GEORGIKON FACULTY
DOCTORAL SCHOOL OF ANIMAL- AND AGRICULTURAL ENVIRONMENTAL SCIENCES
Head of School Professor Dr. Angéla Anda
Supervisors:
PROFESSOR DR. ANGÉLA ANDA DR. PETER WERNER
APHID RESISTANCE IN WHEAT VARIETIES
By:
HENRIETT ELEK
KESZTHELY 2013
Thesis for obtaining a PhD degree Written by:
HENRIETT ELEK
Written in the Animal- and Agricultural Environmental Sciences doctoral school of the University of Pannonia.
Supervisor: Professor Dr. Angéla Anda Dr. Peter Werner
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Acknowledgement 1
Abstract 3
Kivonat 5
Résumé 7
1. Introduction 9
2. Literature review 11
2.1. Aphids 11
2.1.1. Aphid life-cycle 11
2.1.2. Major aphid pests of grain crops 15
2.1.3. Aphid influence on host plants 20
2.2. Plant defences 22
2.2.1. Morphological plant defences 23
2.2.2. Semiochemicals 24
2.2.3. Secondary metabolites and metabolic enzymes 28
2.3. The host selection process and feeding 35
2.4. Resistance: antibiosis, antixenosis, tolerance 36
2.4.1. Schizaphis graminum – Greenbug 37
2.4.2. Diuraphis noxia – Russian wheat aphid (RWA) 38 2.4.3. Rhopalosiphum padi – Bird cherry-oat aphid 39
2.5. Evolution of wheat 39
2.5.1. Sitopsis section 41
2.5.2. Einkorn wheat 42
3. Materials and methods 43
3.1. Plant material 43
3.2. Aphids 47
3.3. Settling test 48
3.4. Fecundity test 51
3.5. Hydroxamic acid analysis by high pressure liquid chromatography 54
3.5.1. HA analysis for different species 56
3.6. Aphid feeding experiment 60
3.8.1. Sucrose solution 65
3.8.2. DIBOA in artificial diet 65
3.8.3. DIMBOA in artificial diet 65
3.9. Electrophoresis of DNA amplification 67
3.10. Apoplast fluid (AF) collection 69
3.11. Hydroxamic acid level from a field experiment 72 3.12. Effect of different environmental condition
on HA levels in the leaf tissue 73
4. Results 75
4.1. Settling test 75
4.2. Fecundity test 81
4.3. Analysis of hydroxamic acids in the leaf tissue 88
4.3.1. Tetraploid and hexaploid 88
4.3.2. B genome diploid species 90
4.3.3. A genome diploid species 95
4.4. Aphid feeding experiment 103
4.5. Honeydew collection 111
4.6. Artificial diet 113
4.6.1. Sucrose solution 113
4.6.2. DIBOA 114
4.6.3. DIMBOA 115
4.7. Unknown compound in the leaf tissue of the
A genome diploid varieties 118
4.8. Apoplast fluid (AF) analysis 121
4.9. Field experiment 128
4.10. Effects of growing medium, temperature and light intensity
on the HAs in the leaf tissue 132
5. Discussion 139
6. Summery 147
7. New scientific results 150
10. References 155
11. Appendix 174
Acknowledgement
I would like to thank my supervisors
Professor Angéla Anda (University of Pannonia) who took me on as a student and helped me throughout my PhD with her advice and guidance.
Dr Peter Werner (KWS UK Ltd.) for his supervision, constant support and for giving me an opportunity to do a PhD and work together with scientists from Rothamsted Research.
The late Dr Miklós Nádasy (University of Pannonia), who accepted me as a PhD student, but sadly passed away in 2010 during the course of this project.
This thesis would not have been possible without the guidance and support of Professor John Pickett CBE DSc FRS (Rothamsted Research). I would like to express my deepest gratitude for his constructive comments, suggestions and encouragement through the project and the thesis, and for giving me the opportunity to be part of his team working on the hydroxamic acid project.
My sincere thanks goes to Dr Ruth Gordon-Weeks (Rothamsted Research) who was the leader of the hydroxamic acid project; without her knowledge, support and advice, this study would not have been possible.
I would like to express my appreciation to Lesley Smart (Rothamsted Research) for her patience, time and support. She was always available when I needed any help especially with establishing the aphid bioassay. I would also like to thank her for her help and comments during the writing of this thesis and the papers.
I am also thankful to members of Rothamsted Research with whom I have interacted during the course of my studies. Particularly I would like to thank to Janet Martin for her help during the aphid bioassay, Dr Shakoor Ahmad for the help and support with the HPLC analysis and to Dr Kim Hammond-Kosack for supplying the Triticum monococcum lines.
I am grateful to the directors of KWS UK Ltd, Professor Chris Tapsell and Amanda Lay for giving me the opportunity, time and the financial support to complete this work;
without the generosity of this company I would not have been able to complete this thesis.
I am also grateful to all my colleagues at KWS UK Ltd especially to Debra Manning and Claire Fremann for helping me improve my knowledge in chemistry and for their support during the project. Claire and Morgane Roth also helped to translate the abstract into French. I would like to thank Mark Greed for making the insect cages; David Bell for IT support; Rachel Loweth for proofreading the manuscripts and organising my endless trips to Hungary; Guillaume Barral Baron and Dr Ed Byrne for their help with the statistical analysis; and finally Louise Rowland and Margaret Turner for their support over all these years.
I’m also grateful to my managers Peter Lakeman and Gillian Covey for their encouragement, patience, understanding, and for the time they provided for me to be able to complete this work.
Last but not least I would like to thank my family for their love, support and encouragement over the last years.
APHID RESISTANCE IN WHEAT VARIETIES
Abstract
Insect pests are ubiquitous and diverse throughout the cultivation zones for wheat, but the most economically significant aphid pest of European cereal crops is the bird cherry-oat aphid (Rhopalosiphum padi). As an environmentally compatible alternative to the use of conventional insecticides to control cereal aphids, we have investigated the possibility of exploiting natural resistance to insect pests in wheat species.
The antixenotic effects of hexaploid, tetraploid and diploid wheat varieties were tested against Rhopalosiphum padi in a settling test. The aphids were given a choice between the test variety and a hexaploid control Solstice. This quick screening method takes account of both the physical and chemical differences that influence aphid host plant selection.
The antibiotic effects of a selected number of the hexaploid, tetraploid and diploid varieties were assessed using a fecundity test. Aphids had no choice but to feed on the experimental variety and effects on nymph production were recorded.
The importance of the secondary metabolites hydroxamic acids (HAs) e.g. DIMBOA in resistance against insects was known from the literature. For this reason, the tissue of seedlings of the different wheat varieties was analysed to discover the reason for negative effects on aphid behaviour and nymph production. During this analysis non HA producing species were identified, which produced, at this point, unidentified compounds, which may have affected aphid behaviour and development.
The changes in the HA levels was studied after aphid feeding, which triggered the defence mechanism.
The toxicity of the aglucone DIMBOA was tested in an artificial diet assay, and found to be lower than levels found in the tested wheat species.
The apoplast fluid of a few varieties was analysed to try to explain why the HA levels in the experimental varieties (which were higher than levels found to be toxic in artificial diet) had no effect in the aphid bioassays.
The effects of aphid feeding on levels of HAs in the different varieties were also recorded under field conditions.
