Host Plant Resistance in Brassicaceae against Aphids

Abstract

This chapter deals with brassica plants and their resistance to sucking pests—aphids. Brassica plants are known to synthesize a number of plant secondary metabolites which impart resistance to insect-pests and diseases. Aphids are known to feed primarily on sieve elements. The sieve elements in vascular bundles of angiosperms are important channels for nutrition. They are the channels of transport of photoassimilates from source to the sink. Because of the high nutrition content of the sap inside sieve elements, they are the target for many insect-pests and bacterial and fungal pathogens. Aphids are one such group of insects which target SE elements of phloem for nutrition. They are among the most important insect pests in agriculture particularly serious in temperate and sub-tropical climates. In addition to direct damage by feeding as well as toxic effects of saliva, the withdrawal of nutrients is detrimental to plant growth and development. In addition to this, aphids also cause indirect damage to plants by acting as vectors of plant pathogenic viruses. Furthermore, honeydew excreted by aphids provides suitable substrate for sooty molds that interfere with normal plant photosynthesis. In this chapter work on host plant resistance in Brassica plants against aphids has been reviewed.

1. Introduction

Brassicaceae family is one of the earliest group of cultivated plants [1] which are a source of vegetables, oilseeds and condiments. Various biotic and abiotic stresses limit the production and productivity of these crops. Out of various insect-pests, aphids are important pests. Turnip aphid alone is known to cause 35.4 to 91.3% reduction in yield with the average yield losses of around 56.2% [2]. At present, systemic insecticides are used to manage aphid pests. Although these insecticides are very effective, but they have the associated problems like residue problem in oil and cake, environmental pollution and development of insecticide resistance. Past two decades have witnessed an increased interest in finding alternate solutions for aphid management. One such strategy is host plant resistance. It is an effective, economical and environment friendly option for pest management. The first step in development of insect resistant cultivar is the identification of source of resistance. In this chapter we have attempted to review literature on screening of plants to find source of resistance and latest developments in host plant resistance in Brassicaceae against aphid pests.

2. Species complex of aphids on Brassicaceae plants

Members of the family Brassicaceae serve as suitable hosts to a number of aphid species. The main aphid species reported to infest Brassica plants are mealy cabbage aphid, Brevicoryne brassicae (L.); turnip/mustard aphid, Lipaphis erysimi (Kaltenbach)/Lipaphis pseudobrassicae (Davis); green peach aphid, Myzus persicae (Sulzer); shallot aphid, Myzus ascalonicus Doncaster; potato aphid, Macrosiphum euphorbiae (Thomas); corn root aphid, Aphis maidiradicis Forbes; and root feeding aphids, namely, bean root aphid, Smynthurodes betae Westwood and cabbage root aphid/poplar petiole gall aphid, Pemphigus populitransversus Riley [3]. Among these, three species viz. B. brassicae, L. erysimi and M. persicae cause serious damage to Brassica crops in one or other part of the world. B. brassicae is native to Europe with worldwide distribution. It is a serious pest of Brassica vegetables in most European countries and results in significant yield losses. It is a specialist pest of Brassicaceae that feeds on phloem sap of its host plants [4]. Although it is a primary pest of Brassica vegetables, it also feeds on other species in genus Brassica [4, 5, 6, 7]. L. erysimi, the most important pest of oilseed Brassica in Indian subcontinent, is native to eastern Asia [3].

Unlike B. brassicae and L. erysimi, peach-potato aphid, M. persicae is a generalist pest and feeds on more than 400 plant species [8] including broccoli, cabbage, carrot, cauliflower, egg plant, lettuce, papaya, peach, peppers, sweet potato, tomato, etc. There are two views about its origin. Many workers believe it to be native of China-the native place of its host plant Prunus persica, while others believe it to have originated from Europe [9]. In addition to direct feeding damage, it is an efficient vector of 100 plant pathogenic viruses including potato virus Y, potato leaf roll virus and various mosaic viruses including western yellows [10, 11]. The pest is polyphagous and cosmopolitan in distribution. It possesses very high genotypic plasticity for color, life cycle, host plant relationships and mechanisms for insecticide resistance.

Initially there were doubts about the origin and identity of Lipaphis pseudobrassicae. Till 1914, it was confused with B. brassicae in North America. Davis [12] recognized it as a distinct species and named it Aphis pseudobrassicae. Later, it was transferred to the genus Rhopalosiphum [13] because of weakly clavate siphunculi and was referred to as Rhopalosiphum pseudobrassicae (Davis) till 1964. In 1932, Börner and Schilder [14] found that species pseudobrassicae should be placed in Lipaphis—a genus erected by Mordvilko [15] for a Brassica feeding aphid, erysimi. While attempting to discriminate pseduobrassicae from erysimi, Hille Ris Lambers [16] could not find any characters that can differentiate the two and stopped short of making it a synonym. However, other workers considered pseudobrassicae as a subspecies of erysimi [17, 18]. Despite this, erysimi continued to be used from 1975 onwards.

