commercialization activities. As a result the discovery or trait moves along
the pipeline often via a start-up or expansion stage company, and at
some stage for biotech traits, the trait is either licensed to a large corporation
or multinational company or the company is bought by such companies for
advanced development, prelaunch and commercial release to growers.
Figure 1 Pathway to commercialization of a biotechnology trait conferring resistance

to plant parasitic nematodes.
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As a product moves along the pipeline the costs of development and

commercialization increase, and it is for this reason that most biotech traits
for large-scale commodity world crops (e.g. soybean, corn, cotton, canola;
possibly wheat and rice in the future), such as nematode resistance, are
necessarily deployed by multinational corporations. Increasingly new traits
will be stacked with other biotech traits, requiring coordination of their
development and introduction into the best available germplasm for a
particular crop.
To estimate the costs of trait deployment, a recent consultancy study by
Phillips McDougall for Crop Life International (September 2011) was undertaken on The cost and time involved in the discovery, development
and authorization of a new plant biotechnology trait. It was based on the
responses of the following multinational companies: BASF, Bayer CropScience, Dow AgroSciences, DuPont/Pioneer Hi-Bred, Monsanto Company and Syngenta AG on costs involved in introducing a new GM crop
trait over the period 20082012. The costs reported are shown in Table 4.
The study revealed that the mean cost associated with the discovery,
development and authorization of a new biotechnology-derived crop trait
introduced in the 20082012 timeframe, including associated international
market approvals required for a grain crop to enter the global grain trade,
was US$136.0 million. However, with deregulation of traits and possible
relaxation of safety and environmental testing based on history of safe usage,
the cost of trait deployment is likely to decrease in the future. Among other
ndings was that the mean time taken for all crops from initial research and
development until commercial sales was 13.1 years.
Table 4 The Cost Involved in Various Stages of Development of a Biotechnology

Trait for Grain Crops
Category
Cost ($ million) No of Responses
Discovery
Early discovery
Late discovery
Total cost
Construct optimization
Commercial Event production & selection
Introgression Breeding & Wide Area Testing
Regulatory Science
Regulatory & Regulatory Affairs
Total
17.6
13.4
31.0
28.3
13.6
28.0
17.9
17.2
136.0
5
5
5
5
6
6
6
6
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Figure 2 Stages and sources of funding for technology commercialization.
7.3 The Funding Gap for Early Stages of Commercialization

A major problem for early stage commercialization of discoveries is known
as the funding gap (Figure 2), and this is particularly relevant to translation
of research from universities. The basic research may be funded by a variety
of government or competitive grants, but without good proof-of-concept
data there is a funding gap in which commercialization of university research
is lacking. However, once that gap is bridged, with suitable evidence of
efcacy, commercial investment is more readily available.
7.4 The Commercial Value of Nematode Resistance Traits

The commercial value of a biotech trait conferring nematode resistance depends on many factors, and on a case-by-case basis the following aspects
need to be considered: the value of the crop, where it is grown (this may
be limited by the robustness of intellectual property (IP) and patenting regimes in a particular jurisdiction), the area grown, the percentage of that
area that is affected by nematodes, the extent of losses caused by specic
nematodes, the degree of protection provided by the biotech trait, the
mode of delivery, the cost of alternative methods of control, the addressable
market, the expected time course of uptake and percentage of the market
that can be accessed, the cost of meeting regulatory requirements and the
added value provided by the trait. Since a nematode-resistance trait will
be delivered via appropriate germplasm, the main value of the germplasm
containing the trait will go to the breeders and seed marketers as is standard,
with the technology developers of the trait receiving a small percentage of
the overall value of seed sales based on the value added by the trait. This
363
may be in the order of 15%, depending on the factors to be considered

indicated above, but in the future may be less, if a nematode-resistance trait
is delivered as one of a set of stacked biotech traits. Conversely, a biotech
nematode resistance trait could differentiate one variety of seeds for a crop
from those of another supplier that lacks them, and so have a greater value
as a result of improved seed marketing.
7.5 Specialist/Small-Scale Commercialization of Nematode

