Plants, herbivorous arthropods, nematodes, or vertebrates make up the greater part of the earth's extant macroscopic biodiversity and biomass. Thus, the association between plants and the different types of herbivores is one of the principal ecological interactions in nature [START_REF] Johnson | Evolutionary ecology of plant defences against herbivores: Plant defences against herbivores[END_REF]. This is because of the direct and indirect impacts herbivory generates on biogeochemical processes affecting ecosystem functioning and general community dynamics [START_REF] Nooten | Patterns of insect herbivory on four Australian understory plant species: Insect herbivory on Australian plant species[END_REF][START_REF] Schowalter | Chapter 12-Herbivory[END_REF].

Herbivores generally have adverse effects on plant fitness through reduced plant growth rate, survival and the ability to reproduce [START_REF] Lehndal | Herbivory Differentially Affects Plant Fitness in Three Populations of the Perennial Herb Lythrum salicaria along a Latitudinal Gradient[END_REF]. In temperate and tropical ecosystems, annual crop biomass losses due to herbivory have been reported to be beyond 15% [START_REF] Johnson | Evolutionary ecology of plant defences against herbivores: Plant defences against herbivores[END_REF]. This is due to herbivore consumption of plant compartments [START_REF] Schowalter | Chapter 12-Herbivory[END_REF]. These herbivores constitute both aboveground (AG) and belowground (BG) heterotrophic communities of different taxonomic categories [START_REF] Sharma | Morphological and chemical components of resistance to pod borer, Helicoverpa armigera in wild relatives of pigeonpea[END_REF][START_REF] Van Dam | Multitrophic interactions below and above ground: En route to the next level: Belowground-above-ground interactions[END_REF]. Tissue chewing herbivores feed on large chunks of leaves, stems, flowers, pollen, seeds, and roots [START_REF] Bennett | Fungal-Mediated Above-Belowground Interactions: The Community Approach, Stability, Evolution, Mechanisms, and Applications[END_REF]. Other herbivores have different feeding mechanisms. For instance, aphids are sap-suckers that siphon plant fluids from the phloem [START_REF] Schoonhoven | Insect-plant biology[END_REF]. Tissue borers and miners feed between plant surfaces and gallforming herbivores feed within plants and give rise to abnormal tissue growths [START_REF] Nooten | Patterns of insect herbivory on four Australian understory plant species: Insect herbivory on Australian plant species[END_REF]. Frugivore herbivores have a preference of feeding on the reproductive plant parts [START_REF] Schowalter | Chapter 12-Herbivory[END_REF].

To reduce herbivore pressure, plants respond with a multitude of defence strategies [START_REF] Nooten | Patterns of insect herbivory on four Australian understory plant species: Insect herbivory on Australian plant species[END_REF]). An array of constitutive and inducible defence strategies is employed to address multiple attacks from both pathogens and herbivores [START_REF] Boots | The evolution of constitutive and induced defences to infectious disease[END_REF]. Constitutive defence response is expressed irrespective of external stimuli [START_REF] War | Herbivore induced plant volatiles: Their role in plant defense for pest management[END_REF]. For example, trichomes in strawberries that limit the movement of the mite, Tetranychus urticae, is a structural constitutive trait [START_REF] Figueiredo | The role of glandular and non-glandular trichomes in the negative interactions between strawberry cultivars and spider mite[END_REF]. Chemical constitutive responses include the production of alkaloids, phenolics [START_REF] Kaplan | CONSTITUTIVE AND INDUCED DEFENSES TO HERBIVORY IN ABOVE-AND BELOWGROUND PLANT TISSUES[END_REF] and terpenoids such as diterpenoids which dissuade feeding of corn borer larvae on Zea mays [START_REF] Schmelz | Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize[END_REF]. Contrastingly, induced defence is only switched on and must develop rapidly to full strength in the presence of herbivore attack. [START_REF] Han | Serotonin Regulates the Feeding and Reproductive Behaviors of Pratylenchus penetrans[END_REF].

Incompatible plant-pathogen responses induce a cell death process at the infection site. This locally induced response is referred to as hypersensitive response (HR). HR then activates a signaling process resulting into systemic acquired resistance (SAR) throughout the plant [START_REF] Jordá | Local and Systemic Induction of Two Defense-Related Subtilisin-Like Protease Promoters in Transgenic Arabidopsis Plants. Luciferin Induction of PR Gene Expression[END_REF]. [START_REF] Agrawal | Induced Responses to Herbivory and Increased Plant Performance[END_REF] found that inducing wild radish (Raphanus sativus L. Brassicaceae) by Pieris rapae increased plant fitness in presence of herbivores compared to un-induced (control) plants. This is because induced plant defences are "immune-like" in nature and deter the performance or preference of herbivores. Several works have illuminated that herbivores of different feeding guilds induce a specific set of plant defence responses [START_REF] Rodriguez-Saona | Molecular, Biochemical, and Organismal Analyses of Tomato Plants Simultaneously Attacked by Herbivores from Two Feeding Guilds[END_REF][START_REF] Moreira | Interactions between plant defence signalling pathways: Evidence from bioassays with insect herbivores and plant pathogens[END_REF]Fernández de Bobadilla et al., 2021). Such responses can be modulated by phytohormones [START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF]. The main defence signalling phytohormones are jasmonic acid (JA), salicylic acid (SA) (Bouarab et al., 2009), ethylene (ET) and Abscisic acid (ABA) [START_REF] Walters | PLANT DEFENSE : Warding off Attack by Pathogens, Herbivores, and Parasitic Plants[END_REF]Pieterse et al., 2014).

SA is crucial for defence against biotrophs such as sap-sucking aphids, and hemi-biotrophs [START_REF] Walters | PLANT DEFENSE : Warding off Attack by Pathogens, Herbivores, and Parasitic Plants[END_REF]. JA is induced by tissue damage or feeding by tissue chewing insects (Wang & Wu, 2013).

Depending on the plant species, these phytohormones can antagonise each other resulting into phytohormonal crosstalk [START_REF] Thaler | Evolution of jasmonate and salicylate signal crosstalk[END_REF]. However, in some plant-herbivore complex like rice-M. graminicola, SA and JA biosynthesis genes are altogether suppressed in the giant cells (Kyndt et al., 2014).

Defence compounds are not only concentrated in localised damaged plant parts but are transported to other undamaged plant compartments for their protection in the future. Such a response can also influence how other organisms feeding on the same plant at other locations perform (Wang & Wu, 2013;[START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF]. Phytohormones have been shown to induce the synthesis of GSL in Brassicas. External application of JA in B. oleracea roots induced aliphatic GSL in shoots and when applied to shoots, indole GSL were induced. SA application to shoots lead to low or no increase in indole GSL levels in shoots [START_REF] Van Dam | Root and shoot glucosinolates: A comparison of their diversity, function and interactions in natural and managed ecosystems[END_REF].

Brassicaceae plant defence constituents

The Brassicaceae family is one of the most economically important plant groups with 338 genera and 3709 species identified worldwide. Brassicaceae contains many important food plants with edible species, such as Brassica oleracea and B. rapa. Other species, for instance, Brassica juncea, Sinapis alba and Erysimum spp are sources of condiments. Brassica napus (canola) is an important oil crop and species like Camelina sativa and Eruca vesicaria are sources of biodiesel [START_REF] Gidik | Study of wild plant species of Brassicaceae family in Bayburt region of Turkey[END_REF].

Brassica nigra (black mustard) is a herbaceious plant that grows in temperate environments worldwide. The plant is rich in phytochemicals such as alkaloids, flavonoids, and glycosides among others. B. nigra has been applied in numerous pharmacological uses including roles as antioxidants, anti-inflammatory and antiepileptic activities [START_REF] Al-Snafi | The pharmacological importance of brassica nigra and brassica rapa grown in iraq[END_REF].

Glucosinolates (GSL)

The Brassicaceae family is known to secret secondary defence metabolites called glucosinolates, which are non-volatile, β-thioglucoside N-hydroxysulphates containing two sulphur atoms (Figure 1).

One sulphur atom originates from the metabolism of amino acid cysteine and the other from phosphoadenosine phosphosulphate with a β-D-glucopyranose moiety, a sulphonated oxime group and a variable aglycone side chain [START_REF] Mewis | Major Signaling Pathways Modulate Arabidopsis Glucosinolate Accumulation and Response to Both Phloem-Feeding and Chewing Insects[END_REF][START_REF] Avato | Nematicidal potential of Brassicaceae[END_REF]. The side chain (R) originates from one of eight amino acids [START_REF] Mewis | Major Signaling Pathways Modulate Arabidopsis Glucosinolate Accumulation and Response to Both Phloem-Feeding and Chewing Insects[END_REF][START_REF] Ahuja | Defence mechanisms of Brassicaceae: Implications for plant-insect interactions and potential for integrated pest management. A review[END_REF]. If derived from methionine, GSL are considered aliphatic. They are benzoyl GSL if the R-group is based on phenylalanine or indolic GSL if derived from tryptophan [START_REF] Chhajed | Glucosinolate Biosynthesis and the Glucosinolate-Myrosinase System in Plant Defense[END_REF]. GSL are expressed both constitutively and upon induction by pathogens, insects and nematodes. They play defensive roles for Brassica plants since they manifest as herbivore repellents or have toxic effects [START_REF] Hol | Heterodera schachtii Nematodes Interfere with Aphid-Plant Relations on Brassica oleracea[END_REF]. Primarily, isothiocyanates (ITCs) are biocides when they interact with proteins and amino acids. Benzyl GSL were found to be 10 folds less toxic than its ITCs after hydrolysis on soil dwelling springtail Folsomia fimetaria [START_REF] Jensen | The toxic effects of benzyl glucosinolate and its hydrolysis product, the biofumigant benzyl isothiocyanate, to Folsomia fimetaria[END_REF]. Besides their toxicity, ITCs also function as gustatory stimulants for specialist insects and help phytophagous insects locate host plants [START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF]. Additionally, ITCs and Nitriles from the hydrolysis of aliphatic and aromatic GSL confer indirect plant resistance as they act as volatile cues for herbivore natural enemies [START_REF] Hopkins | Role of Glucosinolates in Insect-Plant Relationships and Multitrophic Interactions[END_REF].