3. Aphid-plant interactions

Aphids are specialized phloem sap feeders which insert their needle like stylets in the plant tissue avoiding/counteracting the different plant defenses and withdraw large quantities of phloem sap while keeping the phloem cells alive. In contrast to the insects with biting and chewing mouthparts which tear the host tissues, aphids penetrate their stylets between epidermal and parenchymal cells to finally reach sieve tubes with slight physical damage to the plants, which is hardly perceived by the host plant [19]. The aphid stylets play major role in host plant selection [20]. The long and flexible stylets mainly move intercellular in the cell wall apoplasm [21], although stylets also make intracellular punctures to probe the internal chemistry of a cell. The high pressure within sieve tubes helps in passive feeding [19]. During the stylet penetration and feeding, aphids produce two types of saliva. The first type is dense and proteinaceous (including phenoloxidases, peroxidases, pectinases, β-glucosidases) that forms an intercellular tunneled path around the stylet in the form of sheath [22]. In addition to proteins this gelling saliva also contains phospholipids, and conjugated carbohydrates [23, 24, 25]. This stylet sheath forms a physical barrier and protects the feeding site from plant’s immune response. When the stylet come in contact with active flow of phloem sap, the feeding aphid releases digestive enzymes in the vascular tissue in the form of second type of ‘watery’ saliva. The injection of watery saliva (E1) prevents the coagulation of proteins in plant sieve tubes and during feeding the watery (E2) saliva gets mixed with the ingested sap which prevents clogging of proteins inside the capillary food canal in the insect stylets [19]. Though, the actual biochemical mode of action of inhibition of protein coagulation is unknown, the calcium binding proteins of aphid saliva are reported to interact with the calcium of plant tissues resulting in suppression of calcium-dependent occlusion of sieve tubes and subsequent delayed plant response [26, 27]. This mechanism of feeding is more specialized and precise which avoids different allelochemicals and indigestible compounds abundant in other plant tissues [28]. In addition to this, aphid saliva also contains non-enzymatic reducing compounds which in the presence of oxidizing enzymes inactivate different defense related compounds produced by plants after insect attack [24].

The early response of plants to feeding by insects or infection by pathogens share some common events such as protein phosphorylation, membrane depolarization, calcium influx and release of reactive oxygen species (ROS, such as hydrogen peroxide) [29], which leads to activation of phytohormone dependent pathways. In response to infestation/infection different phytohormone-dependent pathways are activated. The ethylene (ET) and jasmonate (JA) pathways are activated by different necrotrophic pathogens [30] and grazing insects [31], while salicylate (SA) dependent responses are activated by biotrophic pathogens [30]. These responses lead to production of various defense related proteins and secondary metabolites with antixenotic or antibiotic properties. In the case of infestation by aphids, a SA-dependent response appears to be activated, while the expression of JA-dependent genes is repressed [32, 33, 34, 35]. All these responses lead to the manipulation of the plant metabolism to ensure compatible aphid-plant interactions.

4. Stages and extent of damage

Damage is caused by both nymphs and adults. Wing dimorphism leads to two different morphs—alatae (winged) and apterae (wingless). Apterae are small to medium sized pale to yellowish, gray or olive green with body covered with small waxy coating (not as waxy as B. brassicae). However, under cold and humid conditions this waxy covering becomes dense coat of white wax. Small to large colonies of L. erysimi suck plant sap from flower buds, flowers, siliquae, pods and underside of leaves which leads to their curling, crinkling and yellowing. Continuous feeding and consequent resource restriction leads to drying of the plant part being fed upon.

Parthenogenetic viviparity limits the need for males to fertilize females and eliminates the egg stage from life cycle. Further, the development of an aphid begins even before its mother’s birth—a phenomenon known as telescoping of generations. Thus, the generation time is considerably reduced to as low as 5–7 days under favorable conditions [36] leading to rapid increase in population growth. Under varying population levels, prevailing agro-climatic conditions and phenological stage of the crop damage by L. erysimi has been reported to vary from as low as 10 to as high as 90% [37, 38, 39, 40, 41, 42, 43, 44, 45, 46]. In addition to direct feeding damage, L. erysimi is also vector of 10 non-persistent viruses including turnip mosaic virus, cauliflower mosaic virus and cabbage black ring spot virus [43, 47]. Like B. brassicae, it is also a Brassica specialist. Brassica rapa and B. juncea are generally better hosts compared to other Brassica species [43]. It is cosmopolitan in distribution and is found wherever Brassica plants are grown. Host range may include many species and genera of Brassicaceae, including Brassica, Barbarea, Capsella, Erysimum, Iberis, Lepidium, Matthiola, Nasturtium, Raphanus, Rorippa, Sinapis, Sisymbrium and Thiaspi [48, 49].