An experiment was set up in a controlled environment to study the effects of temperature, light intensity and growing medium on the HA levels to support the field data.
A BÚZAFAJTÁK LEVÉLTETŰ REZISZTENCIÁJÁNAK VIZSGÁLATA
Kivonat
Europában gazdaságilag kárt okozó rovar a zselnyicemeggy levéltetű (Rhopalosiphum padi), mely ellen jelenleg csak inszekticidekkel tudunk hatásosan védekezni.
Környezetkímélő alternatívaként e kutatási munka során különböző búzafajták rezisztenciáját vizsgáltuk R. padi levéltetvek ellen.
A táplálékválasztási vizsgálat során a hexaploid, tetraploid és diploid búzafajták antixenotikus hatását vizsgáltuk. Ez a módszer választási lehetőséget ad a levéltetvek számára a teszt növény és a hexaploid kontroll között, s egyúttal figyelembe veszi a növény fizikai és kémiai tulajdonságait, ami befolyásolja a levéltetveket a gazdanövény választásban. E vizsgálat során megállapítottuk, hogy a diploid fajták kevésbé bizonyultak kedvelt gazdanövénynek.
Szelektált hexaploid, tetraploid és diploid fajták antibiotikus hatását R. padi levéltetvekre a reprodukció vizsgálaton keresztül teszteltük. A levéltetvek választási lehetőség nélkül a kísérleti növényen táplálkoztak, amely hatással volt az utódprodukcióra.
A másodlagos anyagcseretermékek, a hidroxámsavak (HA), mint pl. a DIMBOA fontossága a rovarok elleni rezisztencia kialakításában az irodalomból már ismert volt. Ez alapján megvizsgáltuk a kísérlet során felhasznált néhány búzafajta levelének HA tartalmát, hogy képet kapjunk arról, van-e összefüggés a HA tartalom és a gazdanövény választás, illetve a csökkent reprodukció között. E vizsgálat során a HA-t nem termelő növények levelében számunkra ismeretlen vegyületeket találtunk, amelyek hatással lehetnek a levéltetvek viselkedésére és fejlődésére.
Figyelemmel kísértük a a levéltetvek táplálkozásának hatására kialakuló HA szintjének változását, ami a növény egyfajta védekezési reakcióját váltotta ki.
Az aglukon (DIMBOA) toxicitását mesterséges diéta alkalmazásával vizsgáltuk, s megállapítottuk, hogy a növények levelében található HA koncentrációnál alacsonyabb koncentráció toxikusnak bizonyult.
A sejt közötti folyadék analizálását abból a célból végeztük, hogy magyarázatot kapjunk arra, hogy a hexaploid és tetraploid növények levelében található HA tartalomnak miért nem volt negatív hatása a levéletetvekre.
A levéltetvek táplálkozásának hatását a HA szintjének változására szántóföldi körülmények között is elemeztük. Megállapítást nyert, hogy a külső környezeti hatások szignifikánsan befolyásolják a növények HA tartalmát.
Kontrollált laboratóriumi körülmények között vizsgáltuk a fényerősség, hőmérséklet és a táptalaj hatását a HA tartalomra, mellyel a szántóföldi körülmények között kapott eredményeket kívántuk alátámasztani.
RESISTANCE AUX PUCERONS DANS LES VARIETES DE BLE
Résumé
Parmi les pucerons ravageurs, le puceron du merisier à grappes (Rhopalosiphum padi) a la plus grande importance économique en Europe. La possibilité d’exploiter la résistance naturelle des variétés de blé afin de réduire l’emploi d’insecticides conventionnels a ainsi été examinée.
Les essais biologiques sur les pucerons utilisant R.padi ont montré des différences entre des accessions aux effets antixénotiques et antibiotiques. Toutefois, peu de preuves ont été trouvées montrant une variation avantageuse dans les variétés hexaploides ou tetraploides testées, même si certaines espèces diploides étudiées présentent une meilleure performance.
D’après la littérature, l’importance des métabolites secondaires, en particulier des acides hydroxamiques (AH) a été mise en évidence. Ainsi, le tissu de plantules de blé a été analysé pour établir un lien entre les niveaux d’AH et une quelconque réduction de colonisation ou de la production de nymphes par R.padi. Nous n’avons pas trouvé de corrélation entre les niveaux d’AH et le comportement des pucerons sur les variétés hexaploides et tétraploides.
Les variations de concentration d’AH ont aussi été étudiées après avoir nourri les pucerons, déclenchant les mécanismes de défense.
Nous avons étudié la toxicité de l’aglucone par l’emploi de différents régimes artificiels, permettant de baisser sa concentration par rapport à celle de pucerons étudiés précédemment. La solution DIMBOA à 2mM présenta une haute toxicité pour les pucerons R.padi, concentration qui est similaire pour les variétés hexaploides et tetraploides.
Le liquide interstitiel (LI) de quelques variétés a été collecté et analysé pour tenter d’expliquer pourquoi les niveaux d’AH dans les variétés testées – qui est plus élevé qu’avec des régimes artificiels – ne rendent pas compte des résultats des essais biologiques des pucerons. Dans la plupart des variétés testées, le LI ne contient que peu de DIMBOA et reste en dessous du niveau toxique.
Les niveaux d’AH ont été testés en champs et en environnement contrôlé ainsi que sous-alimentation artificielle. Ils ont montré que les métabolites sont significativement affectés par le mode de culture.
1. Introduction
Aphids are serious pests worldwide, able to cause severe damage in cereal crops, particularly wheat, Triticum aestivum, by direct feeding and by transmitting plant pathogenic viruses such as barley yellow dwarf virus (BYDV) (Mann et al.1996, Thackray et al. 2009). Aphids can affect the development in the early stages of the crops; long lasting infestation can reduce tillering (Anonymous 1995-2013), number of spikelets and seed (Pike and Schaffner 1985). Aphid damage (Rhopalosiphum padi) in the boot stage can result in 19-31% yield loss; later infestation reduces the yield loss to up to 20% (Voss et al. 1997). Infestation during the grain fill period may result in low grain protein content (Anonymous 1995-2013). With climate change, the importance of cereal aphid pests is increasing because many aphids are able to feed through the winter on cereal crops without recourse to sexual reproduction. Insecticides currently give sufficient protection, but can be expensive and environmentally undesirable. Some products may ultimately be lost due to the development of insecticide resistance. Resistance by aphids to insecticides was first recognised in the 1950s and now it is a global problem (Loxdale 2008). In addition, within the EU, legislation is in place to reduce the range and quantity of pesticide applied to cereal crops. An alternative approach is the development of insect resistant wheat varieties.
Resistance breeding against cereal aphids began in the 1970s. Resistance has been reported in Triticum turgidum conv. durum, Aegilops squarosa syn. Triticum tauschii (Castro et al.1999) and Aegilops speltoides (Castro et al. 2004) against greenbug Schizaphis graminum. Since then a series of resistant varieties were introduced, but due to the development of a new biotype of the pest, which could damage those varieties, resistance research has become more important to find different effective plant defence mechanisms against aphids. The selection pressure to evolve a biotype, which is able to adapt to antixenotic and/or antibiotic plants and break the resistance, is high because aphids are able to produce many generations a year (Basky 2005) and the incoming flux of potentially mutated individuals is very high.
It was previously recognised that ancestral species present good potential sources for further crop improvement through synthetic polyploidisation and introgression into modern wheat cultivars (Nevo et al. 2002).
