Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats

While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9-12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests.     

Drought stress affects response of phytopathogen vectors and their parasitoids to infection‐ and damage‐induced plant volatile cues

1. The response of a phytopathogen vector to pathogen‐induced plant volatiles was investigated, as well as the response of the phytopathogen vector's parasitoid to herbivore‐induced plant volatiles released from plants with and without drought stress. 2. These experiments were performed with Asian citrus psyllid (Diaphorina citri), vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas) and its parasitoid Tamarixia radiata as models. Candidatus Liberibacter asiaticus is the presumed causal pathogen of huanglongbing (HLB), also called citrus greening disease. 3. Diaphorina citri vectors were attracted to headspace volatiles of CLas‐infected citrus plants at 95% of their water‐holding capacity (WHC); such attraction to infected plants was much lower under drought stress. Attraction of the vector to infected and non‐stressed plants was correlated with greater release of methyl salicylate (MeSA) as compared with uninfected and non‐stressed control citrus plants. Drought stress decreased MeSA release from CLas‐infected plants as compared with non‐stressed and infected plants. 4. Similarly, T. radiata was attracted to headspace volatiles released from D. citri‐infested citrus plants at 95% of their WHC. However, wasps did not show preference between headspace volatiles of psyllid‐infested and uninfested plants when they were at 35% WHC, suggesting that herbivore‐induced defences did not activate to recruit this natural enemy under drought stress. 5. Our results demonstrate that herbivore‐ and pathogen‐induced responses are environmentally dependent and do not occur systematically following damage. Drought stress affected both pathogen‐ and herbivore‐induced plant volatile release, resulting in concomitant decreases in behavioural response of both the pathogen's vector and the vector's primary parasitoid.     

Behavioral evidence for contextual olfactory‐mediated avoidance of the ubiquitous phytopathogen Botrytis cinerea by Drosophila suzukii

Herbivorous insects may benefit from avoiding the smell produced by phytopathogens infecting plant host tissue if the infected tissue reduces insect fitness. However, in many cases the same species of phytopathogen can also infect host plant tissues that do not directly affect herbivore fitness. Thus, insects may benefit from differentiating between pathogen odors emanating from food and nonfood tissues. This is based on the hypothesis that unnecessarily staying attentive to pathogen odor from nonfood tissue may incur opportunity costs associated with not responding to other important survival functions. In this study adults of Drosophila suzukii Matsumura, an invasive larval frugivore, showed reduced attraction to the odor of raspberry fruit, a food tissue, when infected with Botrytis cinerea Pers., a ubiquitous phytopathogen, in favor of odors of uninfected raspberry fruit. Moreover, D. suzukii oviposited fewer eggs on infected raspberry fruit relative to uninfected raspberry fruit. Larval survival and adult size after eclosion were significantly reduced when reared on B. cinerea‐infected raspberry relative to uninfected fruit. Interestingly, when the behavioral choice experiment was repeated using Botrytis‐infected vs. ‐uninfected strawberry leaves, a nonfood tissue, in combination with fresh raspberry fruit, odor from B. cinerea‐infected leaves did not reduce D. suzukii attraction to raspberries relative to raspberries with uninfected leaves. These behavioral results illustrate the important role context can play in odor‐mediated interactions between insects, plants and microbes. We discuss implications of our findings for developing a repellent that can be useful for the management of D. suzukii.     

Influence of presence and spatial arrangement of belowground insects on host‐plant selection of aboveground insects: a field study

Abstract 1. Several studies have shown that above‐ and belowground insects can interact by influencing each others growth, development, and survival when they feed on the same host‐plant. In natural systems, however, insects can make choices on which plants to oviposit and feed. A field experiment was carried out to determine if root‐feeding insects can influence feeding and oviposition preferences and decisions of naturally colonising foliar‐feeding insects. 2. Using the wild cruciferous plant Brassica nigra and larvae of the cabbage root fly Delia radicum as the belowground root‐feeding insect, naturally colonising populations of foliar‐feeding insects were monitored over the course of a summer season. 3. Groups of root‐infested and root‐uninfested B. nigra plants were placed in a meadow during June, July, and August of 2006 for periods of 3 days. The root‐infested and the root‐uninfested plants were either dispersed evenly or placed in clusters. Once daily, all leaves of each plant were carefully inspected and insects were removed and collected for identification. 4. The flea beetles Phyllotreta spp. and the aphid Brevicoryne brassicae were significantly more abundant on root‐uninfested (control) than on root‐infested plants. However, for B. brassicae this was only apparent when the plants were placed in clusters. Host‐plant selection by the generalist aphid M. persicae and oviposition preference by the specialist butterfly P. rapae, however, were not significantly influenced by root herbivory. 5. The results of this study show that the presence of root‐feeding insects can affect feeding and oviposition preferences of foliar‐feeding insects, even under natural conditions where many other interactions occur simultaneously. The results suggest that root‐feeding insects play a role in the structuring of aboveground communities of insects, but these effects depend on the insect species as well as on the spatial distribution of the root‐feeding insects.     