5. Bioecology and different control interventions

Many workers have attempted to study the bioecology of the pest in an effort to find weak links in pest’s life cycle so that this information can be used in devising an effective pest management strategy. Though good information has been generated, but keeping a view the changed spectrum of mustard varieties over the time, cultural practices and the global environmental change, there are still many gaps in our knowledge. There is no egg or other resting stage in its life cycle and the mustard aphid is reported to survive on some wild crucifers and some vegetables during summer months [50, 51] particularly in submountaineous regions. On the other hand, in plain region of Rajasthan, Sachan and Srivastava [52] could not locate the pest from July to October on cabbage. Similarly, Lal [53] also stated that this pest is not traceable in plains of India during summer months. Thus, it was hypothesized that aphids migrate from hilly areas to plains of India to avoid extremely low temperatures in winter season. However, this ‘Hills to Plain Hypothesis’ failed to highlight the exact route of aphid migration and the exact source of aphid population. Recently, Ghosh et al. [54] have studied the migration behavior of L. erysimi over Indo-Gangatic plains through 24 h backward air-mass trajectory and found that mountainous regions of Kashmir are the source of aphid migration in North-Indian plains in winter season. Studies on aphid migration and their development on host plants help in developing effective forewarning and forecasting models which have implications in precise timing of control interventions. Generally, the alatae of L. erysimi start appearing on the crop in October when the crop is still young. They generally remain at low levels in winter season and start increasing from December till mid March in different regions of the country (Table 1) after which a decline in population is observed due to maturity of the crop and rise in temperature [52, 65, 66]. Though a number of natural enemies are reported to be associated with L. erysimi (Diaeretiella rapae, coccinellids, chrysopids and syrphids) which increase in abundance with the warming of temperature after winter season, but they are generally ineffective in suppressing the aphid population. There is a lack of phenological synchrony between their peak populations since natural enemies are active late in the season when most of the damage by aphids has already occurred [67, 68].

State	Period of peak activity	Crop	Reference(s)
Rajasthan	End January	B. juncea	[55]
Punjab	Mid February	B. juncea	[56]
	Jan–Feb	B. campestris
	Jan–Mar	B. juncea
	Mar	Brassica napus
		B. carinata	[57]
Haryana	Jan–Feb	Brassicas	[58, 59]
Delhi	Feb	Brassica rapa	[60]
Bihar	Jan–Feb	Rape/mustard	[61]
Orissa	January	Rape/mustard	[62]
Uttar Pradesh	January	B. juncea	[63]

Table 1.

Period of peak activity of Lipaphis erysimi as influenced by different types of cruciferous plants.

Source: Bakhetia et al. [64].

Cold and cloudy conditions are generally favorable for the development of mustard aphid [69], while extreme weather events like sub-zero temperature, fog, frost, rains and thunderstorms and very high temperature are the leading abiotic mortality factors. Mean maximum temperature of 17–18°C favors rapid population multiplication [58] while very low temperature during December and high temperature after March have detrimental effect on its multiplication. Hsiao [70] reported that L. erysimi manifests maximum intrinsic rate of increase, higher net reproductive rate and longer mean generation time at 25°C compared to other range of temperatures tested. In Nagpur, India, Kulat et al. [71] reported that a combination of maximum and minimum temperature in the range of 26.4–29.0°C and 8.4–12.6°C along with relative humidity of 75–85% in January resulted in conditions favorable for L. erysimi population development. On the other hand, a declining trend in population was observed at relative humidity ≤65%.

5.1 Cultural management

5.1.1 Sowing time

Time of sowing has a significant influence on the damage caused by aphids on oilseed Brassica. In India, L. erysimi generally causes maximum damage at flowering stage of the crop [72] which spans from end December/first fortnight of January to mid-February in different parts of the country. Thus, alteration in sowing date can help crop escape from the damage caused by L. erysimi as it leads to phenological asynchrony between the most susceptible crop stage and peak period of aphid activity. Phenological asynchrony can be achieved either through breeding by incorporating genes for earliness or alterations in sowing time. It has been observed that crop sown early (before October 20) escapes damage by L. erysimi in India [73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85]. However, care should be taken to avoid sowing too early especially in dry regions such as Rajasthan as it can result in more damage by painted bug [86]. On the other hand, crop sown late suffers heavy damage byL. erysimi [41, 42, 79, 87, 88, 89, 90].

5.1.2 Fertilizer application

Optimum nutrient application is an essential and often ignored component in both integrated pest as well as disease management. Excessive use of nitrogenous fertilizers can make plants more succulent [91] and susceptible to insect attack [92]. L. erysimi population was 4–8 times more in mustard crop that received 40 and 60 kg ha⁻¹ N as compared to that in the crop which received no fertilizer application [93, 94]. On the other hand, Bakhetia and Sharma [95] reported that increase in nitrogen application upto 80 kg ha⁻¹ had no effect on L. erysimi population development, however a negative correlation was observed between sulfur application and L. erysimi population development [96]. Similarly, increase in K application adversely affected reproduction and honey dew excretion of L. erysimi [97]. Increased application of P and K reduced aphid incidence on mustard plants [98].