We choose the bird cherry-oat aphid, Rhopalosiphum padi for our project work because it has recently become one of the most important pests of cereals in some European countries including England, Scandinavian countries and some areas of Turkey (Leather et al. 1989;
Blackman and Eastop 2000; Legrand and Barbosa 2000). In 1977 McLean and his tem described R. padi as “most serious pest” because of its rapid development and high reproductive rate compared to other cereal aphids (Sitobion avenae, Metopolophium dirhodum) in England and Wales. Although its feeding causes no chlorosis or other visible damage to wheat plants, heavy infestation can also reduce grain quality (protein content), thousand grain weight and even reduce protein assimilation by grazing cattle (Anonymous 2009). Rhopalosiphum padi is able to cause serious damage in cereal crops by direct feeding in the early developmental stage (Pike and Schaffner 1985), it is the main vector of the barley yellow dwarf virus which can cause up to 50% yield loss (Fischl et al. 1995).
The indirect effect of the aphid feeding is the large honeydew productions which reduces photosynthesis and induce sooty mould production and premature leaf senescence which will cause further yield loss.
The aims of this project:
to test a wide range of wheat varieties and ancestral species to look for natural resistance against Rhopalosiphum padi (Bird cherry-oat aphid)
to study the natural host plant defence mechanisms
study a group of secondary metabolites the hydroxamic acids and their effects on R.
padi host selection and fecundity
investigate the level of hydroxamic acids in plants under biotic stress (aphid feeding) in a controlled and a field environment
to help the breeder’s work by providing important information towards the production of marketable R. padi resistant wheat varieties in the future.
2. Literature review
2.1. Aphids
Aphids belong to the Hemiptera, a large order or piercing and sucking insects, which feed on plant sap or on vertebrate blood. Aphids are in suborder Sternorrhyncha, and many important crop pest species are from the family Aphididae and the subfamily Aphidinae.
The subfamily Aphidinae developed in the late Cretaceous Period, with the rapid expansion of herbaceous flowering plants, which provided valuable food supply during the summer for asexual reproduction. These plants became their secondary / summer host.
Aphids remained with their original woody host for sexual reproduction in the autumn and overwintered as eggs on the bark (Pickett et al. 2003). The proliferation of Aphididae may have occurred in the Miocene when primary grasses became widespread (Heie 1996).
Today about 4000 species of aphids are known, classified in 10 families; of these, around 250 species are serious pests for agriculture and forestry as well as an annoyance for gardens. Economically they damage crops by removing photoassimilates and vectoring numerous devastating plant viruses (Smith and Boyko 2007).
2.1.1. Aphid life-cycle
There are six stages in the life history of an aphid: the egg or embryonic stage, four instars, and the adult. Aphids have evolved a range of annual and biennial life cycles and other adaptive stages that often vary within as well as among species. The life cycle is holocyclic if the sexually reproducing generation is present or anholocyclic if the sexual generation is absent (Resh and Cardé 2003).
Anholocyclic life cycle
Under specific circumstances, some aphids can survive throughout the year, reproducing parthenogenetically (Blackman 1974). This can be important in agricultural ecosystems because they are able to survive the winter in a parthenogenetically reproducing form, and to continue feeding and reproducing. The anholocyclic life cycle is found most frequently in areas where the primary host plant is reduced. For example, the increase in the anholocyclic clones of Rhopalosiphum padi (bird cherry-oat aphid) in the UK is associated with a decline in the abundance of its primary host, Prunus padus (bird cherry) (Emden and Harrington 2007). A similar connection was detected in Australia and under Mediterranean conditions between R. padi and its primary host by Pons et al. (1993) and Thackray et al. (2009).
Parthenogenesis
The summer forms, both apterae and alatae, are all females and are viviparous giving birth to live offsprings. They produce female nymphs from unfertilized ova by parthenogenesis.
This method of reproduction enables aphids to multiply rapidly avoiding the time- consuming egg stage. The ova do not have to be fertilized but start to divide and develop into embryos inside a nymph before she is born. The nymph starts feeding as soon as it is born and it takes only one to two weeks to develop to adult. She is ready to give birth almost as soon as she reaches the adult stage.
Only 3% of the aphid species produce their nymphs exclusively by parthenogenesis, all the other species alternate with their sexual form at some stage in their life cycle (Blackman 1974, Basky 2005). In general, in countries with a cold winter, aphids produce a sexual generation, which directly influences survival from one year to the next. They produce cold resistant eggs, which can survive the winter conditions.
There are two types of parthenogenesis in insects. One is termed arrhenotoky, wherein haploid males arise from unfertilized eggs. This is found especially in the Hymenoptera (ants, bees and wasps). The other is the thelytoky, in which only diploid, female offspring are produced. In aphids the parthenogenesis is thelytokous. If thelyotoky involves meiosis,
chromosomes pair and exchange genetic material, and the offspring can inherit a rather different combination of genes from that of their parent; if the development is completely without meiosis then the offspring will all be genetically identical to their parent. Both types of thelytoky are known in other animals, but the process in aphids is still not fully understood. Cognetti (1961) believed that aphid clones are not strictly clones, but there was no conclusive evidence. The maturation of an egg under apomictic parthenogenesis is ameiotic, with the chromosomes undergoing essentially a mitotic division, producing true clones (Sunnucks et al. 1996).
Modern molecular genetic techniques substantiated the clonal nature of aphids by showing the stability of various regions of nuclear DNA during parthenogenesis. However, recently, germ line and somatic mutations were detected with molecular markers in clones of Sitobion avenae, thus confirming the occurrence of mutations in apomictic lineages (Shufran et al. 2003). Aphids can show recombination and non-equal exchange either between homologues or between sister chromatids within rDNA arrays during parthenogenetic reproduction (Hales et al. 2002).
Vanlerberghe-Masutti and Chavigny (1998) carried out a RAPD (random amplified polymorphic DNA) analysis and determined RAPD bands that appeared to be population and host plant specific. It is possible that the variation in host plant preference traits is sometimes generated by rearrangements of the karyotype. For example, Rhopalosiphum maidis collected from barley has 10 chromosomes and the same species collected from maize and sorghum has 8 chromosomes (Blackman et al. 2000).
The chromosome numbers for Schizaphis graminum range from 2n=6 to 2n=8. A higher variation of the karyotype is expected within aphid species compared to other insects, due to their holocentrism. Aphids have holocentric chromosomes, which have diffuse centromeres (Blackman 1974, Rubín de Celis 1997, Blackman et al. 2000). When the sister chromatids separate at mitotic anaphase they move apart along their entire length, instead of appearing to be pulled apart at one point. If a holocentric chromosome breaks into two or more parts the fragments can still move independently into the daughter cells (Blackman 1974), broken chromosomal fragments are still capable of segregating at mitosis, but there is no strong evidence that such damaged chromosomes remain stable (Rubín de Celis et al. 1997).
Variation in parthenogenetic lineages may arise from three sources:
Mutational divergence under asexual reproduction (Loxdale 2008, Sunnucks et al.
1996)
Multiple origins from sexual individuals
Interactions between parthenogenetic and sexual conspecifics such as matings between males and females from parthenogenetic and holocyclic lineages.
Several aphid chromosomal rearrangements are associated with radical new genotypes including insecticide resistance and host specificity (Sunnucks et al. 1996).