Resistance Traits
The costs of deploying a trait in a major grain crop may appear daunting, but
there are small companies, such as the Canadian biotech company, Okanagan Specialty Fruits (www.okspecialtyfruits.com), which is developing
transgenic fruit tree products themselves, at a fraction of the cost indicated
above for major grain crops. Okanagan Specialty Fruits are using RNAi
technology to downregulate expression of a polyphenol oxidase (PPO)
gene in apples to reduce browning. The company has applied for regulatory
approval in the United States and Canada for commercial growth of two
GM apple varieties (Arctic Granny and Arctic Golden). This company
is also using transient gene silencing to modify the expression of genes in
existing apple cultivars: they argue that Transient gene silencing is not genetic modication in the traditional sense. What this means is that they are
developing RNAi-based transgenic apple rootstocks, modied to suppress
the expression of PPO, on which commercial cultivars of apple are grafted.
They state that the trait can be transferred from the donor to the recipient
though small interfering RNA (siRNA) that migrates through the plant.
The result is the production of nonbrowning fruit on the nontransgenic
recipient. They suggest that transient gene silencing of PPO in apples produced on the scion can be triggered in the right conditions.
The strategy of using transgenic rootstocks grafted to nontransgenic scions
is clearly relevant to nematode control, in which the transgenic rootstock is
nematode resistant, whereas the produce harvested from the scion is not.
8. TRANSGENIC NEMATODE RESISTANCE FOR PUBLIC

GOOD
The alternative to commercial deployment of a nematode resistance
trait is to bypass all the issues of IP and costs of patenting a trait, and to provide it for public good, often funded by overseas philanthropic organizations. Nevertheless, signicant funds are still needed to meet safety,
364
human health, environmental and other regulatory requirements before

deployment. One such example of this approach is the work of Atkinson
and colleagues (Atkinson, Lilley, & Urwin, 2012; Atkinson 2014 personal
communication). They employed two biotech approaches to develop
nematode resistance in plants overexpression of cysteine proteinase inhibitors (cystatins) which interfere with intestinal digestion of dietary protein ingested from the plant, and synthetic peptides expressed or secreted
from roots which interfere with nematode chemoreception by binding
to either acetylcholinesterase or nicotinic acetylcholine receptors, which
are both targets in the nematode cholinergic nervous system. The peptide
inhibits nematode chemoreception after uptake by chemosensory sensilla
in the amphid pouches and transports along chemoreceptive neurons to
their cell bodies.
Plant parasitic nematodes cause an average of 12.75% losses to ve staple
crops in Africa (maize, sugarcane, banana and plantain, yam and cassava)
(FAOSTAT, 2012), with particular losses in banana and plantain (up to
70%) in some regions. In the latter case several nematode species are responsible: control is inadequate, nematicides are not appropriate due to cost and
hazards of application. In eld experiments with transgenic plantain events
expressing the 7-mer repellent peptide and/or the cystatin protease inhibitor,
promising resistance has been obtained (8998% reduction in nematodes
recovered), especially for plants expressing the repellent peptide (Tripathi,
Roderick, Babirye, & Atkinson, 2014). Biosafety assessments accompanying
this work indicate safety based on the fact that cystatin is a normal part of the
human diet, is not allergenic and is rapidly degraded by gastric juices: the peptide is too small to be allergenic and is degraded in the human small intestine.
Regarding environmental safety, no adverse effects of cystatin expression
were evident on the range of non-target species tested, the peptide is rapidly
degraded in the soil and does not affect the soil microora or other non-target
species tested. There is therefore no evidence for safety concerns: in addition
the expression of the cystatin and peptide genes could be driven by a rootspecic promoter, limiting their presence to the roots.
In discussing de-regulated release of such nematode-resistant crops,
Atkinson et al. (2012), note that countries with future food security concerns are most likely to adopt transgenic resistance, particularly for crops
like cooking bananas, plantains or yams which cannot be improved readily
using other approaches. For wider acceptance, effective policies must be
developed to engage consumers and the food industry as well as growers
(farmers).
365
9. REGULATION AND PUBLIC ACCEPTANCE OF GM

TRAITS
The issues involving regulation and acceptance of GM crops are well
known, and some of the developing technologies and concepts have the potential to improve public acceptance. For example, there have been a number of
publications arguing for the exemption of cisgenic plants from the scope of
GM regulations (e.g. Jacobsen & Schouten, 2008). In the current debate on
how regulators may deal with newer GM technologies, directed mutation
may be treated like mutagenesis, treatment of cisgenic plants depends on the
method of transformation, treatment of non-GM grafts on GM rootstocks depends on the safety assessment of the GM rootstock; reverse breeding and agroinoculation may be non-GM or exempt (J Dunlop, University of Reading,
personal communication). The regulatory and acceptance aspects of applying
new technologies to nematode control is very important when considering trait
commercialization, since the complexities of regulations and public opinion
affect the cost of deployment. If it is too expensive in relation to the
added value of the trait, then commercial deployment may not be undertaken.
Of particular relevance to nematode control, using RNAi technology,
inserted DNA does not encode message for a functional protein, and so
should be in a lower risk category. Extending this to transgenic rootstocks
with a nematode resistance trait (e.g. RNAi), with harvested produce
from a nontransgenic scion, any risk factors are again reduced. However,
in France vines with transgenic rootstocks were destroyed, in contrast, in
Canada as discussed above, Artic Apples, grown on transgenic rootstocks,
are close to commercialization, and the potential movement of siRNAs
from rootstock to scion needs to be considered.
10. SAFETY OF RNAi-BASED TRAITS