5.1.3 Irrigation

In an agroecosystem, plants encounter multiple stresses that can influence their physiology and chemical composition including plant secondary metabolites. Drought/water stress not only influences plant physiology leading to decreased growth, but also leads to changes in profile of secondary metabolites and allocation of resources [99, 100, 101, 102]. Water stressed mustard plants were reported to support lower population of Brassica specialist L. erysimi [103, 104], while opposite trend was observed for generalist aphid M. persicae [100]. Similarly, Mewis et al. [102] reported rapid growth of M. persicae on water stressed Arabidopsis thaliana plants while B. brassicae performed equally well both on water stressed and well watered plants. However, heavy infestation of B. brassicae on water stressed plants of Brassica napus compared to unstressed plants was reported by some workers [105, 106]. This may partly be due to increase in concentration of amino acids in phloem sap [107] which makes it more nutritious. Miles et al. [108] reported increase in concentration of amino acids in water stressed rape plants leading to enhanced development of B. brassicae.

Besides changes in primary metabolites, water stress also leads to changes in plant secondary metabolites. Variations in glucosinolates may be in part responsible for observed variation in insect performance. Previous studies have reported decrease in glucosinolate levels in water stressed plants [100, 101]. Unlike generalist aphids, specialist aphids may tolerate glucosinolates in their host plants. However, there is general lack of complete understanding w.r.t. to effect of drought stress on secondary metabolite accumulation in relation to impact on plant resistance against aphids with different feeding specializations. Mewis et al. [102] reported a general trend of increase in levels of sucrose, several amino acids such as glutamic acid, proline, isoleucine and lysine while decrease in the levels of 4-methoxyindol-3-yl methyl glucosinolate was observed in water stressed plants. On the other hand, Chadda and Arora [107] observed a reduction in amino acids concentration in water stressed plants which in turn resulted in amino acid imbalance in aphid excretion resulting in reduced fecundity.

Bakhetia and Brar [109] reported a heavy aphid infestation on mustard grown under rainfed conditions with very high damage while irrigated crop maintained a good crop stand despite high aphid pressure partly due to differences in plant vigor.

5.2 Biological control

The term biological control covers a broad range of macro and microorganisms (e.g. parastitoids, predators, bacteria, virus, fungi, etc.), botanical extracts, semiochemicals and secondary metabolites from living organisms. The entomopathogenic fungus Verticillium lecanii has been found promising against L. erysimi [110, 111]. In a 2 years field study, spray of V. lecanii @ 10⁸ cs/ml followed by spray of neem seed kernel extract (5%) resulted in 60% reduction in L. erysimi population on Indian mustard as against 49% increase in aphid population in untreated control [110]. Field efficacy of neem seed kernel extract (5%) and neem leaf extract (5%) against L. erysimi has also been reported by other workers [112]. Many plant based materials have been evaluated against L. erysimi including neem/azadirachtin, nicotine sulphate, rotenone and pyrethrins. Extracts from common plants such as Azadirachta indica, Lantana camara, Melia azedarach, Solanum xanthocarpum exhibited variable toxicity against L. erysimi [113]. Tetrahydroazadirachtin-A, a thermo and photostable derivative of azadirachtin provided superior control of mustard aphid on B. juncea compared to azadirachtin [114]. Despite variable efficacy of botanicals, there is much needed to be done for their commercial exploitation. For example, the application rates of neem seed formulation (0.5–2.0 kg/ha) or fresh leaves (10–20 kg/ha) are too high to be acceptable by growers [115]. In developing countries, the use of biopesticides in pest management is low due to a number of factors such as low efficacy, speed of action, limited spectrum of activity, availability and affordability [116] and there is a need to create awareness among farmers about the ill effects associated with the use of chemical insecticides.

Aphid natural enemies can also be used for its management under field conditions. Like other agricultural systems, Brassica agroecosystem is also prone to pest outbreaks compared to natural ecosystems primarily due to loss of biodiversity [117]. However, Hawkins et al. [118] stated that one or two particularly effective natural enemies are all that are needed for effective pest control. Bakhetia and Sekhon [38] reported six Coccinellid species, 16 Syrphids, one chamaemyiid, hemerobiid (predators), four species each of parasitoids and entomopathogenic fungi and one predatory bird to be associated with L. erysimi as its natural enemies in India. Coccinellids are the predominant predators of this aphid species, among which Coccinella septempunctata, C. repanda, transversalis, Brumoides suturalis, Menochilus sexmaculatus and Hippodamia variegata are abundant in Brassica agroecosystem. Kumar [67, 119] observed that these natural enemies generally become active very late in the season when most of the damage by aphids has already occurred. Thus, despite their abundance these natural enemies fail to provide satisfactory control of mustard aphid due to phenological asynchrony between the peak activity period of L. erysimi and its natural enemies [67, 68]. Limited efficacy of C. septempunctata @ 5000 beetles ha⁻¹ and V. lecanii @ 10⁸ conidial spores ml⁻¹ against L. erysimi was reported on Indian mustard upto 10 days after release [111]. Although a number of syrphids are known to predate on mustard aphid, but they are generally very low in number to provide effective population suppression. The common associated syrphids are Episyrphus balteatus, Sophaerophoria scutellaris, Metasyrphus adligatus, M. corollae, Eristalis obscuritarsis, E. tenax, Xanthogramma scutellaris, Syrphus serarius and S. issaci. Similarly, the green lacewings (Chrysoperla carnea and Chrysopa scaslastes) have limited scope for use in population suppression of insect-pests.