The glucosinolate-myrosinase (GSL-MYR) defence system GSL can be found in vacuoles of various plant cells. In plants, GSL and myrosinase (MYR) are spatially separated and are stored in inactive glucosylated forms to keep the plant safe from toxic effects of its defence system [START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF]. Myrosin cells occur at various locations in stems, leaves, seeds, seedlings, petioles and roots [START_REF] Ahuja | Defence mechanisms of Brassicaceae: Implications for plant-insect interactions and potential for integrated pest management. A review[END_REF]. These cells contain myrosin grains which form a continuous reticular system called the myrosin body. Myrosinase enzymes also refered to as βthioglucoside glucohydrolase, EC 3.2.3.1 are stored within the myrosin grains [START_REF] Pontoppidan | Infestation by cabbage aphid ( Brevicoryne brassicae ) on oilseed rape ( Brassica napus ) causes a long lasting induction of the myrosinase system: Cabbage aphid infestation of oilseed rape[END_REF]. Injury to plant tissues links glucosinolates and myrosinase, hence, allowing the degradation of glucosinolates by myrosinase and the subsequent release of biologically active compounds for instance, isothiocyanates, nitriles, and epithionitriles [START_REF] Ahuja | Defence mechanisms of Brassicaceae: Implications for plant-insect interactions and potential for integrated pest management. A review[END_REF][START_REF] Szczygłowska | Use of Brassica Plants in the Phytoremediation and Biofumigation Processes[END_REF] (Figure 2). Isothiocyanates are always released in a higher concentration while nitriles in a lower concentration upon GSL hydrolysis. Nitrile production requires plants to express Epithiospecifier proteins for their formation at the expense of isothiocyanates [START_REF] Halkier | Biology and biochemistry of glucosinolates[END_REF].

The coming together of glucosinolates and myrosinase after tissue damage is referred to as the glucosinolate-myrosinase defence system or the 'mustard oil bomb' [START_REF] Avato | Nematicidal potential of Brassicaceae[END_REF]. This defence system is a two-edged sword. Generalist herbivores that are affected by the toxicity of GSL breakdown products are warded off on one side and on the other side, plants become more vulnerable to specialist feeders such as Brevicoryne brassicae which sequesters GSL for its defence against predation [START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF]. Therefore, it is beneficial for plants to respond to pressure by such a specialist herbivore through rapid regrowth or compensation rather than by overproduction of glucosinolates (Sotelo et al., 2014). 1.3. The cabbage aphid, Brevicoryne brassicae: Feeding behaviour and coping with glucosinolates Aphids are part of the over one million plant-feeding insect species that have been described so far [START_REF] Jaouannet | Plant immunity in plant-aphid interactions[END_REF]. They are important pests in both oilseed and vegetable Brassicas [START_REF] Cole | Comparison of feeding behaviour of two Brassica pests Brevicoryne brassicae and Myzus persicae on wild and cultivated brassica species[END_REF][START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF] and are the main agricultural pests in northern Europe that cause damage to both field and greenhouse crop production [START_REF] Ahuja | Defence mechanisms of Brassicaceae: Implications for plant-insect interactions and potential for integrated pest management. A review[END_REF]. In contrast to herbivores with chewing mouthparts, aphids are phloem sap-sucking pests [START_REF] Goggin | Plant-aphid interactions: Molecular and ecological perspectives[END_REF][START_REF] Dinant | Phloem sap intricacy and interplay with aphid feeding[END_REF][START_REF] Nalam | Plant defense against aphids, the pest extraordinaire[END_REF]. Albeit small, they can cause major plant losses through reduced plant growth, wilting and eventual death of the plant in severe cases of infestation [START_REF] Will | How phloem-feeding insects face the challenge of phloem-located defenses[END_REF]. Some aphids are general feeders and infest plants belonging to a vast range of families. However, most aphids are specialised and are restricted to feeding on a single-family or few closely related plant species [START_REF] Blackman | Taxonomic Issues[END_REF].

Brevicoryne brassicae is a host specific herbivore that feeds on plants in the Brassicaceae family [START_REF] Cole | Comparison of feeding behaviour of two Brassica pests Brevicoryne brassicae and Myzus persicae on wild and cultivated brassica species[END_REF][START_REF] Lankau | Specialist and generalist herbivores exert opposing selection on a chemical defense[END_REF]. This aphid's life stages overlap through 15 generations throughout the crop season. This is due to their rapid development time of 8-12 days from the first instar nymph to adult, asexual reproduction, and an extended reproductive life-span of 30 or more days at 4-6 nymphs per day [START_REF] Pal | Biology and ecology of the cabbage aphid, Brevicoryne brassicae (linn.) (homoptera : aphididae)[END_REF]. Climate determines the type of life cycle of B. brassicae and adult females reproduce sexually and asexually. During warm climates or mild winter, they are anholocyclic and reproduce parthenogenetically. In the course of this period, females are viviparous and give birth to nymphs. As the temperature and photoperiod decrease during autumn, it is holocyclic. Winged males and apterous oviparous females appear and sexual reproduction occurs resulting in laying of overwintering eggs. Overcrowding and a decline in plant quality leads to appearance of winged morphs. The older nymphs and adults then leave the plant and move to other plants or other parts within the same plant. Photoperiod and temperatures below 10-15°C for at least 24h also cause development of winged aphids [START_REF] Gabrys | Cabbage Aphid, Brevicoryne brassicae (L.) (Hemiptera: Aphididae)[END_REF]. B. brassicae prefers young leaves and stems in spite of the high glucosinolate content in these parts of brassica plants. This is as a means to limit competition for nutrition [START_REF] Hopkins | Influence of increasing herbivore pressure on modification of glucosinolate content of swedes (Brassica napus spp. rapifera)[END_REF]. The piercing and sucking mouthpart (stylet) penetrates plant cells in an intercellular manner before reaching the phloem to facilitate feeding [START_REF] Miles | Aphid saliva[END_REF]. This implies that additional mechanical injury may not induce plant defence in contrast to tissue chewing herbivores (Zhu-Salzman et al., 2005). Consequently, GSL are not hydrolysed and the aphid is not challenged by harmful products of aromatic and aliphatic glucosinolate hydrolysis. The hydrolysis of indole GSL does not require the presence of myrosinase due to their instability, hence, can pose negative impacts on the aphid [START_REF] Hopkins | Role of Glucosinolates in Insect-Plant Relationships and Multitrophic Interactions[END_REF]. Nonetheless, the aphid's stylet may rupture the cells of the epidermis, mesophyll, and parenchyma while probing and inturn trigger plant defence responses [START_REF] Tjallingii | New structure in cell puncture activities by aphid stylets: A dual-mode EPG study[END_REF][START_REF] Will | How phloem-feeding insects face the challenge of phloem-located defenses[END_REF]. This structural damage brings inactive precursors into contact with myrosinase enzyme to hydrolyse GSL [START_REF] Züst | Mechanisms and evolution of plant resistance to aphids[END_REF].

B. brassicae can ingest and selectively sequester GSL intact and dodge their toxicity [START_REF] Goodey | Aphids Pick Their Poison: Selective Sequestration of Plant Chemicals Affects Host Plant Use in a Specialist Herbivore[END_REF][START_REF] Nalam | Plant defense against aphids, the pest extraordinaire[END_REF]. Aliphatic glucosinolates are sequestered mostly due to their capability for defense against predation compared to Indole GSL which may be toxic to the aphid [START_REF] Kos | Prey-mediated effects of glucosinolates on aphid predators[END_REF].

With the aid of a coevolved myrosinase enzyme called thioglucosidase [START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF], the sequestered glucosinolates are hydrolysed into toxic products which protect the aphid against its natural enemies [START_REF] Goodey | Aphids Pick Their Poison: Selective Sequestration of Plant Chemicals Affects Host Plant Use in a Specialist Herbivore[END_REF][START_REF] Kumar | Plant secondary metabolites (PSMs) of Brassicaceae and their role in plant defense against insect herbivores -A review[END_REF]. When these aphids are crushed, they release volatiles which can alarm other aphids in the colony [START_REF] Ahuja | Defence mechanisms of Brassicaceae: Implications for plant-insect interactions and potential for integrated pest management. A review[END_REF]. Disturbed aphids can as well release alarm pheromones that warn conspecifics of predators. For example, isothiocyanates together with (E)-βfarnesene serve as alarm signals in the case of mustard aphids (Lipaphis erysimi) [START_REF] Pontoppidan | Infestation by cabbage aphid ( Brevicoryne brassicae ) on oilseed rape ( Brassica napus ) causes a long lasting induction of the myrosinase system: Cabbage aphid infestation of oilseed rape[END_REF].