Holocyclic life cycle
Holocyclic aphids have life cycles in which sexual forms and eggs are produced regularly every year.
At the beginning of the spring when buds of the winter host start to swell aphid’s eggs hatch and the nymphs start to feed immediately. In temperate regions the young nymphs are resistant to frost as soon as they start to feed and they develop quickly (Blackman 1974). After 3 moults they become an adult called a fundatrix (Basky 2005), which becomes the first individual of a parthenogenetic line (Blackman 1974).
Each fundatrix can produce up to 70 nymphs, which after 4 moults will become wingless fundatrigeniae. This generation produces 40-50 nymphs some of which will be alata (Basky 2005) that leave the winter host because it is no longer suitable for them. These are the spring migrants that start colonies of apterous virginoparae on a summer host. During the summer period if conditions become poor further generations of alate virginoparae are produced to allow the aphids to migrate to colonise new plants. Some of them will find another host that is acceptable. Once settled they quickly start to produce a new colony of apterous virginoparae (Blackman 1974).
For aphids in the Aphidinae subfamily, at the end of the summer short days stimulate adults to give birth to the autumn migrants (Powell and Hardie 2001), these are called gynoparae and are the parents of the sexual females. They fly to the primary overwintering
host plant and there, they give birth to the sexual, egg-laying females, called oviparae. The colonies on the summer host then start to produce winged males, which also fly to the winter host where mating occurs. After mating, the oviparae lay eggs, which do not hatch until the following spring. The embryos inside have to go through a cold period before they can complete their development (Kuroli 1994, Delmotte et al. 2001, Basky 2005).
Host-alternating aphids in Anoeciinae, Erisomatinae and Hormaphidinae subfamilies produce only one return migrant form sexuparae, which give birth to oviparae and apterous males on the primary host plant (Powell and Hardie 2001).
2.1.2. Major aphid pests of grain crops
Bird cherry-oat aphid – Rhopalosiphum padi (Linnaeus)
The bird cherry-oat aphid is one of the most serious pests of cereals worldwide and one of the main vectors of Barley yellow dwarf virus (BYDV) (Thackray et al. 2009). It is most common in wheat in the autumn and winter (Johnson and Townsend 1999). The adult aptera is 1.2 – 2.4mm long. It has a rounded body shape and is brownish olive green, with two short dark tubes (siphunculi or cornicles) at the rear end surrounded by a rusty red coloured area (Figure 1.). The apical ends of the siphunculi are swollen slightly and finish with a strong flange preceded by a distinct constriction. The tail (cauda) is pale and shorter than the siphunculi. The alate is also 1.2 – 2.4mm long with a pale to dark green abdomen.
Figure 1. Rhopalosiphum padi (photo: Rothamsted Research)
Bird cherry-oat aphids can be found after cereal seedling emergence in the autumn in the UK, but are most common from February onwards. In southern England, an R.padi population overwintering anholocyclically was first recorded in the 1970s on Gramineae (Hand 1989).
The primary host is the bird cherry tree (Prunus padus), the secondary hosts belong to the Poaceae. Pons and colleagues (1993) studied the winter populations of cereal aphids in the Mediterranean climate between 1983 and 1991.They observed that when the primary host of R. padi is absent no overwintering eggs could be found on other Prunus species R. padi were able to develop and reproduce on the primary host through the winter (Pons et al.
1993).
Damage
Bird cherry-oat aphids attack all small grains, and heavy populations may cause a golden yellow streaking on the leaves. The aphid prefers to infest lower leaves and the stem moving to higher leaves only when the population increases. Although it can remove considerable amounts of liquid and nutrients, and strong infestations can sometimes lead to controtion of leaves, the direct effect on grain is generally only slight, especially if plants are young at the time of infestation. This aphid causes most damage by transmission of BYDV and often provides the primary source of infection in early sown winter cereals.
Russian wheat aphid (RWA) – Diuraphis noxia (Mordvilko)
The genus Diuraphis is of possible palearctic origin. Diuraphis noxia originated from Russia where the first serious outbreak was noted on barley in 1912 (Blackman and Eastop 2006). It was not well known until the 1970s when it started to colonize the main wheat and barley crops in East Asia and South Africa (Emden and Harrington 2007).It first appeared in the United States in 1986, in Chile in 1990 and in Argentina in 1992 (Castro et al. 2005).
It is a small pale green insect with an elongated, spindle-shaped body that can be covered with powdery wax (Figure 2.). It is easy to recognize from the supracaudal process, which is located above the cauda and looks like a twin tail. In the summer, RWA feeds on grasses and in the late fall migrates to cereals. It is tolerant of cold weather and can survive sub- freezing temperatures.
Figure 2. Diuraphis noxia (photo: www.uniprot.org)
It prefers late sown crops on poor soil. In Europe and Asia it has a sexual phase, without host alternation, on wheat and barley. In North America, a small genetic variation was found between populations and males have not been found, which could indicate anholocyclic reproduction (Basky 2005, Emden and Harrington 2007).
Damage
RWA injects toxin into the plant during feeding, which is responsible for most of the damage symptoms. The most characteristic symptom is white longitudinal streaks on the leaves. Mainly apical leaves are attacked, which curl up (Heie 1992), and if the ear gets stuck in the curved flag leaf, the head becomes distorted and looks like a fish-hook.
RWA is able to transmit the barley yellow dwarf virus, sugarcane mosaic virus and barley stripe mosaic virus (Damsteegt et al. 1992)
Greenbug – Schizaphis graminum (Rondani)
Greenbug is a palearctic aphid, possible of Middle eastern or Central Asian origin (Emden and Harrington 2007). It was recognized as a major pest of small grains over 150 years ago by Rondani. Schizaphis graminum was first recorded in 1889 in Argentina and was the first aphid to cause serious damage to wheat in North America.
It is a small (1.3-2.1mm) elongate oval shaped aphid; the head and first part of the thorax are straw to pale green coloured and the abdomen is light to medium green (Figure 3.). It has a darker green stripe in the middle of the dorsal surface of the abdomen, which is most visible on adults. The cornicles are pale with darkened tips.
Figure 3. Schizaphis graminum (photo: www.ipm.ucdavis.edu)
Damage
Greenbug feeding causes yellow or red leaf spots. Continued feeding leads to general yellowing or reddening, leaf and root death and plant death, because greenbug injects a toxin into the plant causing the tissue to die. As a result of greenbug feeding plant size and yield are reduced.
Greenbug is the vector of maize dwarf mosaic virus (Fischl et al. 1995), barley yellow dwarf virus, sugarcane mosaic virus (Basky 2005).
Grain aphid – Sitobion avenae (Fabricius)
Grain aphid overwinters in egg form in Europe, but also continues to reproduce parthenogenetically, when the winter is mild enough, on winter cereals (Emden and Harrington 2007). It is 2-2.8mm long, with coloration varying from yellow to green, red- brown and purple (Figure 4.). Antennae, siphunculi, and leg joints are black, and the cauda is pale. The siphunculi are twice as long as the cauda.
Figure 4. Sitobion avenae (photo: www.iranwheat.ir)
It usually appears later than the other cereal aphids.
The primitive life cycle of the genus involves host alternating just like Macrosiphum avenae - primary hosts belong to the Rosaceae family and the secondary hosts to the
Poaceae – but the majority of Sitobion species no longer have host alternation (Emden and Harrington 2007).