In addition to predators, small aphid parasitoids, Diaeretiella rapae and Encyrtus sp. are also associated with mustard aphid. Just like predators, the parasitoids also appear late in the season (around mid February). Atwal et al. [120] reported that D. rapae causes more than 70% aphid parasitization in Punjab, India. However, recently Kumar [119] reported only 15.6% aphid parasitization on B. rapa. Biological control has the potential to offer sustainable solution for pest problems in agriculture [121] but its success rate is very low compared to chemical control [122]. With globalization and intensification of agriculture, the pest problems will increase resulting in increased use of pesticides [123]. For sustainable pest management, the expectations from biological control agents will increase.

5.3 Chemical control

At present, there is no stable resistant cultivar available against aphid pests in rapeseed-mustard. Thus, in their absence, insecticides are and will continue to be the major component of any pest management programme. In a developing country like India, farmers use them as the primary method of pest management as they find it easily available, economical and effective method of pest management. However, in an ideal pest management programme, insecticides should be used as the last option when all other alternative methods fail to provide satisfactory control since there are many problems associated with their use including environmental pollution, insecticide resistance and resurgence and pesticide residues in oil and cake. In India, pesticides are extensively used in rapeseed-mustard, but their application is mostly erratic. The fields requiring pesticide application are left unsprayed while other fields are sprayed indiscriminately and unnecessarily [124]. Even in a developed country like UK, the indiscriminate use of insecticides of vegetable brassicas was common due to lack of economic thresholds for many pests in the 90s [125]. The introduction of neonicotenoids played a significant role in pest management, but these were not introduced on a large scale in brassica crops unlike other crops [126]. In European Union, the ban on use of neonicotenoid insecticides as seed treatment on crops that attract pollinators, implemented in December 2013, adversely affected the pest control in oilseed rape [127, 128]. There were serious crop losses in 2014, 2015 and 2016 due to flea beetle, Psylliodes chrysocephala and peach-potato aphid, M. persicae which were already resistant to the alternative pyrethroid insecticides [129]. However, Blacquière and van der Steen [127] argue that decline of honey bees and wild pollinators is not likely caused by use of neonicotenoids and suggested for comprehensive studies on interactions with non-pesticide stressors. In India, neonicotinods are still used for pest management. Single application of imidacloprid provided 99% L. pseudobrassicae control [130]. Although, very high level of aphid control is obtained by use of synthetic insecticides, but high fecundity and short generation time of aphids leads to rapid population growth to levels similar to those in untreated fields within just 2–3 weekds [131]. To avoid indiscriminate use and prophylactic application of insecticides, the pesticide application decisions should be based on action (economic) threshold. But economic threshold levels are available for only a few major insects and there is need to calculate the same for other pests as well.

5.4 Integrated pest management

The sustainable solution to pest problems revolves around amalgamation of all the available and viable pest management strategies. However, in developing countries including India farmers are largely dependent on the use of synthetic chemicals due to their easily availability and quick results. The well known example of control failures of diamondback moth, Plutella xylostella was attributed to widespread and indiscriminate use of insecticides. This not only disturbed the natural control by parasitoids and predators in Brassica ecosystem, but also exerted high selection pressure on insect population leading to development of insecticide resistance to almost all groups of insecticides [132, 133]. In northern India, early sowing of rapeseed-mustard in October is advocated to create phenological asynchrony between the peak activity period of L. erysimi and flowering stage of the crop. However, growers in most parts of the North-Western India particularly Punjab and Haryana are unable to sow their crop in October due to late harvesting of rice crop in the preceding season. Action thresholds for control decisions are available, but control interventions are rarely made based on these thresholds. In the developing countries including India, there is a functional extension system to educate growers about importance of IPM and ill effects of insecticides, but farmers do not follow the advice of extension personnel. They follow the recommendations only if they are made into law [49].

Even in the developed country like UK, guidelines to manage aphids and insecticide resistance management have been published [134, 135], but insecticides are not selected on the basis of being less harmful to aphid natural enemies [136, 137]. A well established pest monitoring and forecasting system also exists in UK. In contrary to developing countries, it is supposed that growers in UK will follow the advice of extension functionaries—but this is not true. Most of the Brassica growers do not follow the recommendations of extension functionaries and go for prophylactic application of insecticides [136].

Though, aphid natural enemies are active in Brassica ecosystem but their peak activity lags behind the peak aphid activity [67] and they fail to provide effective aphid control. Rapeseed-mustard cultivars with less susceptibility/tolerance to aphids are available, but the resistance levels are still not high enough to induce growers to use them as a sole control measure. At present, semiochemicals are not applied to disrupt aphid pests or to attract their natural enemies in the agroecosystem. Thus, there are very limited control options available that can be made component of integrated pest management module. A resistant cultivar can serve as the core component of IPM module which will not only reduce reliance on insecticides but will also reduce pest management cost in addition to reduction in environment pollution and pesticide residues in oil and cake.