Aphid probing behaviour determines host selection as the aphid samples for sucrose and glucosinolates as chemical cues on the leaf surface [START_REF] Gabrys | Cabbage Aphid, Brevicoryne brassicae (L.) (Hemiptera: Aphididae)[END_REF], in myrosin cell vacuoles, and within the apoplast or phloem [START_REF] Cole | Comparison of feeding behaviour of two Brassica pests Brevicoryne brassicae and Myzus persicae on wild and cultivated brassica species[END_REF]. Feeding is usually accompanied by ejection of saliva containing effectors into different cells (Figure 3(B1)). The stylet punctures the mesophyll cells, more saliva containing effectors is ejected (Figure 3(B2)) and the aphid determines the suitability of the host plant. As a result, the plant responds to elicitors contained in the saliva [START_REF] Miles | Aphid saliva[END_REF]. Plants respond by clogging the phloem with callose (Figure 3(B3)) to block the flow of phloem contents from reaching the stealthy aphid [START_REF] Will | Physical and chemical interactions between aphids and plants[END_REF]. Protein plugs first seal the sieve plates then callose is deposited to occlude the sieve elements [START_REF] Nalam | Plant defense against aphids, the pest extraordinaire[END_REF] (Figure 3(B4)). Both gelling (egested during penetration) and watery saliva (egested during substrate ingestion) are secreted by aphids. Gelling saliva forms a sheath around the stylet and minimises damage to the tip of the stylet during probing [START_REF] Louis | Arabidopsis thaliana-Myzus persicae interaction: Shaping the understanding of plant defense against phloem-feeding aphids[END_REF]. The sheath also plugs any plant tissue wounds due to probing, hence, limiting any wounding defences by the plants. The watery saliva contains calcium-binding proteins which antagonise wound healing making the plant a suitable host. The aphid then feeds continuously from a single sieve tube element by reversing the phloem occlusion [START_REF] Will | Aphid watery saliva counteracts sieve-tube occlusion: A universal phenomenon[END_REF].

Plant secondary metabolites are secreted (Figure 3(B5)) and transported to the phloem tubes (Figure 3(B6)). The aphid ingests these metabolites which are then absorbed into the haemocoel (Figure 3(B7)) and excreted into the hind gut [START_REF] Nalam | Plant defense against aphids, the pest extraordinaire[END_REF][START_REF] Züst | Mechanisms and evolution of plant resistance to aphids[END_REF] (Figure 3(B8)). The metabolites maybe exuded together with honeydew (Figure 3(B9)) and molecules in honeydew may alter plant defence responses [START_REF] Jaouannet | Plant immunity in plant-aphid interactions[END_REF][START_REF] Züst | Mechanisms and evolution of plant resistance to aphids[END_REF]).

Nematode Herbivory: Feeding and invasion mechanism

Nematodes are recognised as the most abundant soil-dwelling multicellular animals [START_REF] Lazarova | Functional Diversity of Soil Nematodes in Relation to the Impact of Agriculture-A Review[END_REF]. Most nematodes are free-living and feed primarily on bacteria and fungi while others are parasitic to plants. They are referred to as plant-parasitic nematodes (PPNs). They may also be referred to as 'eelworms' and have detrimental effects on plants, resulting in stunted growth and early senescence. PPNs are of considerable importance and cause a great loss of agricultural produce estimated at about 80 billion USD annually [START_REF] Sato | Plant Immune Responses to Parasitic Nematodes[END_REF]. In dire nematode infestations, it can cause total crop losses [START_REF] Perry | Introduction to Plant-Parasitic Nematodes; Modes of Parasitism[END_REF]. In the same wayas the aphids, PPNs are serious pests with piercing-sucking feeding habits. They either invade the root systems and obtain nourishment from the root cortex or live in soil and feed on rhizodermalcells of plants [START_REF] Avato | Nematicidal potential of Brassicaceae[END_REF]. They inject effector-containing esophageal secretions into their hosts. This allows them to tune several plant metabolic processes to their advantage. Different nematode species inject specific salivary compounds and the plant may recognise and use this to respond accordingly (Ghaemi et al., 2020).

Most PPNs are polyphagous and are grouped into three feeding modes; ecto-parasitic, endo-parasitic and semi-endoparasitic. Ecto-parasitic PPNs do not invade the plant roots but remain in the soil and use their stylets to puncture the plant cells. Some are motile while others have long stylets and form permanent attachments to the plant roots. The entire life cycle occurs outside the host. Endoparasitic nematodes completely move inside the root. They can cause significant damage during migration (Pratylenchuspenetrans), feeding (root-knot nematodes) or at both infection stages (cyst nematodes).

The last type is the semi-endoparasitic nematodes which insert only the anterior part in the roots to feed while the posterior region remains in the soil (Perry & Moens, 2006;[START_REF] Sato | Plant Immune Responses to Parasitic Nematodes[END_REF].

Root-knot nematodes: Meloidogyne incognita

Root-knot nematodes (RKNs) are endo-parasitic, polyphagous and obligate pathogens in nature. They produce galls/knots on roots and disrupt the plant's physiology [START_REF] Abad | Plant Nematode Interaction: A Sophisticated Dialogue[END_REF]. M. incognita reproduces by obligate parthenogenesis and the males are completely excluded in this process [START_REF] Sato | Plant Immune Responses to Parasitic Nematodes[END_REF]. The infective growth stage, second juvenile stage (J2), uses its stylet to probe the growing tip of the root in the elongation zone during invasion. J2 moves in an intercellular pattern through the root and reaches the vascular cylinder. Here, it secrets effectors that modify the host plant's target cell's physiology to induce and maintain giant feeding cells during the pathogen's establishment stage [START_REF] Mitchum | Nematode effector proteins: An emerging paradigm of parasitism[END_REF]. The J2 then becomes sedentary as it feeds and moults through three cycles to develop into adults. Adult M. incognita females remain sedentary and the vermiform males are motile and exit the root. On or below the root surface, the adult female lays an egg mass that is protected by a gelatinous matrix [START_REF] Abad | Plant Nematode Interaction: A Sophisticated Dialogue[END_REF]. Giant cell formation serves to provide nutrition for the nematode's growth and production of eggs (Figure 4). Regions with induced giant cell formation enlarge as the juveniles become adults. The female expands in size as it is filled with developing eggs and the roots are enlarged further. Eggs are released following a rupture of the roots and a new cycle begins [START_REF] Tattar | Nematodes[END_REF]. During the establishment of parasitic life, enzymes such as xylanase and polygalacturonase are secreted to facilitate migration by weakening or breaking down host plant cell walls [START_REF] Perry | Introduction to Plant-Parasitic Nematodes; Modes of Parasitism[END_REF]. Other enzymes that are produced include; ENGs(Endoglucanases), PELs(Pectate lyases), CBPs(Cellulose binding proteins)and enzymes that target the shikimate pathway, specifically, CM(chorismate mutase). Additionally, M. incognita secretions with immunosuppressive activity can manipulate host plant defence systems. The MiMsp40 specific gene in M. incognita suppresses PTI and/or ETI initiation to establish a parasitic life [START_REF] Niu | Msp40 effector of root-knot nematode manipulates plant immunity to facilitate parasitism[END_REF].

Root lesion nematodes (Pratylenchus penetrans)

In comparison to other parasitic nematodes such as the cyst and RKNs, root lesion nematodes have been studied less. This is because unlike cyst and RKNs, Pratylenchus sp remain mobile throughout development and feed both endo and ecto-parasitically on root hairs of the host plant [START_REF] Jones | Molecular biology of root lesion nematodes ( Pratylenchus spp.) and their interaction with host plants: Molecular biology of root lesion nematodes[END_REF]. This results in unique damage dynamics that may trigger different plant responses in comparison to that by sedentary RKNs (Holet al., 2016). Adult females lay eggs either inside infested roots or deposit eggs singly or in groups outside of the host's roots in adjacent soil [START_REF] Han | Serotonin Regulates the Feeding and Reproductive Behaviors of Pratylenchus penetrans[END_REF].

The nematode develops within the egg to form the first juvenile (J1) stage which moults into J2s. J2 hatches from the egg and together with the adults, can enter and leave the roots (Figure 5) [START_REF] Jones | Molecular biology of root lesion nematodes ( Pratylenchus spp.) and their interaction with host plants: Molecular biology of root lesion nematodes[END_REF]. Both juvenile and adult stages are infective and depending on the temperature, the life cycle may last for 4 weeks [START_REF] Van Dam | Root herbivory reduces growth and survival of the shoot feeding specialist Pieris rapae on Brassica nigra[END_REF]. P. penetrans primarily penetrates roots in the region of root hair development and the J2s feed on cortical cells in the elongation zone [START_REF] Zunke | Observations on the Invasion and Endoparasitic Behavior of the Root Lesion Nematode Pratylenchus penetrans[END_REF]) (Table 1). In a similar to M. incognita, P. penetrans ejects a repertoire of parasitism related digestive and metabolic secretions for its successful penetration and invasion of the host. Infection results in the formation of root lesions, necrosis, reduction in root growth and eventual cell death [START_REF] Siddiqi | Tylenchida. Parasites of Plants and Insects, 2nd edn[END_REF][START_REF] Vieira | Cellular and Transcriptional Responses of Resistant and Susceptible Cultivars of Alfalfa to the Root Lesion Nematode, Pratylenchus penetrans[END_REF].

Study objectives

As defence chemicals in Brassicas, glucosinolates remain a significant research area with its roles in the plant-herbivore associations [START_REF] Nguyen | Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities[END_REF]. Plants face an increasing and various herbivore and pathogen pressure throughout their life cycle and it is important to understand the dynamic nature of the GSL defence system as influenced by herbivores. It has been established that individual GSL concentrations and proportions alter as induced by several pest species, specifically, turnip root fly [START_REF] Hopkins | Influence of increasing herbivore pressure on modification of glucosinolate content of swedes (Brassica napus spp. rapifera)[END_REF], flea beetles and diamondback moth (biting insects), nematodes [START_REF] Hol | Heterodera schachtii Nematodes Interfere with Aphid-Plant Relations on Brassica oleracea[END_REF] as well as due to mechanical wounding [START_REF] Pontoppidan | Infestation by cabbage aphid ( Brevicoryne brassicae ) on oilseed rape ( Brassica napus ) causes a long lasting induction of the myrosinase system: Cabbage aphid infestation of oilseed rape[END_REF]. Generally, herbivores with biting mouthparts cause relatively large quantities of disruptions to cells around the feeding sites.