Grain aphid is found on the leaves and stem of barley and oats, but it also colonises the ears on wheat.
Damage
Direct damage is caused by feeding on the developing ears, and it is also an efficient vector of BYDV (Johnson and Hershman 1996, Emden and Harrington 2007). Heavy infestations can reduce the number of grains per ear resulting in yield loss. Grain aphid is also vector of the bean yellow mosaic virus and the pea mosaic virus (Basky 2005). The aphids produce a lot of honeydew, which is a good medium for the growth of sooty moulds, which inhibit photosynthesis.
2.1.3. Aphid influence on host plants
1. Yield loss
By direct feeding, aphids are able to drain the plants of its nutrient which leads to reduced leaf area, slow growth, yield loss and the premature death of the plant (Capinera 2008).
2. Salivary toxins
Some aphid saliva contains toxins which can cause:
Discoloration / yellowing around the area where the aphid has been feeding e.g.
Schizaphis graminum
Deformities such as leaf-curls (Diuraphis noxia), galls (Pemphigus sp.)
This can negatively affect plant growth and reduce the yield of the crop (Capinera 2008).
3. Honeydew excretion
Aphids ingest a large amount of plant sap in order to acquire sufficient protein. The excess sap, which is carbohydrate, is eliminated from the body as honeydew. This sticky substance can coat the host plant and become a medium for sooty mould fungus, which can cause an economic problem on fruit and vegetables (Capinera 2008).
4. Virus transmission
The major danger to plants is the indirect effect of aphid feeding, pathogen transmission.
Aphids are able to take up viruses with the plant sap and during probing and transfer them to other plants, thus acting as a vector (Capinera 2008).
Types of virus transmission
Non-persistent virus transmission
Aphids take the virus from the plant during the first probe, when the stylets reach the mesophillum. If the plant is infected with Poty virus (eg. potato virusY, plum pox virus, turnip mosaic virus), the aphids also take the virus’s helper protein with the plant sap, and this sticks the virus to the stylet wall. They are able to transfer the virus from the first moment of infection. When the aphids feed on a healthy plant they need less than 10 seconds to transfer the virus. They are infectious to healthy plants only for a short time period and without further feeding it is no longer than 30 hours until the virus loses virulence (Basky 2005).
Semi-persistent virus transmission
This group of viruses (eg. beet yellows Closterovirus (Basky 2005)) are concentrated in the host plant’s sieve elements and this means it takes longer for a feeding aphid to take up the viral particles. There is no period of incubation and the aphid is able to infect another host plant from the minute it acquires the virus. The virus retention time is no more than a few
days. These viruses cannot be detected in the haemolymph (Lapierre and Signoret 2004, Basky 2005).
Persistent virus transmission
The persistent virus transmission has two types: circulative and propagative transmission.
A circulative virus such as barley yellow dwarf virus is vector specific and is acquired by aphids from the xylem or phloem. The period of incubation is 24-48 hours, after that the virus is present in the aphid’s saliva and haemolymph (Basky 2005) but does not multiple in the aphid body.
The propagative viruses for example Maize mosaic virus, Maize stripe virus and Rice stripe virus are transmitted by plant hoppers and able to multiple in the host plant and reduce the life expectancy of the infected insects.
Persistent viruses do not lose virulence after a moult and the aphids are able to infect plants throughout their lifetime (Basky 2005).
2.2. Plant defences
Plant defence against herbivory or pathogens describes a range of adaptations evolved by plants, which improve their survival and reproduction by reducing the impact of herbivores. Plants use several strategies to defend against damage caused by herbivores, some of them are present at all times, and others are induced only in response to herbivore feeding or pathogen infection. Many plants produce secondary metabolites, known as allelochemicals, which influence the behaviour, growth, or survival of herbivores. These chemical defences can act as repellents or toxins to herbivores, or reduce plant digestibility (Frost et al. 2008).
2.2.1. Morphological plant defences
Trichomes
The first plant organs contacted during the preliminary stages of settling on a host are surface hairs or trichomes. Trichomes are epidermal appendages of diverse forms and structures, such as non-glandular hairs, scales, or pelt hairs. Trichomes affect the insect behaviour in general by providing a barrier that prevents small arthropods from landing directly on the plant surface and thereby interfering with movement and feeding. The densities of trichomes on the buds and leaf surface of some cultivars also deter feeding and sometimes oviposition.
Leaf trichome density and position may act as a physical obstacle to aphid feeding. The high trichome density on the leaf veins could prevent the aphid from finding a suitable feeding site (Oberholster 2002-2003).
Surface waxes
Plant leaves are protected against desiccation, insect predation and disease by a layer of surface waxes over the epicuticle. Epicuticular waxes affect the feeding behaviour of insects, particularly the settling of probing insects, acting as either phagostimulants or feeding deterrents. (Al-Ayedh 1997).
The chemistry of wax differs from plant to plant, but the most commonly occurring components are alkanes, primary and secondary alcohols and ketones, sometimes small amount of sugars and amino acids (Bernays and Chapman 1994).
Alkanes are amongst the commonest constituents of all plant waxes. Insects possess the sensory apparatus to detect these chemicals by contact or olfaction. (Al-Ayedh 1997).
2.2.2. Semiochemicals
Semiochemicals are molecular signals mediating interactions between organisms, either of the same species (pheromones) or between different species (allelochemicals) (Hooper and Pickett 2004).
Pheromones
Pheromones are important in the familiar mating response and they can affect insect behaviour as well (Hooper and Pickett 2004).
The main groups of aphid pheromones are:
Sex pheromones
Alarm pheromones
Aggregation pheromones
Aphid sex pheromones are released by the sexual females to attract males and increase the success of mating. The pheromones are produced in glandular epidermal cells on the tibiae of the hind legs of the sexual females. The olfactory receptors are located on the third forth and sometimes on the fifth antennal segments of the male aphids (Hardie et al.
1999).
Aphid alarm pheromones are released when, for example, they are disturbed by a predator, and the response behaviour ranges from removal of mouthparts from the plant and moving away, to running, dropping off the plant and even attacking the predator with frontal horns, although not all aphids in a group respond. Nymphs in the early developmental stages do not respond to alarm pheromones because the risk of predation is lower than the risk involved in ceasing to feed and dropping from the plant.
Droplets secreted from the cornicles contain two types of material; a minor volatile, which rapidly vaporizes comprises the alarm pheromone and a rapidly setting waxy fraction, which is an irritant to predators and parasitoids and can interfere with moulting (Hardie et al. 1999).
Aggregation pheromones are behaviour modifying odours produced by winged aphids; it is attractive to other winged forms but repellent to wingless forms. Such behaviour may be due to host plant characteristics, which determine suitable feeding sites (Hardie et al.
1999).
Kairomones are aphid pheromones that are used by parasitoids and predators. For example, the aphid sex pheromones attracts not only males of certain aphid species, but also female parasitoid wasps and the alarm pheromone indicates the presence of aphids in the local area and stimulates searching behaviour in predators and parasitoids (Hardie et al.
1999).
Allelochemicals
The attraction of insects to plants and other organisms involves detection of specific semiochemicals or specific ratios of these usually volatile semiochemicals. The avoidance of unsuitable hosts can involve the detection of specific semiochemicals associated with non-host taxa (Pickett et al. 2005, Hooper and Pickett 2004).