Since RNAi technology has been used widely in functional genomics
studies of nematode effectors or vital genes, and is a potential route for
commercialization of research ndings, the safety of food and feed with
RNAi-based traits needs particular attention. A review on this subject
relating to human and animal health (Petrick, Brower-Toland, Jackson, &
Kier, 2013) considered these aspects in relation to molecular mediators of
RNAi long dsRNAs, small interfering RNAs (siRNAs) and micro
RNAs (miRNAs). They reviewed available data including that on comparative safety assessments, mice fed on wheat with RNAi-mediated traits, the
366
fundamental differences between biotech crops expressing heterologous

proteins and those with RNAi-mediated gene suppression cassettes, the
potential for unintended effects, a long safe history of ingestion of naturally
occurring dsRNAs in plants and foods, reports of plant-derived miRNA in
mice after oral ingestion (Zhang et al., 2012), mammalian and human studies
on siRNA uptake, RNA molecule specicity, half-life, secretion and the
barriers to oral ingestion. They concluded that available data strongly support the conclusion that biotechnology-derived crops employing RNAmediated gene regulation are safe for human and animal consumption.
11. GENOME-ENABLED DEVELOPMENT OF NOVEL

CHEMICAL NEMATICIDES
As has been discussed by Jones and Fosu-Nyarko (2014) in the short to
medium term at least, it is unlikely that a transgenic or equivalent biotechnology-based approach can be deployed to protect all commonly grown
crops from nematode attack. This view is based both on the costs of developing and implementing such approaches, and public acceptance considerations. Nevertheless, information on genes whose products are vital for
different processes of nematode root location, invasion, host defence
evasion, general metabolic and developmental processes, and feeding or
feeding site formation, can be used to inform the development of new, environmentally friendly nematicides (Danchin et al., 2013). The approach was
to use a bioinformatics pipeline to lter potential gene targets based on
genomic information from M. incognita. It involved the application of a stepwise set of rigorous criteria in which all the genes present in the genome, of
known or unknown function, were assessed, for example to reduce the possibility of off-target effects and sequences potentially common to non-target
organisms, candidates from multigene families and known effectors with
deleterious RNAi phenotypes. This strategy led to a shortlist of high-quality
target genes, which had the potential to serve as leads for development of
new chemical nematicides. Functional analysis was in the form of feeding
experiments in vitro, in which siRNAs designed to target each candidate
gene was assessed for its effect on phenotype or ability of the nematode to
infect host roots. Once appropriate vital nematode target genes have been
identied, then targeted development or screening for chemicals which
can inhibit such functions can be undertaken to develop novel nematicides.
367
12. ECTOPIC DELIVERY OF dsRNA NONTRANSGENIC

RNAi
Using a similar approach to identify target genes for nematode control,
there is also the exciting possibility of using ectopic delivery of dsRNA (i.e.
by spraying) onto plants. This strategy is being followed in the development
of BioDirect Technology as a nontransgenic alternative mode of delivery
of RNAi for crop protection against herbicides, insects and viruses (http://
www.monsanto.com/investors/documents/whistle%20stop%20tour%20vi
%20-%20aug%202012/wst-biodirect_posters.pdf). The challenge here for
nematode control is to develop stable forms of dsRNA and their mode of
delivery, such that they are taken up systemically when sprayed onto crop
plant leaves, and move via the plant vasculature to the roots where they
can be ingested by nematodes when feeding, resulting in inhibition of a vital
process and control of the nematodes.
13. OTHER NEW NEMATODE CONTROL AGENTS