6. Traditional approaches in breeding for aphid resistance

Brassica plants are among the oldest cultivated plants with documented records dating back to ca. 1500 BC [138]. Their domestication over the years has lead to narrowing of genetic base. The breeding efforts were focussed on high yield and quality traits (low glucosinolates and erucic acid). Thus, little/no attention was paid to maintain insect and/or disease resistance. Thus, over the course, the defense related genes in ancestral Brassica plants were lost. However, in the recent times, there is renewed interest in remobilizing these defense related genes. All this requires, rigorous screening of large Brassica germplasm for insect resistance and an efficient screening technique is the very prerequisite for this.

6.1 Screening methodology

Screening for source of resistance is the first step in development of an insect resistant cultivar. A very large number of attempts have been made in the past to identify sources of resistance in primary gene pool of crop brassica species [139, 140, 141, 142, 143]. The literature on the screening techniques for aphid resistance has been reviewed extensively by Bakhetia and Sekhon [38]. The different techniques used for screening are discussed in the following text.

6.1.1 Seedling stage screening

Screening at seedling stage is more desirable than field screening at adult plant stage because of the cost and efforts involved. However, resistance at seedling stage may not express at adult plant stage and no serious effort has been made to correlate seedling stage resistance with the adult plant resistance. Earlier, Bakhetia and Bindra [144] had attempted to develop seedling stage screening method against L. erysimi which is compatible with adult plant evaluation. It is based on seedling mortality at a defined aphid population level. For efficient resistance screening, population levels of 11, 20, 20, 30 apterae and, 1 and 3 ml aphids (1 ml = ∼600 numphs + apterae) per plant are optimum for resistance screening at cotyledonary, 2-leaf, 4-leaf, flower bud initiation and flowering stages, respectively [143]. Despite the advantages of seedling stage screening, this method is not widely used for screening of Brassica germplasm against aphids.

6.1.2 Adult plant stage screening

Contrary to the seedling stage screening, this is the most widely used method in screening for aphid resistance in Brassica germplasm, since it reflects the actual resistance exhibited by plants under field conditions. Though, it is laborious and time consuming method, but it does not undermine its usefulness. It is based on the injury symptoms exhibited by aphid feeding which range from yellowing, curling and crinkling of leaves to drying of floral buds, flowers and shriveling of pods. Different grading systems have been adopted by different workers, but the one suggested by Bakhetia and Sandhu [145] is the most practical and widely accepted for mustard aphid screening.

Based on the aphid injury level, different injury grades for field screening are given to the plants as follows:

Aphid infestation index (AII)	Description
0	Free from aphid infestation. Even if a single wingless aphid is present, the plant is considered infested. Plants showing excellent growth.
1	Normal growth, no curling or yellowing of the leaves, except only a few aphids along with little or no symptoms of injury. Good flowering or pod setting on almost all the branches.
2	Average growth, curling and yellowing of a few leaves. Average flowering and pod setting on all the branches.
3	Growth below average, curling and yellowing of the leaves on some branches. Plants showing some stunting, poor flowering and little pod setting.
4	Very poor growth, heavy curling and the yellowing of leaves, stunting of plants, little or no flowering and only a few pods forming. Heavy aphid colonies on plants.
5	Heavy stunting of plants; curling, crinkling and yellowing of almost all the leaves. No flowering and pod formation. Plants full of aphids.

Bakhetia and Sandhu [145].

Based on the degree of damage, an injury grade is given to every observed plant. The Aphid Infestation Index (AII) is calculated by multiplying the number of plants falling under each injury grade with their respective grade number. AII is calculated at pre-flowering, flowering and pod formation stages as:

$$\text{Aphid Infestation Index}=\frac{\left(0\times \text{a}\right)+\left(1\times \text{b}\right)+\left(2\times \text{c}\right)+\left(3\times \text{d}\right)+\left(4\times \text{e}\right)+\left(5\times \text{f}\right)}{\text{a}+\text{b}+\text{c}+\text{d}+\text{e}+\text{f}}$$

where a, b, c, d, e, and f are the number of plants falling under each injury grade.

The different test entries are classified into different resistance categories based on the AII as:

Aphid infestation index (AII)	Reaction
0.00–1.50	Resistant
1.51–2.50	Moderately resistant
2.51–3.50	Susceptible
>3.50	Highly susceptible

Higher the AII, lower is the level of resistance in an entry.