An undamaged plant maintains a baseline level of volatile metabolites that are released from the surface of the leaf or from accumulated storage sites in the leaf. These chemicals reserves can include monoterpenes, sesquiterpenes and aromatics, which are stored in specialized glands or trichomes. Emissions of volatiles are often synchronized with the light cycle generally showing low emission at night and a high level during the periods of maximal photosynthesis. The volatiles released by an insect damaged plant are different from those of undamaged or mechanically damaged plants. Around the damaged area, systemic volatiles are synthesized and a mobile chemical messenger transmits information to the distal leaves and activates several defence responses in the host plant (Paré and Tumlison 1999). Semiochemicals are also used by parasitic insects and other predators of aphids to locate the host plants of their aphid prey. For example, Aphidius rhopalosiphi, the parasitoid wasp of cereal aphids, is attracted by wheat and this attraction is stronger if the plant is infested by R. padi (Gonzáles et al. 1999).
A high population density of aphids (9 aphids/cm2) causes wheat seedlings to produce volatiles that have a repellent effect on apterous R. padi individuals (Quiroz et al.1997, Gonzáles et al. 1999).
Jasmonic acid
The plant stress hormone jasmonic acid activates many defence responses in the plant.
Jasmonic acid levels rise steeply in response to damage caused by a variety of herbivores and triggers the formation of many different kinds of plant defences besides proteinase inhibitors, including terpenes and alkaloids (Taiz and Zeiger 2002).
Jasmonic acid can act internally as a plant hormone associated with a damage/stress response, but, when methylated (methyl jasmonate), can be released by the plant and, whether naturally or not, will certainly have an effect on intact plants by up-regulating defence related and other genes (Pickett et al. 2006).
Cis-jasmone, which is also involved in the jasmonate pathway is a metabolic product of jasmonate and was thought originally to represents a sink for this pathway. Cis-jasmone affects plants by increasing attraction and searching by aphid predators (Pickett et al.
2005).
Salicylic acid
Methyl salicylate is biosynthetically related to salicylic acid, a signal molecule associated with systemically acquired resistance. This may indicate that the host plant defence pathways associated with hormonal activity of salicylate could present difficulties for colonization by aphids (Pickett et al. 2006). Methyl salicylate was found to deter aphids from colonizing plants and to increase foraging by parasitoids and predators attacking herbivorous insects and mites (Pickett and Poppy 2001). Prunus padus the winter host of R. padi releases a large amount of methyl salicylate, which stimulates the production of winged migrants on the winter host (Glinwood and Pettersson 2000).
Salicylic acid is also an internal stress signal (Pickett and Poppy 2001). Over 40 insect species from five separate Orders have been identified that have olfactory receptors for the methylated form methyl salicylate (Chamberlain et al. 2000, Pickett et al. 2003). The cereal aphids R. padi, S. avenae and Metopolophium dirhodum have a specific olfactory neuron on the sixth antennal segment to detect methyl salicylate (Pickett et al. 2006).
Ethylene (ET)
Ethylene is a plant hormone which plays an important role in plant defence. Ethylene biosynthesis is increased by stress conditions such as drought, flooding, chilling or mechanical wounding which triggers the stress responses such as abscission, senescence, wound healing and increased disease resistance. In combination with jasmonic acid is required for the activation of several plant defence genes (Taiz and Zeiger 2002). Insect herbivory is also known to stimulate an increase in ET emission. For example after Schizaphis graminum (biotype C) and Rhopalophum padi feeding on resistant barley a higher ET level was measured than in the susceptible barley (Argandona et al. 2001).
Harfouche et al. (2006) showed the armyworm larvae growth rate was significantly lower on ET producing maize plants than on plants treated with ET inhibitor which indicates ET is involved in the insect defence mechanism as well.
Abscisic acid (ABA)
ABA controls the resistance against pathogens and also plays a key role in the tolerance response to abiotic stress, and has been reported to act as a systemically transported signal from the roots to shoots (Erb et al. 2009). Above ground Diabrotica virgifera virgifera feeding induced the accumulation of abscisic acid in the above and below ground area of the plant which upregulated the hydroxamic acid pathway which resulted a higher level of DIMBOA in the shoot. Erb et al. (2009) concluded that D. virgifera induced production in the leaves is mediated by ABA.
Response to semiochemicals
Adult aphids can detect olfactory cues and use them for host location, escape responses and mate recognition. The olfactory receptor system of the host-alternating aphids is not only species- and sex-specific, but also morph-specific. The nymphal alatiform virginopara antenna is morphologically different to the adult’s antenna, which has three different types of olfactory receptor organs, proximal and distal primary rhinarium, and numerous secondary rhinaria. The primary rhinaria are presented in all developmental stages the
secondary rhinaria appear only in specific morphs of the adult stage and they become sex pheromone receptors in males.
The nymph’s olfactory system is not as developed as an adult because it does not require a specific sensory system for host selection (Park and Hardie 2003).
Aphids can detect semiochemicals with the primary rhinaria, which are on the fifth and sixth antennal segments (Pickett et al. 1997), and the contact chemosensory sensillae, which contact the plant surface during walking and are located at the tip of the antenna (Powell et al. 1999).
2.2.3. Secondary metabolites and metabolic enzymes
The avoidance of non-hosts by many insects is based on volatile metabolites, which can be detected at a distance or by initial attempts at colonization or feeding, which are terminated due to, lack of appropriate physiology, nutritional aspects or by the detection of potentially toxic secondary metabolites (Agelopoulos et al. 1999).
Secondary metabolites are those compounds, which tend not to be so essential for the basic growth of plants, and for which function is not always known (Bernays and Chapman 1994).
Cyanogenic glycosides
Various nitrogenous protective compounds other than alkaloids are found in plants (Taiz and Zeiger 2002). Approximately 2500 plant species produce hydrogen cyanide (HCN).
HCN is a general respiratory poison, which is stored in the plant in a nontoxic form, often combined with a sugar to form a cyanogenic glycoside. When the tissue is damaged the vacuolar glycoside is contacted by the cytoplasmic hydrolyzing enzymes and the damaged plant releases HCN (Bernays and Chapman 1994)
Cyanogenic glycosides cause serious problems to numerous herbivores since they are widely distributed and generate toxic HCN as the plant tissues are masticated or ingested.
Cyanogenesis is especially dangerous for generalist phytophagous insects because the cyanide, in a process similar to carbon monoxide, nitrogen oxide or azides replaces oxygen binding to heme units of the terminal cytochrome oxidase and blocks electron transport through the respiratory chain. Thus the generalists usually do not feed on plants rich in the cyanogenic glycosides and in this role cyanogenesis might play an important role in plant chemical defence. Monophagous insects that are specialized to feed on cyanogenic plants have developed specific enzymes: cyanoalanine synthase, sulphur transferase (rhodanase) and/or linamarase that allows them to detoxify the highly toxic cyanide.
A very high content of cyanogenic glycosides is found in the youngest leaves of bird cherry when the first fundatrices of R. padi appear. While the aphid population starts to build up, the cyanogenic glycoside level decreases rapidly until almost all the aphids have left the primary host (Leszczyński et al. 2003).
Polyphenoloxidase
Polyphenoloxidase (PPO) plays an important role in plant resistance to insects, and in the detoxification of phenolic compounds taken in the nutrient components.