With the loss or reduced use mandates of more than two-thirds of the
classical nematicides, because of their toxicity, persistence in the environment or capacity to deplete the ozone layer, industry has been active in
developing the next generation of nematode control agents. These are
mainly targeted at horticultural crops, since a genetic/biotech strategy is
more likely to be developed for major broadacre crops such as soybean,
corn and cotton. Although there is substantial literature on the search for
new phytochemical control agents (Chitwood, 2002; Taylor, Belton,
Beta, & Duodu, 2014), very few have shown promise the reasons for
this include low activity, phytotoxicity, poor stability, leaching to groundwater, too expensive to produce or mammalian toxicity.
In some cases industry has brought in new technologies, with the intention of applying integrated pest management programs. New chemical
nematicide discovery usually involves synthesis of new compounds, modifying existing compounds or testing compounds identied for other activities, running them through a high-throughput screen to identify hits,
then lead compounds, then glasshouse and eld assessments for efcacy,
environmental aspects, cost of production, meeting market demand, etc.
Alternatively a target-based approach can be employed, using data such as
that gained from gene silencing experiments (Danchin et al., 2013) to design
368
molecules that will t the active site of established target gene products and
inhibit their vital function.
One focus is on an integrated pest management strategy to control nematodes, which may combine both biological and chemical agents, agents such
as Bayer CropSciencess Bacillus rmus (Votivo), Paecilomyces lilacinus (Bioact)
combined with chemical control, e.g. uopyram (Velum/Verango), or using Pasteuria technology (e.g. CALARIVA) with AVICTA (Syngenta) and
other new products on the market. These can be combined with more
restricted methods of delivery, such as via drip irrigation or seed coating.
These delivery methods are more suited to nematode control in horticultural crops, and have the benet of providing control agents only where they
are needed, thus enabling more expensive and less stable chemicals to be
applied, so reducing costs and any detrimental environmental effects.
14. CONCLUSIONS
The exploitation of natural resistance genes using genotype screening
and marker-assisted selection will continue to be the standard approach to
improving resistance and/or tolerance to nematodes for many horticultural
Figure 3 Number of approved releases agronomic traits by phenotype (permits and

notications). http://www.isb.vt.edu/release-summary-data.aspx.
369
and staple food crops. However, for many crops this approach is slow, and in
most cases adequate broad-spectrum resistance is not available. This in part
accounts for the lag in approved release of nematode resistance traits
compared to other important agronomic traits as compiled by Information
Systems for Biotechnology (Figure 3, http://www.isb.vt.edu/releasesummary-data.aspx). With advances in transgenic technologies the release
of commercial varieties of major crops with transgenic nematode resistance
traits can be predicted to arrive soon. Already maize with eight stacked genes
(SmartStax Geniutycorn, with two herbicide tolerances and six Bacillus
thuringiensis-based insect resistance traits) is commercially available, and varieties with similar stacked traits will soon be available for cotton, soybean and
other crops. The addition of a nematode resistance trait would differentiate
the rst producer of such a trait, and would provide a competitive advantage. Such a biotech trait could be based on RNAi or on expression of other
synthetic resistance genes, and be added to genotypes with the best conventional nematode resistance. In addition, synthetic resistance genes with
different modes of action could be stacked to increase the efcacy and durability of the nematode resistance trait, and their expression limited to roots to
improve acceptability and unnecessary expression in harvested parts of the
plant.
As emphasized by Atkinson et al. (2012), crop losses to nematode infestation is often greatest in developing countries, where the need for food
security is greatest, and nematodes attack both commodity and staple crops.
In these countries it may not be a commercial proposition to develop nematode-resistant transgenic crops: rather a humanitarian approach may be
adopted in which such crops are developed for the public good. However,
following this approach will still require detailed studies to be undertaken to
ensure the food is safe for humans and the environment, and the political and
public support for their implementation, and in country support for their
further development and deployment.
Since two-thirds of the worlds population already lives in countries
where GM crops are grown, and concern for world food security is
now increasing, it is likely that the pace for adoption of transgenic
crops will increase as one of the mix of measures that will be needed
to feed over 9 billion people in a sustainable manner. Transgenic nematode resistance traits will undoubtedly be used to help ensure global food
security in the future, although the path to reach this stage will have
some issues to address, particularly in relation to public perception rather
than science.
370
That is not to say that there have not been practical successes in
nematode control, as can be seen when considering advances in control
for different crops and nematode pests on a case-by-case basis. For
example, real benets have been derived through conventional plant
breeding practices by introduction of the H1 major resistance gene
against the potato cyst nematode Globodera rostochiensis (FinkersTomczak et al., 2011) and the Mi gene conferring host resistance to
root knot (Meloidogyne) species in tomato (Williamson & Kumar, 2006),
which has been transferred to many different tomato varieties throughout
the world. A notable success has arisen from the study of natural genes for
resistance to the cereal cyst nematode (H. avenae), in which effective control has been developed for wheat initially using large-scale screening of
genotypes with resistance to H. avenae, followed by identication of
NBS-LRR type resistance genes from wheat and its wild relatives, and
their implementation in breeding programs. A series of 11 Cre genes
have been identied and introgressed into wheat cultivars (in particular
the genes Cre 1, Cre3 and Cre 8) to confer resistance, particularly in
Australia (Lewis, Matic, & McKay, 2009; Ogbonnaya, Eastwood, &
Lagudah, 2009). Marker-assisted selection is now used to pyramid some
of these resistance genes, and this approach is used routinely in many
wheat breeding programs worldwide (Ogbonnaya et al., 2009) to provide
effective control of H. avenae.
Marker-assisted selection can also be used to pyramid and combine
different desired traits into improved germplasm, and this has been an
ongoing activity undertaken by plant breeders over many years. In
addition to major gene resistances, minor (multigene-based) resistance
has been used incrementally to improve host resistance to nematode
infestation in many crops, such as the rht genes in soybean improving
resistance to the soybean cyst nematode (H. glycines), and a range of
minor genes contributing to increased resistance to root lesion nematodes
(Pratylenchus spp.) in cereals (Jones & Fosu-Nyarko, 2014). The more
conventional application of biotechnology to nematode control by
marker-assisted selection is now being complemented by the developing
biotechnological approaches of transgenic plants, genome editing, developing new nematode control agents and new modes of delivery of control
agents. The commercial implementation and delivery of these biotechnology-based technologies will undoubtedly contribute to future global
food security.
371
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Application of Biotechnology For Nematode Control