Consequently, this readily induces plant glucosinolate defense response. However, phloem feeders such as B. brassicae have been reported to avoid glucosinolate induction due to its intercellular mechanism of feeding which minimises tissue damage (Zhu-Salzman et al., 2005). GSL require structural damage to bring inactive precursors into contact with a spatially separated activating myrosinase enzyme [START_REF] Züst | Mechanisms and evolution of plant resistance to aphids[END_REF].

Root feeding PPNs have been reported to generally affect production of shoot metabolites [START_REF] Wondafrash | Plant systemic induced responses mediate interactions between root parasitic nematodes and aboveground herbivorous insects[END_REF][START_REF] Hol | Heterodera schachtii Nematodes Interfere with Aphid-Plant Relations on Brassica oleracea[END_REF] and also influence reproduction of B. brassicae [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF]. [START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF] established that through systemically induced phytohormone responses (SA and JA), B. brassicae feeding AG is confronted by nematodes feeding BG. B. nigra infested by M. incognita, showed enhanced B. brassicae performance while infestation by the cyst nematode, H. schachtii, showed negative results. Therefore, regardless of B. brassicae host specificity and ability to sequester glucosinolates, its performance and reproduction can still be affected by nematode feeding.

Most of the aforementioned studies on AG-BG signalling interactions with aphids and PPNs have involved a single nematode species of either root-knot, root-lesion or cyst nematodes. Here, the study looks into B. brassicae performance and life history when confronted by either RKNs or RLNs and a combination of both nematodes feeding BG. When roots emerge out of a seed, it is faced with numerous soil herbivores and pathogens among which nematodes form part. Plant roots are frequently parasitized by PPNs in mixed populations of two or more genera and species. In the dynamic PPN communities, there's a constant interaction with each other and the nature of this interaction dtermines how plants respond to multi-herbivores. The first hypothesis is that the observed aphid performance and life history will depend on the BG nematode species. This is on the basis of the differences in the feeding patterns employed by the two nematode species as has been reported in studies by [START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF] and [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF].

This research also finds it important to understand how GSL profiles and concentrations vary with root damage resulting from feeding by RKNs and RLNs, and how it correlates with the observed aphid performance and life history parameters. Migratory endo-parasites are also generally considered as less advanced parasites in comparison to sedentary endo-parasites such as M. incognita. This is due to the ability of M. incognita to develop a complex relationship which alters its host plant's physiology. This can inturn affect host suitability of the for the migratory endo-nematode. On the contrary, P. penetrans has a simpler relationship with the plant and can suppress M. incognita due to disruption of feeding sites created by its migratory pattern depending on the host plant (Khan,1993). Therefore, the second hypothesis is that nematode induced changes in glucosinolate concentration would result from the effect of a more dorminant species between the two nematodes when in combination. This is because both these species occupy the same feeding site and employ different feeding behaviours with different impacts on the surrounding feeding site (Table 1).

In Glucosinolates extraction and detection.

GSL were extracted following the protocol by [START_REF] Grosser | A Straightforward Method for Glucosinolate Extraction and Analysis with Highpressure Liquid Chromatography (HPLC)[END_REF]. In brief, a column rack for the Pasteur pipette column was setup and the columns were labelled in accordance with the sample numbers (Figure 7). A block holding 2mL microcentrtifuge tubes was also setup. 0.5 mL of dextran gel, type G-25 gel (column material) was prepared and pipetted into each column and flashed down the column with 1 mL of ultrapure water. Plant materials (roots and leaf samples) were freeze dried at -80°C and then finely ground under liquid nitrogen. The plant materials were then weighed (50mg per sample) and extracted with 1 mL of 70% MeOH (methanol) solution and boiled in a hot water bath at 90°C for 5 minutes. The samples were then placed in an ultrasonic bath for 15 min and centrifuged at 2,700 x g in a benchtop centrifuge for 10 min at room temperature (RT). The resulting supernatants were then added to the labelled columns and five sinigrin columns were setup as references (Figure 6).

Each column was then washed twice with 1 mL of 70% MeOH to remove apolar compounds from the extracts. The MeOH was flushed out by 1 mL of ultrapure water in each column. 1 mL of 20mM

NaOAc (Sodium acetate) buffer was pipetted twice into each column to create optimal conditions for sulfatase performance. The rack was then placed over the block containing the microcentrtifuge tubes and 20 μL of sulfatase solution was added to each column. 50 μL of NaOAc buffer was added into each column to ensure that the sulfatase reaches the surface of the column material and left to stand overnight. The next day, 2 x 0.75 mL of ultrapure water was added into each column to elute the desulfoglucosinolates released by the activity of sulfatase. The reaction tubes were then removed and placed in a -80 °C freezer for 30 min before freeze drying for 24 h. After freeze drying, each residue was re-dissolved in 1 mL of ultrapure and transferred to HPLC vials and analysed with HPLC. GSL were separated on a reversed-phase C 18 column (4.6 x 150 mm, 3 μm, 300 Å) on High-pressure Liquid Chromatography (HPLC) with an acetonitrile-water gradient from 0 to 30 min and a flow of 0.75 mL/min. Detection was performed using ultraviolet(UV) detector.

Statistical analysis

All data was analysed using RStudio Version 1.3.959 software. Parametric tests were done using ANOVA, and the means were compared using the post-hoc Tukey's honestly significant difference (HSD) tests. When variances did not meet the assumptions of ANOVA, the data was transformed for variances to meet normality and when it failed, non parametric tests were performed. 3. Results To check the effect of time and nematode feeding on the total concentration of glucosinolates in the roots, it was hypothesized that allowing more feeding time for nematodes would significantly affect glucosinolate concentration. This was on the basis of differences in induced responses initiated as the nematodes penetrate cells, feed and establish a parasitic life. An analysis of variance (ANOVA) was used to test this hypothesis. The results revealed that both nematode treatment (F(3,24) = 1.250, p=0.313) and time (F(1,24) = 0.615, p=0.440) did not cause a significant change in the total concentration of GSL in the roots. However, the main effect on GSL concentration in roots was the interaction between time and nematode feeding (F(3,24) = 6.342, p=0.012). A post hoc Tukey test showed that Pp treatment at 10dpni significantly increased glucosinolate concentrations by 2.4 fold compared to 7dpni (TukeyHSD, p=0.043). No other statistically significant nematode treatments with timewas observed. However, the general trend is a decrease with time except the Pp treatment group that increased at 10dpni (Figure 7).

Aphid performance and life history

Looking into the GSL classes, indole GSL levels did not show statistically significant differences due to effects of either time (Anova, F(1,24 ) = 0.565 , p=0.460), nematode treatment (Anova, F(3,24) = 2.159 , p=.119) or their interaction (Anova, F(3) = 0.929, p=0.442). However, the trend followed a decrease with time except for treatment Pp that produced an increase of 1.4 folds (TukeyHSD,p= 0.983) between the two time points (Figure 8(A)).

Nonetheless, individual indole GSL were marginally affected by treatments. Nematode impacts on 4methoxyglucobrassicin (4MeOH) levels showed a distinct tendency towards statistical significance (Anova, F(3,24)= 2.645, df=3, p=.07). Time was not a significant determining factor (Anova, F(1,24)= 1.640, df=1, p>0.05). Pp treatment (TukeyHSD, p>0.05) and MP (TukeyHSD, p>0.05) showed statistically non-significant increases in levels of 4MeOH by 1.9 fold and 1.27 fold respectively at 10dpni. Mi treatment was as well not statistically (TukeyHSD, p>0.05) (Figure 9 Sinigrin, as the predominant Aliphatic glucosinolate in Brassicas [START_REF] Nguyen | Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities[END_REF] was strongly influenced by time-nematode treatment interaction (Anova, F(3) = 5.4164, p=0.005). It was also the most consistent GSL with high concentrations. After 10dpni, Sinigrin levels increased with Pp treatment by 3.8 fold (TukeyHSD, p=0.028). No statistically significant results were observed for other treatments groups. However, the trend also followed a decrease with time (Figure 9 (B)).

Both nematode treatment (Anova,F(3,24) =3.502, p=0.0308) and the interaction with time (Anova, F(3,24) = 3.419, p=0.033) produced significant effects on levels of Benzenic glucosinloates in roots.

NAS/Gluconasturtiin was the only detected Benzenic GSL. Pp treatment decreased NAS levels by 2.7 folds compared to Ctrl at 7dpni (TukeyHSD, p=0.023). MP plants 10dpni also showed a decrease in NAS levels when compared to Ctrl at 7dpni (TukeyHSD, p=0.029). All other nematode treatment groups and interaction with time were not significant enough to cause any changes on the level of NAS (Figure 9(C)).

Systemic induction of glucosinolates in young uninfected leaves after nematode treatment

The total glucosinolate concentration in young uninfected leaves was not affected by nematode treatment (Anova, F(3,24) = 0.363, p>0.05), time (Anova, F(1,24) = 0.937, p>0.05) or their interaction (Anova, F(3,24) = 0.593 , p>0.05) (Figure 11).