Polyphenoloxidase is a widespread enzyme found in plant cells, located in the chloroplast thylacoid membranes. Oxidation of phenolic compounds in plant cells is responsible for initiating the browning reaction of the tissues and is characteristic of the pathogen factor or of pest feeding. Within the tissues liable to damage by feeding insects there is an increased concentration of phenolic compounds and moreover, PPO induces metabolization of these phenolic compounds into more toxic forms. Some aphid resistant wheat varieties, for example spring wheat Eta, have markedly decreased PPO activity which is caused by aphid feeding. That is maybe because too high degree of phenol oxidation may induce a rapid loss of toxicity by further transformation into harmless polymers or be induced by insertion of oxidases in the aphid saliva into tissues of the host plant (Chrzanowski et al.
2003). Some varieties have a different reaction to aphid feeding; for example in Tugela DN, which expresses the Dn1 gene for resistance to the RWA the PPO activity was more than double than in the susceptible variety (Mohase and Van der Westhuizen 2002).
Hydroxamic acid (HAs)
Hydroxamic acids are the main group of secondary metabolites involved in the resistance of certain cereals against bacteria, fungi and several insects including aphids (Thackray et al. 1991, Nicol et al. 1992, Rustamani et al. 1996). They were first discovered in 1955 in rye in relation to fungal diseases (Virtanen and Hietala 1955) and later found in maize where they were associated with resistance to the European corn borer, Ostrinia nubilalis (Niemeyer et al. 1992) and corn leaf aphid Rhopalosiphum maidis (Beck et al. 1983). HAs can be found in the cultivated monocotyledons, in maize, wheat (Nicol et al. 1992) and rye but are not present in barley, however DIBOA can be found in the wild Hordeum species (Barria et al. 1992). HAs are not present in the seed (Cambier et al. 2000).
Hydroxamic acids are concentrated in the mesophyll protoplasts, the vascular bundles (Givovich and Niemeyer 1995) and in the sieve elements. The compounds are present in the plant as glucosides, which are enzymatically hydrolysed by endo-β-glucosides to DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) when the tissue is injured (Hofman and Hofmanová 1969, Givovich et al. 1994) (Figure 5). DIBOA (2,4-Dihydroxy- 1,4-benzoxazin-3-one) is the main hydroxamic acid in rye, whereas DIMBOA is the predominant form in wheat and maize (Frey et al. 1997, Figure 6.). The HA level is highest in the early seedling stage and decreases rapidly as the seedling matures (Klun and Robinson 1969). The accumulation of these compounds may be influenced by the growing environment. High light intensity (Manuwoto and Scriber 1985), long photoperiod, elevated growth temperature (Epstein et al. 1986) and soil moisture can reduce the HA level in the seedling (Richardson and Bacon 1993).
β-glucosidase
Figure 5. Aphid feeding triggers the conversion from glucoside to the main aglucone in the cell. Blue arrows show the punctured cells, 1-3 injected watery saliva during probing and feeding, 4 taken up phloem sap
which contains DIMBOA-glucoside through the stylet during feeding (Tjallingii 2006).
Biosynthesis of hydroxamic acids has several steps. Fray et al. (1997) showed that five genes are required for the biosynthesis of 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) from indole-3-glycerol phosphate. Leighton, Niemeyer and Jonsson’s (1994) work showed that the glucosylation of hydroxamic acids would be the last step of the pathway (Cambier et al. 2000) (Figure 6.).
DIMBOA-glucoside DIMBOA
DIMBOA-glucoside
Figure 6. Hypothetical pathway of hydroxamic acids in maize (Frey et al. 1997 modified by Gordon-Weeks et al. 2010.)
Studies by Niemeyer and co-workers suggest that genes on chromosome 4A and 4B are involved in the transformation of DIBOA into DIMBOA. Comparison of the HA levels in tetraploid and hexaploid wheat shows that the incorporation of the D genome into the latter leads to a considerable decrease in HA accumulation (Niemeyer and Jerez 1997).
bx1 mutant should be defective in the production of free indole and DIMBOA (Frey et al. 1997)
bx3 mutant (homozygous for the recessive mutant allele) no DIMBOA was detected in the maize seedlings (Frey et al. 1997)
DIMBOA-glucoside can be converted into HDMBOA-glucoside, which could be important in the defence against insect and disease (Cambier et al. 1999). HDMBOA- glucoside accumulation was noted after stem rust infection, jasmonic acid treatment (Oikawa et al. 2002) and armyworm feeding (Oikawa et al. 2004). Artificial diet studies showed HDMBOA-glucoside is toxic. A diet containing 2mM HDMBOA-glucoside significantly reduced the number of Metopolophium dirhodum after 2 days (Cambier et al.
2001). The aglucone of HDMBOA-glucoside is HDMBOA which is produced by hydrolysis (Oikawa et al. 2001) (Figure 7.), this molecule however proves to be very unstable and degrades rapidly into MBOA which is also the degradation product of DIMBOA (Cambier et al. 2000).
Figure 7. Hypothetical pathway leading to the release of MBOA (Oikawa et al. 2004)
DIMBOA is the main HA aglucone in wheat. Extracts of DIMBOA produce antibiosis, feeding deterrence, decreased performance and reduced reproduction in aphids. It has mutagenic effects and affects the level of genetic polymorphism in aphid populations (Figueroa et al. 2004). Hydroxamic acids have also been associated with detoxification of triazine herbicides (Hamilton 1964, Frey et al. 1997). The maximum recorded HA level in cultivated wheat is between 1.4 – 10.9 mmol/kg fresh weight (Copaja et al. 1991). Thus, in the ancestors of the hexaploid wheat the highest level of HAs was found in the B genome species Aegilops speltoides 16 mmol/kg fresh weight by Niemeyer (1988) and nearly 40mmol/kg fresh weight was found in wild rye (Nicol et al. 1992, Nicol et al. 1993).
Nicol and team confirmed the potential of HAs as aphid resistance factors in modern cultivars based on seedling antixenosis bioassays that tested the suitability of the host plants to the aphids (Nicol et al. 1992). Givovich and Niemeyer published similar results where they showed R. padi preferentially settled on wheat seedlings with lower DIMBOA levels in a choice test (Givovich and Niemeyer 1991). In artificial diet the increased level of DIBOA and DIMBOA concentration decreased the survival rate of cereal aphids (Barria et al. 1992, Niemeyer et al. 1992)
As the concentration of HA increases, aphids need a longer time to search for suitable phloem vessels (Givovich and Niemeyer 1995, Givovich and Niemeyer1994), because they feed from a single sieve tube and the HA concentration can be different between sieve tubes (Givovich et al. 1994). They also spend a longer period ingesting xylem fluid (Ramìrez and Niemeyer1999), which doesn’t contain HAs (Givovich and Niemeyer 1995).
HA levels affect virus transmission as well because aphids takes a longer time to reach the phloem and are therefore less able to transmit the BYDV (Givovich and Niemeyer 1991, Nicol et al. 1992).
2.3. The host selection process and feeding
Host-selection behaviour is based on plant chemistry. Aphids are weak flyers and they use plant specific cues to increase the chance of finding a suitable host plant.
The landing response of a flying aphid is affected by attraction to non-specific visual stimuli, the colour or the form of the host plant (Powell and Hardie 2001) and plant volatiles, which are detected by antennal olfactory sensillae (Powell et al. 2006). The olfactory signal is the indicator of an appropriate host, stimulating the insect to take off and move towards the source of the odour (Bernays and Chapman 1994).