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All in-text references underlined in blue are linked to publications on ResearchGate,

Available from: Michael G.K. Jones

Application of Biotechnology for

Advances in Botanical Research, Volume 73

2015 Elsevier Ltd.

John Fosu-Nyarko and Michael G.K. Jones

Application of Biotechnology for Nematode Control in Crop Plants

2. EARLY SELECTION FOR PLANTS WITH NEMATODE

The introgression and combination of natural

Disruption of sensory functions

Wall-degrading enzymes may be required for

Major or minor natural resistance

Nematode migration in the

Migration in the root

Avoiding host defences

Disruption of feeding site formation

Table 1 Biotechnology-Based Strategies for Nematode Control

Marker-assisted breeding for

New approaches for genome editing now available

Make use of basic work on nematode effectors and

Delivery of toxic compounds to the

Develop new nematicides and

Disrupt expression of genes vital for the nematode

Modify genes for host plant

Overexpression of host genes with

Disrupting vital genes

RNAi downregulation of expression

Application of Biotechnology for Nematode Control in Crop Plants

John Fosu-Nyarko and Michael G.K. Jones

nematodes can also exhibit varying degrees of tolerance to infestation, when

3. BIOTECHNOLOGICAL APPROACHES TO PLANT

4. NATURAL RESISTANCE APPROACH TO PLANT

Application of Biotechnology for Nematode Control in Crop Plants

screening for resistance in germplasm from wild ancestors or progenitors of

Ballvora et al. (1995), Leister et al. (1996), Kreike

Examples of QTLs on 2BS, 6DS and 6DL, 6D,

Thompson, Brennan, Clewett, Sheedy, and

QRlnt.lrc-6D.2, QRlnt.lrc-6D.1 Wheat

Major QTLs Identied on Chromosomes

Garcia, Stalker, Shroeder, and Kochert (1996), Chu

Root Lesion Nematodes

Mi-1 and Mi-9 on chromosome 6

Mae, Mag, Rma

Root Knot Nematodes

Application of Biotechnology for Nematode Control in Crop Plants

John Fosu-Nyarko and Michael G.K. Jones

4.1 Transfer of Natural Resistance Genes to Different Species

Application of Biotechnology for Nematode Control in Crop Plants

example, transfer of the Mi gene to eggplant confers resistance to M. javanica

5. TRANSGENIC APPROACHES TO PLANT PARASITIC

John Fosu-Nyarko and Michael G.K. Jones

promoters alone is sufciently tightly expressed in the feeding cells to link

5.2 Overexpression of Host Genes with Modied Expression

5.3 RNAi-Based Nematode Resistance

Application of Biotechnology for Nematode Control in Crop Plants

Mitochondrial stress-70 protein

Secreted peptide (16D10)

Yadav et al. (2006)

>90% reduction in established

Ibrahim et al. (2011)