6.1.3 Other screening methods

Recently, Dhillon et al. [146] evaluated twig cage, whole plant cage, plot cage and uncaged plants methods to look for efficient screening method against L. erysimi. They concluded that no-choice twig cage method is the most appropriate of all for field screening of Brassica genotypes. However, there were many flaws in their methodology followed. The authors infested the test plants artificially with pieces of infested Brassica twigs pinned to the plant. Pinning of host plants inflicts mechanical injury which activates the myrosinase-glucosinolate defense system in Brassica plants which in turn interferes with expression of natural resistance. Further, twig cage alters the microclimate leading to physiological and nutritional deviation from naturally grown plants. Generally, caged twigs/plants exhibit abnormal growth which may interfere with their natural expression of resistance. Furthermore, authors have worked out both Aphid Population Index (API) and Aphid Damage Index (ADI) each on 0–5 scale. Aphid Resistance Index (ARI) is worked out after taking mean of the two. While, ADI is based on the degree of damage done to host plants, API is based on the aphid population—higher the pest numbers, more the API will be. Some plants may harbor high aphid population without exhibiting significant damage and vice versa. Thus, inclusion of API in calculations of Aphid Resistance Index does not represent the true resistance exhibited by host plant. While, Bakhetia and Sandhu [145] recorded both aphid population and injury grade at same point of time (though they have not used population data for calculation of Aphid Infestation Index), Dhillon et al. [146] recorded API after 21 days of artificial infestation and ADI at completion of pod formation, which puts another question mark on the methodology followed.

In addition to this, aphid population at a particular stage and an increase in population during a given time interval can also be used in germplasm screening [38]. Kumar et al. [147] attempted to screen a diverse array of wild crucifers based both on the adult plant resistance and effect on aphid demographic parameters (survival, development and fecundity) and reported one wild Brassica fruticulosa to be resistant to L. erysimi. Only limited attempts have been made to develop screening technique based on aphid biology, despite its significance in identifying sources of resistance. It is possible to develop such a criterion for aphid screening since nymphal survival, fecundity, longevity and reproduction are similar at all the plant growth stages [144]. Singh et al. [148] and Malik [149] have also reported fecundity to be inversely related to resistance.

6.2 Conventional breeding

The three modalities of resistance include antixenosis, antibiosis and tolerance. Although, antixenosis does not exert any selection pressure on insect population and there is no risk of biotype development, it is rarely effective under no choice conditions as insects can learn to feed on less preferred host plant. In contrast, antibiosis exerts high selection pressure on the insect population leading to high risk of biotype development, a danger not applicable to tolerance. Insect population can be allowed to feed on the crop and growers would not need to control them, but they would breed population to infest their neighbors’ crops. Thus, an ideal resistance is a combination of all the three mechanisms [150].

Earlier workers have attempted to develop resistant cultivars using different breeding methods viz. intervarietal hybridization, induced mutagenesis or autotetraploidy. B. napus strains and colchicine induced tetraploid toria (B. rapa) were found to be resistant to mustard aphid as compared to diploids with antibiosis mechanism of resistance [148, 151, 152, 153, 154]. However, these were cytogenetically unstable. Many workers have attempted to artificially synthesize B. napus and B. rapa x Eruca sativa alloploids [155] but these were not resistant.

In an attempt to develop aphid resistant cabbage variety, Lammerink [156] made selections from F₃ generation of the cross (Broad Leaf Essex rape x Colder Swede) x giant rape. In addition to this, he also made recurrent selection in the crosses involving purple top white globe and Sjodin turnip. Kumar et al. [147] screened a diverse array of wild crucifers and found one wild B. fruticulosa to be resistant to L. erysimi. They further attempted to introgress the resistance gene to B. juncea background. In addition to L. erysimi, B. fruticulosa has been earlier reported to be resistant to mealy cabbage aphid, B. brassicae [5, 6, 157, 158]. It was reported to possess antixenosis and antibiosis mechanisms of resistance against L. erysimi along with the B. juncea-fruticulosa introgression lines [159]. Further, monitoring of feeding behavior of B. brassicae by electrical penetration graph (EPG) revealed large reduction in duration of passive phloem uptake in B. fruticulosa compared to Brassica oleracea var. capitata cv. ‘Offenham Compacta’. Aphids either showed quick withdrawl of stylets from sieve tubes or there was disrupted phloem uptake [5]. The mechanism of resistance was a combination of both antixenosis and antibiosis [157]. In addition to resistance against aphid pests, B. fruticulosa has also been reported to possess resistance (antibiosis) against Delia radicum [160].

In addition, efforts have also been made to induce mutation in B. juncea for resistance against aphid pests both by chemical [161] and physical mutagens [162, 163] but no significant results were obtained. Recently, Agrawal et al. [164] have attempted to use γ-irradiation on a set of introgression lines to optimize the introgressed segment.

6.3 Use of transgenic technology

Transgenic technology has emerged as an alternative breeding strategy to conventional breeding. Different strategies such as expression of protease inhibitors, RNA interference (RNAi), antimicrobial peptides and repellents can be employed for sap sucking insects such as aphids. Since aphids are phloem feeders, thus, phloem specific promoters can be used for expression of defense related compounds against them. This would lead to target specific expression of defense compounds with little/no effect on non-target insects. Further, it will also limit the GM-associated resource investment by plants to the plant tissues that are not attacked by the insect. The SUC2 promoter that regulates the AtSUC2 sucrose-H⁺ symporter gene is restricted to plant phloem which produces aphid toxic proteins. This protein is transferred through sieve tubes to actual aphid feeding site [165].