After landing, olfaction still remains important, together with contact chemoreception, mechanoreception and vision. The highest concentration of leaf odour can be detected in the air close to the leaf surface, which is called the boundary layer (Bernays and Chapman 1994). The first plant organs contacted during landing are trichomes. The density and the exudates of the trichomes can affect the settling behaviour (Powell et al. 1999). Plant wax is important in host selection because it contain chemicals that are characteristic of the plant (Bernays and Chapman 1994).
The aphid walks across the plant surface to detect chemical cues (Powell and Hardie 2001, Powell et al. 2006). During this process the aphid’s antennae are waving backwards and forwards. The chemosensory hairs on the tip of the antenna contact with the substrate, which enable it to detect gustatory cues. Epicuticular waxes, trichome exudates, substrate texture, topology and colour may all affect aphid behaviour before probing (Powell et al.
2006).
If the aphid detects a suitable substance from the array of tactile and olfactory cues, it proceeds to make exploratory probes. Those probes are limited to the epidermis and stylet penetration of the plant tissue is initially made up of regular brief cell punctures, allowing assessment of the internal plant chemistry (Powell et al. 2006).
If the aphid finds the substrate satisfactory the stylet penetration will persist longer than 30s allowing penetration into mesophyll and parenchyma tissues for further ingestion of plant sap for gustatory discrimination (Powell et al. 2006).
Stylet penetration of the phloem sieve element is followed by injection of watery saliva to disable the defensive phloem-sealing mechanisms present in most host plants.
If the aphid continues to feed from the phloem for longer than 10 minutes this generally indicates complete acceptance and feeding will normally continue for several hours (Powell et al. 2006).
Saliva
During feeding, aphids secrete rapidly gelling sheath saliva and watery, digestive saliva (Tjallingii 2006). Sheath is composed primarily of proteins, phospholipids, and conjugated carbohydrates. Watery digestive saliva is a more complex mixture of enzymes and other components capable of eliciting plant defence signals (Smith and Boyko 2007). Among aphids, specificity of elicitation could be mediated by differences in the composition of saliva, which may generate different profiles of plant allelochemicals (Messina et al.
2002).
Two enzymes found in the grain aphid’s saliva, are polyphenol oxidase and peroxidase.
Both enzymes are present in the gelling saliva but only polyphenol oxidase activity is found in the watery saliva. The secretion of these enzymes was associated with the probing during penetration of the epidermal and mesophyll tissues (Urbanska et al. 1998).
2.4. Resistance: antibiosis, antixenosis, tolerance
The three well-known mechanisms of plant defence against aphids are antibiosis, antixenosis and tolerance.
Antibiosis: the ability of the plant to reduce or stop the growth and or development of the insect (El Khishen et al. 2009), the insect accepts the plant as a host, but relative growth rate; body weight and the number of offspring are decreased.
Antixenosis: reduces the probability of contact between plants and the pest by influencing insect choice (El Khishen et al. 2009). The insect does not accept or recognise the plant as a host, and will avoid it because of one or more morphological or biochemical attributes of the plant.
Tolerance: is the mechanism by which plants maintain similar levels of yield under vastly different levels of insect pressure (El Khishen et al. 2009). The aphid survives and grows, but does not damage the plant during feeding (Basky 2005, Wratten et al. 1991).
Aphids are able to produce 16-18 generations a year and for these reasons have a huge reproductive potential and therefore are able to evolve novel biotypes which are adapted to the antixenotic and/or antibiotic aspects of plants and thereby break the resistance (Basky 2005).
2.4.1. Schizaphis graminum - Greenbug
Effective resistance breeding programs began in the 1970s against this aphid. Resistance to greenbug has been reported in Triticum turgidum conv. durum, Aegilops squarosa syn.
Triticum tauschii (Castro et al. 1999) and Aegilops speltoides (Castro et al. 2004). A series of resistant varieties were introduced, but each time a new aphid biotype appeared that could damage those varieties. By 1986, 5 biotypes of aphid were known to be pests on the resistant varieties, and today there are 8 aggressive biotypes (Castro et al.1999). These biotypes are distinguishable by their ability to overcome different genes for host plant resistance, which show clear differences in mtDNA (Emden and Harrington 2007).
The first greenbug resistant wheat cultivar, TAM 110 was released in 1997 (Smith et al.
2004).
Twenty-two genes have been characterized in various Gramineae, which express resistance to greenbug (Smith et al. 2004). Six genes have been introduced into wheat to control greenbug. The resistance genes Gb2 and Gb6 derived from rye are located on the wheat/rye chromosome 1RS/1AL. The gene Gb3 in Largo derived from Aegilops tauschii it is located on chromosome 7DL. The gene Gb5 derived from Ae. speltoides was located on chromosome 7S (Castro et al. 2004) and in hexaploid wheat has been introgressed on chromosome 7AL. Mapping positions of Gb1 and Gb4 are unknown (Castro et al. 2005).
2.4.2. Diuraphis noxia – Russian wheat aphid (RWA)
The first D. noxia resistant wheat cultivar was reported by Francoise Du Toit in 1987.
However the first registration of the D. noxia resistant cultivar ‘Halt’- which was a hard red winter wheat - happened in the US in 1996 (Quick et al. 1996). Since then, resistance has been identified in several wheat lines (Qureshi et al. 2005) and today about 300 varieties are known to contain resistance genes.
Previous studies on RWA – wheat interactions showed that the resistance response was associated with the hypersensitive response, including local and systemic accumulation of chitinases, β-1,3-glucanases and peroxidases. The induction of local resistance and systemic resistance is generally accompanied by elevated levels of salicylic acid in dicotyledonous plants. In the defence response of wheat to the RWA, salicylic acid plays an important role by inhibiting catalase activity leading to an accumulation of hydrogen peroxidase (Mohase and Van der Westhuizen 2002 b).
Eleven genes have been characterized and introduced to control RWA. Resistance in Halt arises from a single dominant gene called Dn4 (Randolph et al. 2005, Qureshi et al. 2005), which is located on 1DS chromosome (Arzani et al. 2004, Castro et al. 2004). In 2003 they discovered a new biotype of D.noxia, which breaks down resistance in cultivars which have Dn2 and Dn4 resistance genes (Castro et al. 2005, Qureshi et al. 2005).
Novel resistance-breaking aphid biotypes can evolve when antibiosis is the major component of resistance. Plant resistance to D. noxia is largely based on antibiotic effects, but combining different categories of resistance would be more effective than individual resistance. (Qureshi et al. 2005)
Van der Westhuizen concluded that Dn1 based resistance to D. noxia hinged on the production of pathogenic proteins produced by the plant after aphid damage (Qureshi et al.
2005). Dn1 (Mclntosh et al. 1998), Dn2 (Zheng-Qiang et al. 1995), Dn5 (Heyns et al.
2006), Dn6 (Randolph et al. 2005) and Dnx dominant genes are tightly linked to each other on 7DS near the centromere (Castro et al. 2004, Liu et al. 2005), while 7DL is the location of Dn3 and Dn8. (Castro et al. 2005). Dn9 is located on 1DL (Castro et al. 2004, Castro et al. 2005). Other resistance genes are Dn7, which was derived from Secale cereale (Castro et al. 2005) and Dnk.