Allowing more nematode feeding time 10dpni did not cause a significant result on the total concentration of indole GSL in young leaves (Anova, F(1,24) = 0.007, p>0.05). Nematode treatment (Anova, F(3,24) = 1.066, p>0.05) and its interaction(Anova, F(3,24) = 1.159, p>0.05) with time was not strong enough to cause a statistically significant result either (Figure 12 (A)). In spite of this, the trend shows an increasing amount of indole glucosinolates with nematode treatment compared to control plants.

The main glucosinolate (sinigrin) was not significantly affected by either nematode treatment (Anova, F(3,24) = 0.366, p>0.05) , nematode feeding time (Anova, F(1,24) = 0.943, p>0.05) or the interaction between time and treatment groups (Anova, F(3,24) = 0.601, p>0.05) (Figure 12(B)).

Neither nematode treatments (Kruskal-Wallis, df = 3, p-value = 0.097) nor 3 days of aphid feeding (Kruskal-Wallis, df = 1, p-value = 0.612) resulted into a statistically significant change in NAS levels (Figure 12(C)). NAS levels in young uninfected leaves was not obervered with Pp treatment 10dpni.

However, when compared to the same treatment at 7dpni, the difference was not significant (Dunnett test, Z=1.30, p= 0.899) or with the control plant 10dpni (Dunnett test, Z=1.93, p= 1). From the control plants, the general trend follows a decreasing pattern with nematode treatments except the MP group.

This shows an increase in NAS levels 10dpni by 5 fold compared to plants at 7dpni. However, this was also statistically insignificant (Dunnett test, Z=-1.47, p= 1). Mi treatments similarly did not produce statistically significant results between the two time-points (Dunnett test, Z=-0.09, p= 0.928). Identical letters represent no statistical significant differences between groups with time and treatment.

3.4. Induction of glucosinolates in infected leaves by nematodes and aphids.

Nematode infestation 10dpni (Anova, F(3,24) = 0.079, p>0.05) and aphid feeding 3dpai (Anova, F(1,24) = 0.36, p>0.05) did not cause a significant effect on the total concentration of glucosinolates in infected leaves in nematode free plants (Ctrl) and nematode inoculated plants. The aphid-nematode interaction was as well insignificant on the total concentration (Anova, F(3,24) = 1.388, p>0.05) (Figure 13).

In Figure 14 (A), an Analysis of variance revealed that total indole GSL concentrations varied with nematode treatment (F(3,24) = 9.268, p<0.001) and nematode-aphid interaction (F(3,24) = 2.795, p=0.062). Aphid presence was statistically insignificant (F(1,24) = 4.341, p>0.05). With a post hoc Tukey test, it was observed that in the absence of aphids, Pp treatment increased indole GSL levels by 3 fold compared to Ctrl plants(p=0.080). An increase of 2.9 fold was also noted with Pp groups compared to Mi plants (TukeyHSD, p=0.091). Nematode-aphid interaction was only notable with MP groups when compared to Ctrl-aphid plants. This resulted in an increase of 5.5 fold with MP groups (TukeyHSD, p=0.005). MP-aphid treatment also increased levels by 3.8 fold when compared to Ctrl in the presence of aphids (TukeyHSD, p=0.014). No other groups showed (marginally) statistically significant results.

Neither nematode treatment (Anova, F(3,24) =0.079, p>0.05) nor inoculating aphids and allowing them to feed for 3 days (Anova, F(1,24) =0.340, p>0.05) caused a statistically significant effect on SIN concentration differences. Aphids and nematodes did not yield a significant interaction effect either (Anova, F(3,24) =1.399, p>0.05). However, the general trend is an increasing pattern with both Mi, MP and a decreasing trend with Pp and control plants (Figure 14 (B)).

Analysis of NAS concentration did not reveal a significant influence by nematode treatment (Kruskall-Wallis analysis on ranks, p-value = 0.097). Nematode treatment resulted in a decreased pattern compared to the control plants and no amount could be detected with Pp plant groups.

Likewise, aphid feeding did not significantly cause an interaction effect on NAS levels (Kruskal-Wallis analysis analysis on ranks, p-value = 0.403). The trend is also a decreasing pattern compared to control plants without nematodes. Pairwise comparisons using Dunn's test did not indicate any differences when aphids were inoculated especially with Pp plants (dunnTest, Z=-0.946, p=1) since NAS was only observed after aphid feeding. No treatments were statistically significant (Figure 14 (C)).

Systemic induction of GSLs in young uninfected leaves

Nematode treatment had no statistical significance on the total glucosinolate concentration in young leaves (Anova, F (3,24) =0.834, p>0.05). The interaction between nematodes and aphids did not yield a significant result either (Anova, F(3,24)=0.265, p>0.05). Aphid presence however appeared to increase total GSL slightly with Mi and Pp treatments while decreasing amounts in control and MP plants.

However, this was not significant statistically (Anova, F(3,24)=0. 001, p>0.05) (Figure 15). 4. Discussion.

Glucosinolates are constitutively present in all tissues and alter upon damage by herbivores and pathogens. Root herbivory can cause both local and systemic alterations in the concentrations of glucosinolates [START_REF] Van Dam | Root and shoot glucosinolates: A comparison of their diversity, function and interactions in natural and managed ecosystems[END_REF] Plants exposed to M. incognita yielded an intermediate average aphid population value (66.5+SE) that lay between control (69.0+SE) and P. penetrans groups. These aphid performance variations could be explained by differences in the feeding strategies employed by nematodes as shown by [START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF]. [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF] suggested that lower aphid fecundity (daily average number of nymphs produced by each reproductive female) in P. penetrans infested plants caused a lower number of aphids on such plants. This also relies on density-dependent effects in aphid populations. The aphid performance experiment employed 10 one day old nymphs per plant which became adults and increased to 47+SE and 66.5+SE aphids in P. penetrans and M. incognita groups respectively in 10 days (by the end of the set-up). On the other hand, in the aphid life history study, three nymphs were allowed to reach the reproductive adult stage per plant in all treatment groups. As newly born nymphs were removed daily, it left plants with a low density of aphids than would normally occur in natural aphid-plant settings. So in line with [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF], this difference in aphid densities could have induced defences in the aphid performance set up than those in the life history experiment in which the aphid numbers were lower.

Different tissues contain different amounts of nutrients and GSLs. The youngest parts such as leaves usually have a higher capacity for growth and are more protected from herbivores and pathogens.

Hence, they contain more defensive secondary metabolites compared to older leaves [START_REF] Cao | Nutrition versus defense: Why Myzus persicae (green peach aphid) prefers and performs better on young leaves of cabbage[END_REF]. However, B. brassicae usually prefers the youngest tissues. The reason for this is that these plant parts contain more nutrients and this also reduces competition within the population [START_REF] Hopkins | Influence of increasing herbivore pressure on modification of glucosinolate content of swedes (Brassica napus spp. rapifera)[END_REF]. In a study by [START_REF] Cibils-Stewart | Feeding location affects demographic performance of cabbage aphids on winter canola[END_REF], they reported that cabbage aphids had higher fecundity while feeding on the reproductive parts (flowers) of B. napus compared to mature leaves.

However, the longevity and development time were also reduced. Another study by [START_REF] Cao | Nutrition versus defense: Why Myzus persicae (green peach aphid) prefers and performs better on young leaves of cabbage[END_REF] found that Myzus persicae also prefered the young cabbage leaves and performed better in terms of body weight and population growth. The similarities of performances between the two aphids can be attributed to the higher amount of amino acids and sugars present in the younger leaves compared to older leaves. So it is more likely that in this study system, the adult aphids obtained more of nutrients than glucosinolates and this could account for the non significance of the observed aphid reproduction (average of nymphs produced daily) (Table 3). This is because when aphid nymphs are born, they usually stay within the same place(leaf) compared to adults that move and choose preferred feeding sites [START_REF] Cao | Nutrition versus defense: Why Myzus persicae (green peach aphid) prefers and performs better on young leaves of cabbage[END_REF]. During the daily aphid nymph count, some adults were also found habiting the youngest leaves on the plants together with their nymph (data not shown). On the basis of the performance (Table 2) and maturation (Table 3) results and the trend of observations, this study accepts the first hypothesis that aphid performance and life history is affected by BG nematode species with the observed results.

Nematode induction of glucosinolates in roots

B. nigra roots can be colonized by both Pratylenchus spp and Meloidogyne spp [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF].

Locally in the roots (Figure 7), GSL were differentially induced by PPNs. However, M. incognita did not cause a significant change in the total GSL concentration in the roots over the two time points.

This could depend on the namatode's life cycle stages. The invasive stage during which the J2s penetrate cells and cause defence responses occurs after 5dpni. The galling and reproductive stages occur at 15 dpni and 30 dpni respectively [START_REF] Mbaluto | The impact of Spodoptera exigua herbivory on Meloidogyne incognita-induced root responses depends on the nematodes' life cycle stages[END_REF]. Since the set-up was not run beyond 10dpni, the juveniles would not yet have developed to the galling stage to cause a difference in levels of plant GSL between the two time-points. Different M. incognita life stages were found to induce different responses. Specifically, SA pathway was induced during the entire period of infection with the galling and reproductive stages eliciting gene expression of glycoalkaloids and ABA respectively in tomato plants [START_REF] Mbaluto | Induced Local and Systemic Defense Responses in Tomato Underlying Interactions Between the Root-Knot Nematode Meloidogyne incognita and the Potato Aphid Macrosiphum euphorbiae[END_REF]. The insufficient induction of significant differences can also result from the mode of migration of RKN J2s through plant roots. The intercellular pattern through which the juveniles move to reach the vascular cylinder limits chances of cell destruction [START_REF] Mitchum | Nematode effector proteins: An emerging paradigm of parasitism[END_REF]. This implies that the GSL-MYR system is not triggered or is only activated to a limited extent.