Likewise Protease Inhibitors (PIs) can also be targeted to confer resistance in transgenic plants to insects, which inhibit/reduce the activity of enzymes involved in protein digestion (proteases). Toxic effects of PIs on insect-pests have been well demonstrated particularly those from order Coleoptera, Lepidoptera and Orthoptera [166]. In aphids, PIs ingested along with plant sap inhibit protein digestion in insect gut leading to disruption in amino acid assimilation subsequently leading to adverse effect on insect growth and its ability to cause plant damage. Successful attempts have been made to express PIs such as trypsin inhibitors and chymotrypsin inhibitors in phloem of transgenic plant [167, 168]. Barley cystein proteinase inhibitor, HvCPI-6 is reported to inhibit performance of M. persicae and Acyrthosiphon pisum in artificial diet [169] while, cysteine protease inhibitor, oryzacystatin I (OC I) inhibited growth of M. persicae, A. gossypii and A. pisum [170]. M. persicae fed on transgenic B. napus plants expressing (OC I) suffered reduction in adult weight, biomass and fecundity in comparison to those fed on control plants. Thus, protease inhibitors have a good potential to be used as an effective strategy to confer aphid resistance in plants.

In addition to PIs, lectins also exhibit high toxicity against sap sucking insects including aphids. Lectins are proteins that selectively bind to carbohydrates and carbohydrate moieties of glycoproteins leading to poisonous effect on the insect. The poisonous effects of lectins have been demonstrated on a number of insects, especially the sap sucking insects [171, 172]. A number of genes coding for different lectins have been introduced in B. juncea that confer resistance against L. erysimi such as wheat germ agglutinin from Triticum spp. [173], agglutinin ACA from Allium cepa [174], fusion lectin ASAL from Allium sativum and ACA from A. cepa [174]. Laboratory bioassays have confirmed significant toxic effect of these transgenic plants on L. erysimi.

RNAi is gaining increased attention as another potential strategy to confer resistance against insects. It involves suppression of genes at the level of RNA (posttranslational RNA-mediated gene silencing). Transgenic plants that delivered dsRNA to M. persicae resulted in inhibition of Rack1 protein located in gut and C002 protein located in the salivary glands of the aphid [175]. The transformed tobacco and A. thaliana plants resulted in adverse effect on aphid fecundity with upto 60% silencing in aphids that fed on these plants. Although salivary and gut proteins are the promising targets for sucking insects including aphids, the other targets can be transporters in the bacteriocyte plasma membrane required for transport of nutrients between aphids and symbionts, Buchnera aphidicola.

7. Induced resistance

Plants are known to increase the level of many defense related compounds post insect infestation. This induction of resistance after insect feeding has also been reported in Brassica plants. During infestation of plants by insects, major defense related plant hormones are salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) which are involved in induced resistance of many plants against insects [176]. Aphid feeding is known to activate SA signaling pathway in a number of host plant species [35, 177, 178]. However, no involvement of SA signaling against aphids was reported in Arabiopsis [179]. Kettles et al. [180] reported an increase in M. persicae population in ET-insensitive Arabidopsis mutant ein2, indicating the role of ET in conferring resistance to aphids. Kerchev et al. [181] reported that resistance of Arabidopsis to aphids also depends upon ABA biosynthesis and signaling. Recently, Palial et al. [182] have reported in induction in glucosinolates content in B. fruticulosa and total phenols content in B. juncea-fruticulosa introgression lines after L. erysimi feeding.

8. Conclusion

The continuous coevolutionary history of aphids and members of family Brassicaceae have enabled these plants to evolve an array of defense related genes. However, plant breeding efforts have largely focused on selection for yield related and quality traits such as low glucosinolates and erucic acid traits with little attention to retain the adequate levels of insect and disease resistance. This lead to the loss of defense related genes in these crops over the time. Further, availability of chemical control measures at that time downgraded the importance of host plant resistance since chemical control was thought to be satisfactory and invulnerable. However, later it was realized that though insecticides can provide a short term pest control and host plant resistance can provide effective, economical and environment friendly pest management option. Thus, early plant breeders focused on host plant resistance as a single component of pest management and laid more emphasis on screening for virtual immunity to aphids. Immunity/high level of resistance can result from very high level of toxic substance (toxic to aphids) in host plant which can exert high selection pressure on aphid population leading to the development new biotypes, possible side effects on non-target organisms including honeybees and yield drag. Thus, partial resistance has potential role in sustainable pest management as varieties with partial resistance can be integrated with other pest management methods. At present, there is no effective IPM strategy against aphids due to lack of aphid resistant variety. Although, various workers have developed Brassica transgenics that offer some degree of resistance against aphids, but they have primarily evaluated under laboratory settings and field testing of such transgenics is still awaited.

To maintain sustainability of pest control and production systems, IPM should be seen as the best approach and host plant resistance can serve as core component of any IPM module. Rather than complete resistance, it is partial resistance that has greater potential to maintain such sustainability.