Contrary to this observation, a significant outcome in total root glucosinolate concentration between both time points was noted 10dpni with P. penetrans. Again on the basis of the life cycle stages, all P.

penetrans are infective besides the eggs and J1(first juvenile stage) when compared to M. incognita with only the J2s as an infective stage. Also, the root tissue invasion pattern by P. penetrans involves mechanical robust thrusts by the stylet which damages cells activating the GSL-MYR defence system.

This group of PPNs also remain mobile throughout development [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF]. All these differences can cause unique damage patterns observed in the roots and possibly explain the differences in root GSL induction as being dependent on the feeding pattern of the nematodes.

As observed in Figure ( 7), a combination of both nematodes did not show statistical significance in alterations of GSL concentration in roots with time. In natural systems, multiple herbivores simultaneously attack plants. Thus, plants are tasked with recognising specific herbivores and fine tuning specific responses according to the feeding guild, position and effectors present in ejected saliva of the feeding herbivores [START_REF] Züst | Mechanisms and evolution of plant resistance to aphids[END_REF]. In vegetative B. nigra, the glucosinolate profile and concentration is usually highest in roots compared to shoots. The root system is usually met by various pathogens/herbivores. This high pathogen pressure does not usually cause a strong change in thconstitutive concentration in roots since roots may be dependent on high GSL levels for defence [START_REF] Van Dam | Root and shoot glucosinolates: A comparison of their diversity, function and interactions in natural and managed ecosystems[END_REF]. However, plant response could also depend on the nature of interaction between the two nematodes. This may be determined by various factors when both species are feeding on the same host plant. For instance, migratory endo-parasites are generally considered as less advanced parasites in comparison to sedentary endo-parasites such as M. incognita. This is due to the ability of M. incognita to develop a complex relationship which alters its host plant's physiology.

P. penetrans has a simpler relationship with the plant and can suppress M. incognita due to disruption of feeding sites created by its migratory pattern [START_REF] Khan | Nematode Interactions[END_REF]). This interaction nature would therefore imply that the plant will mostly respond to the presence of P. penetrans when in a combination treatment (MP). In this study system, this doesnt seem likely as the root total GSL levels induced by MP plants are lower than that by either Mi or Pp treatment groups. It therefore implies that the two nematodes could rather exhibit a mutually antagnonistic effect in a short time experiment. This mutual antagonism between the two nematodes was also reported in tomato plants [START_REF] Khan | Nematode Interactions[END_REF].

Glucosinolate induction in infected leaves

The cabbage aphid sequesteres glucosinolates from the host plants and uses it for its own defence against predators. Mainly aliphatic and not Indolic GSL are sequestered. This is probably due to the higher efficiency of aliphatic glucosinolates for defense against natural enemies. This would then imply that the non-sequestered indole GSL might pose toxic effects on this specialist aphid.

In Figure 14 (A), Indole GSL levels were highest with aphid presence in MP and Pp compared to Mi and control plants. This increase is less likey to be induced by aphids since aphid presence did not show statistically significant results. Therefore, the plant could simply be responding to the presence of aphids after being primed by nematode feeding for 7 days. These plants also showed that aphid development required the most amount of time (days) to reach the adult reproductive stage, precisely, 11.4+SE days (Table 3). Aphids do not only sequester GSL at the adult stage but throughout the developmental stages continuously from first nymphs to adults [START_REF] Kos | Prey-mediated effects of glucosinolates on aphid predators[END_REF]. In the study of [START_REF] Kos | Prey-mediated effects of glucosinolates on aphid predators[END_REF], it was discovered that adult aphids contained higher concentrations of aliphatic GSL while the highest concentrations of indole glucosinolates were found in the nymphs. Also, aliphatic GSL were sequestered in higher concentrations from the third instar nymph to the adult stage as opposed to sequestration of indole GSL. This difference in GSL sequestration was suggested to be dependent on insect transporters for glucosinolate uptake that are rather specific. For example, different sawflies were reported to sequester different classes of GSL [START_REF] Kos | Prey-mediated effects of glucosinolates on aphid predators[END_REF]). Since the aphid's mode of feeding follows an intercellular pattern, the aphid will limit detectable tissue disruption and ingest aliphatic GSL intact without the action of plant myrosinases. This would then imply that the observed slow developmental time of nymphs in Pp and MP plant groups was caused by the presence of indole GSL and not aliphatic or benzenic GSL. Indole glucosinolates are hydrolysed into toxic bioactive products and this does not rely on the presence of myrosinase. This results from the fact that indole GSL are unstable on their own as a result of the replacement of the functional group lost as the thiocyanate ion (SCN -), with various nucleophiles [START_REF] Agerbirk | Indole glucosinolate breakdown and its biological effects[END_REF]. Hence, B.

brassicae is met by detrimental indole GSL isothiocyanates.

Analysis of aliphatic GSL levels in aphid inoculated leaves did not reveal significant effects by nematodes or aphids although aphid presence appeared to increase sinigrin levels with Mi and MP treatment groups (Figure.14(C)). Other studies have found an increase [START_REF] Mewis | Major Signaling Pathways Modulate Arabidopsis Glucosinolate Accumulation and Response to Both Phloem-Feeding and Chewing Insects[END_REF]Kim and Jander, 2007) and a decrease (Kim and Jander, 2007) in the levels of aliphatic GSL with other Brassica plants. This difference could be caused by the low number of aphids used for inoculation in this study as the above mentioned studies employed higher aphid populations of the same or different species.

The benzenic GSL, 2-phenyl-ethylglucosinolate (NAS), is dominant in the root system and only present in low concentration or absent in the shoots. This could be attributed to its low volatility and high hydrophobic nature of its hydrolysis products (van Dam et al., 2008). However, NAS levels were not observed in leaves of plants exposed to P. penetrans not until after 3 days of aphid feeding. It is unlikely that aphid presence induced NAS levels in the Pp leaves and more likely that the HPLC could not detect the peaks due to low concentration levels in the leaves.

As regards the second hypothesis, it is not exactly clear how the two nematodes are behaving to influence systemic glucosinolate concentrations in infected leaves. The results only showed statistical significance with indole GSLs than aliphatic or benzenic GSL. The level of indole GSL was not highest with MP plants when compared to Mi and Pp groups, though not statistically significant.

Therefore, this research rejects the second hypothesis since it can not be supported by the observed GSL changes in infected leaves. However, it is only clear that aphid maturation was slowest with MP treatment and this may as well be attributed to other factors such as plant nutrient availability under multi-herbivore systems.

Induction of GSL in uninfected leaves

During herbivore/pathogen infestation, signalling phytohormones elicit induced chemical resposnes.

This causes a change in the chemical profiles of uninfected or undamaged plant tissues. For example, Infection of tomato plants by Pseudomonas syringae induced resisteance to Helicoverpa zea (Stout et al., 1999). Systemic defences were also shown to be caused in Arabidopsis spp response to nematode ascoriside application to leaves or roots. This is because ascorisides migrate from roots to leaves and induces mobile signals in roots that reach the shoots to activate plant responses [START_REF] Manosalva | Conserved nematode signalling molecules elicit plant defenses and pathogen resistance[END_REF]. In this set up, neither nematode caused a statistically significant influence on GSL concentration in uninfected leaves when allowed to feed for 10 days (Figures 11 and 12). Likewise, the presence of, in the presence of aphids, no statistically significant changes could be observed (Figures 15 and 16). It was suggested that glucosinolate transport from roots to shoots could change the profile [START_REF] Brown | Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana[END_REF] but the work by Kim and Jander, (2007) showed that even with detached Arabidopsis leaves with petioles immersed in water, GSL changes were observed upon M. persicae feeding. Therefore, transport between different plant parts may not be a essential for such observed changes. This implies that the non statistical significances in this study is caused by low number of inoculation of nematodes and aphids. The GSL analysis set up utilised 200 all stages but eggs of P. penetrans, while 200 J2s were used for the M. incognita treated plants and MP received 100 of each nematode which were lower than that found in the soil surface in natural environments [START_REF] Gamalero | The Use of Plant Growth-Promoting Bacteria to Prevent Nematode Damage to Plants[END_REF].

Conclusion and perspectives

This study shows that not only B. brassicae life history but the performance as well can be affected by nematodes. The results of this study only refelect effects on a short term plant-herbivore interaction.

The outcome of such experiments involving multi-herbivore interaction systems usually depend on host plant, herbivore feeding pattern, inoculation method, time and sequence of attack. The analysis of glucosinolates revealed only indole glucosinolates to differ with aphid and nematode treatment groups in infected leaves compared to other classes of glucosinolates. This suggests that in such interactions involving plants as mediators between aboveground specialists and belowground herbivores, change in GSL profiles could either lower, increase or have neutral effects on herbivores. Therefore, in the presence of such a specialist, it would be beneficial for Brassicas to produce less glucosinolates than more of it. Albeit the limitation of the glucosinolate defense system to the Brassicacea family, understanding this system and its applications will enable managemnt of plant herbivores in an ecologial manner. This can lessen the use of chemical pestcides that have toxic environmental impacts.

It could be possible that these defence compounds can be transered to non glucosinolate producing plants to build up herbivore/dieases resisitance.

FiguresFigure 1 .

 1 Figures

  Figure 1. Common structure of Glucosinolates (GSL).. ..........................................................................

Figure 2 .

 2 Figure 2. The main glucosinolates side chain classifications...................................................................

Figure 3 .

 3 Figure 3. A schematic overview of the aphid-plant interaction .............................................................

Figure 4 .

 4 Figure 4. Root-Knot nematode life cycle and host plant invasion .........................................................

Figure 5 .

 5 Figure 5. Root-lesion nematode life cycle and host invasion. ...............................................................

Figure 6 .

 6 Figure 6. Glucosinolates extraction ........................................................................................................

Figure 7 .

 7 Figure 7. Figure 7. Total glucosinolate concentrations in nematode infested roots...............................

Figure 8 .

 8 Figure 8. Total concentrations of different glucosinolate classes in roots. ............................................

Figure 9 .

 9 Figure 9. Different indole glucosinolates in roots. .................................................................................

Figure 10 .

 10 Figure 10. Total glucosinolate concentrations in uninfected leaves ......................................................

Figure 11 .

 11 Figure 11. Total concentrations of different glucosinolate classes in uninfected leaves. ......................

Figure 12 .

 12 Figure 12. Total glucosinolate concentrations in infected leaves. .........................................................

Figure 13 .

 13 Figure 13. Total concentrations of different glucosinolate classes in infected leaves. ..........................

Figure 14 .

 14 Figure 14. Total glucosinolate concentration in uninfetced leaves. .......................................................

Figure 15 .

 15 Figure 15. Total concentrations of different glucosinolate classes in uninfected leaves. ......................

Figure 1 .

 1 Figure 1. The main glucosinolates side chain classifications (R) and the products of glucosinolate hydrolysis by the enzyme myrosinase (picture taken from Meschede et al., 2020).

Figure 2 .

 2 Figure 2. Common structure of Glucosinolates (GSL). Precursors of amino acids in the R-group form the basis of the distinguishable GSL classes. (R = indole, alkyl, aryl). The picture is taken from Avato et al., (2013).

Figure 3 .

 3 Figure 3. (A) B. brassicae infestation on a young stem of a B. nigra plant. Overlapping aphid generations resulting from a single adult female 21days post inoculation. (B) A schematic overview of the aphid-plant interaction.

Figure 4 .

 4 Figure 4. Root-Knot nematode life cycle and host plant invasion. Plants are invaded by the RKN J2s in the root's elongation zone. The target vascular cells are reprogrammed through activation of cell mitosis and expansion to form giant cells which serve as feeding sites. J2s develop into adults and the adult RKN female lays eggs in a gelatinous mass of eggs. Giant cells intensify the flow of solutes from the vascular vessel to the nematode (Picture source: Jones &Goto, 2011)

Figure 5 .

 5 Figure 5. Schematic representation of Pratylenchus life cycle and host plant infection. Developmental stages from eggs to J1 and finally adults. Jones &Fosu-Nyarko, (2014).

  or in soil Extracted from Jones &Fosu-Nyarko, (2014).

  summary, the two research questions are; (a) How is B. brassicae performance and life history affected by a combination of different plant parasitic nematodes feeding belowground ? (b) Do glucosinolate profiles alter according to nematode species and how does it influence B. brassicae performance and life history ?To answer these questions on the signalling interactions between aphids and nematodes, the study system comprises of wild Brassica nigra (black mustard) as a host plant, a Brassicas specialist aphid, Brevicoryne brassicae, aboveground and two belowground generalist nematodes, root-knot (M. incognita) and root lesion (P. penetrans) nematodes. Brassica nigra plants were subjected to different treatments groups with and without aphids and nematodes. Plant samples were collected and glucosinolates were extracted and their profiles and changes in concentrations measured. Aphid performance was determined by considering an average of the observed aphid population under different treatments. The life history was determined by daily observation of the aphid nymphs until maturity and reproduction.2. Materials and Methods2.1. Nematode cultures.A starting culture of M. incognita was obtained from Bejo, the Netherlands and was maintained on Solanum lycopersicum cv. 'Moneymaker' in the glasshouse at the Botanical garden, Leipzig, Germany. Pratylenchus penetrans was received from the Plant Science Research Unit of the Research Institute for Agriculture, Fisheries and Food (ILVO), Merelbeke, Belgium. The cultures were maintained in carrot discs at 23±1°C. Following the Baermann technique(van Bezooijen, 2006), second stage juveniles of Meloidogyne incognita and all stages but eggs of Pratylenchus penetrans were extracted and used for inoculation. The extraction set-up was left to run for 24 hours before the nematodes were harvested. The concentration of nematodes was obtained by preparing aliquots containing 100 nematodes mL -1 under a stereomicroscope. Tween-20 solution as a surfactant (20μL per 50ml) was added during inoculation.2.2. Brevicoryne brassicae aphid cultureB. brassicae were obtained from the Max Planck Institute for Chemical Ecology, department of Biochemistry, Jena, Germany. A starting colony was transferred and maintained in B. nigra in insect cages in a growth chamber with a photoperiod of 16h:8h (light: dark), temperature of 22 °C:18 °C day: night, relative humidity of 40/70% and light intensity of 225/0 μmoles/m2/s. This was to obtain a culture of synchronised aphid ages for the experiments.2.3. Brassica nigra growth conditionWild B. nigra seeds collected from the Netherlands were cultured following the protocol described by[START_REF] Van Dam | Differences in Hormonal Signaling Triggered by Two Root-Feeding Nematode Species Result in Contrasting Effects on Aphid Population Growth[END_REF]. Briefly, the seeds were surface sterilised with 5% bleach followed by repeated rinsing with dH2O.The seeds were then germinated on water-soaked glass beads in plastic containers (15X10 cm) which were covered with a transparent lid and placed in a climate chamber. The plant growth temperature was maintained at 20•C:16•C (day: night) and a photoperiod of 16h: 8h (light: dark). The plants were allowed to germinate for 10 days. B. nigra seedlings were then transplanted into 2L pots filled with heat-treated river sand (>80°C for 1 hour) and supplied with 200ml tap water.Each transplanted seedling was then supplied with 50ml of Hoagland solution of strength 50% and grown in a glasshouse. The relative humidity was maintained at ~30 % and the photoperiod at 16h:8h light:dark. The temperature was kept at 26 °C:23 °C (day: night). The plants were then supplied with water or Hoagland solution after every two days to maintain the moisture content of the sand at 15% throughout the experiment. The universal BBCH scale[START_REF] Lancashire | A uniform decimal code for growth stages of crops and weeds[END_REF] was used to determine the developmental stages of plants before subjecting them to different treatments.B. brassicae and finally, (8). M. incognita + P. penetrans + B. brassicae in combination. Nematode treatment of B. nigra plants was performed by inoculating 200 eggs 2mL -1 of M. incognita, 200 all stages (except eggs) of P. penetrans 2mL -1 for the Mi and Pp treatments respectively. The MP treatment received a combination of both nematodes (100 nematodes per species) and Tween 20 solution was added during inoculation. After 7dpni, only plants with aphid treatment as above were challenged with 10 adult apterous synchronised aphids and left to feed for three days. Plant sample harvest was done at two time-points. At time-point A (7dpni), the youngest distal uninfected leaves before aphid treatment) and nematode infected roots were harvested. The second harvest was done at time-point B-10dpni/3dpai (days post aphid inoculation). Young distal uninfetced and Infected leaves by aphids were then harvested for GSL extraction and analysis. Infected leaves represent the 4 th and 5 th fully unfolded leaves where aphids were inoculated during the experiment.

Figure 6 .

 6 Figure 6. Presentation of a prepared rack for with labelled Pasteur pipettes set up for glucosinolates extraction. Height: 150mm, distance between shifted holes: 30 mm (x-axis) x 15 mm(y-axis). (Picture source: personal collection).

  (A)). Neoglucobrassicin (NEO) levels were not significantly altered by nematode treatment (Kruskal-Wallis chi-squared = 2.1439, df = 3, p-value >0.05) or time (Kruskal-Wallis chi-squared = 0.56818, df = 1, pvalue >0.05). MP treatment was not significant (Dunnett test: Z= 0.03, p= 0.89) but reduced NEO concentrations by 0.23 fold. Likewise, the Pp treatment did not show any significance but rather increased NEO levels by 1.5 fold at 10dpni compared to 7dpni (Dunnett test: Z= 0.34, p=1) (Figure 9(B)).

Figure 7 .

 7 Figure 7. Total glucosinolate concentrations (mean ± SE) in nematode infested roots at time-points A (7dpni) and B (10dpni). Letters indicate significant differences between groups with time (Anova, Tukey test, N=5, except for Pp and MP where n=3).

Figure 8 .

 8 Figure 8. Total concentrations (mean ± SE) of different glucosinolate classes in roots under different nematode treatments at time-point A (7dpni) and B (10dpni). Letters indicate significant differences between groups with time. (A) Indole GSL (Anova, Tukey test, P>0.05), (B)Aliphatic GSL (Anova, Tukey test, P<0.01 and (C) Benzenic GSL(Anova, Tukey test, P<0.05).( N=5 except for Pp and MP where n=3).

Figure 9 .

 9 Figure 9. Levels (mean ± SE) of different Indole glucosinolates (A)4-methoxyglucobrassicin (4MeOH) (Anova, Tukey test, p>0.05), (B)Neoglucobrassicin (NEO) (Kruskal, Dunnett test, p>0.05) in roots infected with different nematodes at time-point A (7dpni) and B (10dpni). Letters indicate significant differences between groups with time (n=5, except for Pp and MP where n=3).

Figure 10 .

 10 Figure 10. Total glucosinolate concentrations (mean ± SE) in young distal leaves at time-points A (7dpni) and B (10dpni) with different nematode treatment groups. Identical letters do not indicate significant differences between groups (Anova, Tukey test, p>0.05, N=5, except for Pp and MP where n=3).

Figure 11 .

 11 Figure 11. Total concentrations (mean ± SE) of different glucosinolate classes in young (distal) leaves on nematode infested plants at time-point A (7dpni) and B (10dpni). (A) Indole GSL (Anova, Tukey test, P>0.05), (B)Aliphatic GSL (Anova, Tukey test, P>0.05 and (C) Benzenic GSL (Kruskal-Wallis, Dunnett test, p >0.05) (N=5 except for Pp and MP where n=3).Identical letters represent no statistical significant differences between groups with time and treatment.

Figure 12 .

 12 Figure 12. Total glucosinolate concentrations (mean ± SE) in aphid infected leaves 3dpai and 10dpni. The same letters do not represent statistically significant differences between groups (ANOVA, Tukey test, P ≥ 0.05, n=5, except for Pp and MP where n=3).

Figure 13 .

 13 Figure 13. Total concentrations (mean ± SE) of different glucosinolate classes in aphid infected leaves under different nematode infested plant groups (10dpni). (A) Indole GSL (Anova, Tukey test, p<0.05), (B)Aliphatic GSL (Anova, Tukey test, p>0.05 and (C) Benzenic GSL (Kruskal-wallis, Dunnett test, P>0.05) (N=5 except for Pp and MP where n=3). The same letters represent no statistical significant differences of concentration with treatment between groups.

  Indole glucosinolates were marginally significantly influenced by nematode treatment (Anova, F(3,24)= 2.548, p=.079). The interaction between nematodes and aphids also yielded an almost significant result (Anova, F(3,24)= 2.948, p=0.0531). A post hoc Tukey test showed that only Pp-aphid treatments almost differed significantly from Ctrl-aphids (p=0.065), as well as from Mi-aphid (p=0.0816) treatment groups by 4 fold and 3.8 fold increases respectively (Figure15(A)).Sinigrin levels in young distal leaves was not affected by either nematode treatment (Anova, F(3,24)=0.839, p>0.05), Aphid feeding(Anova, F(1,24)=0.002, p>0.05) or their interaction (Anova, F(3,24)=0.255, p>0.05) (Figure15(B)). NAS levels did not did not show any changes either with Nematodes feeding (Kruskal-Wallis chisquared = 1.3735, df = 3, p-value = 0.7118) or aphid feeding (Kruskal-Wallis chi-squared = 6.5326, df = 7, p-value = 0.479) (Figure15(C)).

Figure 14 .

 14 Figure 14. Total glucosinolate concentration in distal leaves (D) under different nematode (10dpni) and aphid (3dpai) treatment groups (mean ± SE). Identical letters represent no statistically significant differences between groups (ANOVA, Tukey test, P ≥ 0.05, n=5, except for Pp and MP where n=3).

Figure 15 .

 15 Figure 15. Total concentrations (mean ± SE) of different glucosinolate classes in distal young leaves (D) 3dpai on infected leaves and 10dpni. (A) Indole GSL (Anova, Tukey test, p<0.05), (B)Aliphatic GSL (Anova, Tukey test, p>0.05 and (C) Benzenic GSL (Kruskal-wallis, Dunnett test, P>0.05) (N=5 except for Pp and MP where n=3). The same letters represent no statistically significant differences of concentration with treatment between groups.

  Table1. Features of Root-Knot (e.g Meloidogyne incognita) and Root lesion (e.g Pratylenchus penetrans) nematodes association with host plants

	Characteristics	Root-Knot nematodes	Root Lesion Nematodes
	Host specificity	In general, a broad host range, most	Broad host range, most vascular
		vascular plants	plants
	Invasion	Root tips, zone of elongation,	Root tips, zone of elongation, entire
		secondary infections in galls	root surface; all mobile stages can
			enter and leave roots
	Migration	Intercellular	Intracellular
	Feeding cells	Giant cells, formed by repeated	Root hairs, non-specific cortical cells
		mitosis without cytokinesis from	which die after nematode feeding
		initial/pro-vascular cells

Table 3 .

 3 Influence of nematodes on Brevicoryne brassicae life history

Table 4 .

 4 List and full names of the 11 detected glucosinolates

*Glucosinolates not included during analysis due to their very low concentrations and inconsistency.

Table 2 .

 2 Influence of Nematodes on Brevicoryne brassicae performance 3.2. Local induction of glucosinolates in roots by nematodes.

  . This study aimed at determining how AG B. brassicae life history is influenced by alterations in GSLs caused by BG nematodes of different species. B. brassicae was B. nigra plants infested by M. incognita, P. penetrans or a combination of both nematodes. The study found that nematode feeding did not only induce glucosinolate concentration changes in the roots but systemically to aboveground tissues as well.4.1. Aphid performance and life history.The experimental results on aphid performance demonstrated nematode species specific influence on aphid population growth. These results were not significantly different (Anova, F(2,7)=1.476, P=0.29) but showed a general trend of impacts caused by different BG PPN species (Table2). After 7dpni and 3dpai, the least average number of aphids was observed on plants challenged by P. penetrans (47+SE).

Brevicorynebrassicae performance experiment

Plants of similar ages (BBCH 32) were set-up into three different treatment groups; Control plants which were only mock inoculated with a water-Tween 20 solution, M. incognita and P. penetrans treatment groups. The nematodes were inoculated before aphids. Plant groups exposed to M. incognita received 200 eggs of the nematode while those meant for P. penetrans treatment were inoculated by 200 all stages but eggs of this nematode. All three groups were left to run until 7dpni when 10 synchronised one day old aphid nymphs per plant were inoculated on the top of the leaves. The aphids were allowed to feed for 10 days after which their average population growth was taken into account.

Control and M. incognita utilised 3 plants each except P. penetrans where n =4 per treatment group.

Brevicorynebrassicae life history experiment

Aphid life-history analysis was performed according to a modified protocol from Tariq et al. (2013). Succinctly, B. brassicae was reared as described above on Brassica rapa. A cohort of 20 four-weeks old Brassica nigra grown in 1.5L heat-treated sand were used in the experiment. The plants were randomly assigned into four groups of five and subjected to different treatments, i.e, B. brassicae only(Control), B. brassicae + M. incognita, B. brassicae + P. penetrans and B. brassicae+ M. incognita + P. penetrans. The Control group was mock inoculated with a 1mL solution of water and Tween 20. Each of the plants subjected to different nematode treatments groups was inoculated with 100 M. incognita J2s mL -1 and 100 P. penetrans per mL -1 respectively. The nematode combination treatment (M. incognita + P. penetrans) received 50 M. incognita J2s and 50 P. penetrans per 500uL.

Nematode inoculation was done through a pre-generated hole into the sand close to the rhizosphere.

On the seventh day (7dpni-days post nematode inoculation), the plants were transferred to individual insect cages (40cm X 40cm X 60cm). Three adult apterous female aphids were released on top of two fully unfolded leaves using a wet fine brush. The aphids were left to feed for 20hrs and the adult females were removed the next day, leaving only three one-day old instar nymphs per plant. Aphid life history was determined over the next 17 days by recording the number of nymphs born daily and removing them immediately. The following parameters were recorded ; Maturation= pre-reproductive time/development time (birth to first reproduction).

Reproduction= average number of nymphs per apterous aphid per day [START_REF] Hol | Root-Lesion Nematodes Suppress Cabbage Aphid Population Development by Reducing Aphid Daily Reproduction[END_REF]

Glucosinolate analysis experiment

The biological materials were prepared as described above. Grown in 2.5L heat-treated sand, fourweek old B. nigra plants at the BBCH 32 scale were randomly selected and divided into eight treatment groups. i.e, [START_REF] Khan | Nematode Interactions[END_REF]. Control (Ctrl-which was mock-inoculated with water and tween 20 solutions), (2). M. incognita (Mi) treatment only, (3).B. brassicae only, (4). Pratylenchus penetrans (Pp) only, (5). M. incognita + P. penetrans (MP), (6). M. incognita + B. brassicae, (7). P. penetrans +

Abstract

The Brassicaceae plant family is known to biosynthesise a defined group of secondary metabolites, the glucosinolates (GSL), from amino acids. Over 130 different structures of glucosinolates have been identified. They contain sulphur and nitrogen.

Enzymatic hydrolysis leads to rearrangement of the sulfated aglucone to form isothiocyanates or nitriles. These hydrolysis products are defensive compounds mostly against generalist herbivores as opposed to specialist phloem feeders such as B.

brassicae. This specialist herbivore has developed a means of circumventing the toxic effects of glucosinolates. This is due to the presence of a co-evolved myrosinase enzyme which enables it to sequestrate GSL. Regardless of host specificity and ability to sequester glucosinolates, its performance and life history can still be affected by nematode feeding. This research investigated the influence of the belowground migratory root-lesion nematode, P. penetrans, and the sedentary root-knot nematode, M. incognita, on the performance and life history of B. brassicae aboveground. Brassica nigra (black mustard) was used as the host plant and exposed to nematode treatments before aphids were inoculated. The nematode treatments groups included plants challenged by single nematode species and simultaneous inoculation of both nematodes. At the end of the experiment, plant samples were harvested and glucosinolates extracted and their concentrations measured. The maturation time of B. brassicae was mostly increased in treatments that included P. penetrans. The aphids developed slower on these plants and faster on plants exposed to M. incognita or control groups. This was attributed to the differences in nematode feeding pattern which resulted in different indolic GSL levels.

Key words : Aboveground-belowground interactions, Brevicoryne brassicae, Meloidogyne incognita, Pratylenchus penetrans, Glucosinolates.