Host activity and wasp experience affect parasitoid wasp foraging behaviour and oviposition on nematode‐infected larvae of the forestry pest Hylobius abietis

1. Entomopathogenic nematodes (EPN) are currently being used as introduced biological control agents against the larvae of the native European forestry pest Hylobius abietis L. which develop under the bark of stumps and roots of newly dead conifer trees. 2. The potential for resource competition between gregarious ectoparasitoid Bracon hylobii Ratz and EPN by recording oviposition and related behaviours of B. hylobii females on EPN‐infected H. abietis larvae was investigated. Wasps did not parasitise EPN‐infected host larvae that were dead when presented, but naÏve and experienced wasps parasitised live EPN‐infected hosts. NaÏve wasps parasitised live EPN‐infected hosts significantly less frequently than healthy hosts only when the infected larvae were close to death (i.e. died during 24‐h trial). Parasitism by experienced wasps was unaffected by host infection. 3. Wasp probing and oviposition were positively associated with the amount of host movement. Preventing H. abietis larvae from chewing on bark significantly reduced parasitism by naÏve, but not experienced wasps. 4. The number of eggs per clutch was not affected by bark chewing or EPN‐infection of H. abietis larvae. 5. NaÏve and experienced B. hylobii parasitised two abnormal hosts (larvae of coleopteran Rhagium bifasciatum Fabricius and lepidopteran Galleria mellonella L.), both of which moved and chewed on bark during trials. 6. It was concluded that B. hylobii can use vibrational cues generated by host movement and feeding to locate hosts at short range and accepts unsuitable (EPN‐infected or abnormal) hosts as long as these create such cues. The implications for competition between B. hylobii and EPN and possible ways of minimising it when applying EPN are discussed.     

Infection of an Insect Vector with a Bacterial Plant Pathogen Increases Its Propensity for Dispersal

The spread of vector-transmitted pathogens relies on complex interactions between host, vector and pathogen. In sessile plant pathosystems, the spread of a pathogen highly depends on the movement and mobility of the vector. However, questions remain as to whether and how pathogen-induced vector manipulations may affect the spread of a plant pathogen. Here we report for the first time that infection with a bacterial plant pathogen increases the probability of vector dispersal, and that such movement of vectors is likely manipulated by a bacterial plant pathogen. We investigated how Candidatus Liberibacter asiaticus (CLas) affects dispersal behavior, flight capacity, and the sexual attraction of its vector, the Asian citrus psyllid (Diaphorina citri Kuwayama). CLas is the putative causal agent of huanglongbing (HLB), which is a disease that threatens the viability of commercial citrus production worldwide. When D. citri developed on CLas-infected plants, short distance dispersal of male D. citri was greater compared to counterparts reared on uninfected plants. Flight by CLas-infected D. citri was initiated earlier and long flight events were more common than by uninfected psyllids, as measured by a flight mill apparatus. Additionally, CLas titers were higher among psyllids that performed long flights than psyllid that performed short flights. Finally, attractiveness of female D. citri that developed on infected plants to male conspecifics increased proportionally with increasing CLas bacterial titers measured within female psyllids. Our study indicates that the phytopathogen, CLas, may manipulate movement and mate selection behavior of their vectors, which is a possible evolved mechanism to promote their own spread. These results have global implications for both current HLB models of disease spread and control strategies.     

Huanglongbing pathogen Candidatus Liberibacter asiaticus exploits the energy metabolism and host defence responses of its vector Diaphorina citri

Asian citrus psyllid Diaphorina citri is the vector of the citrus Huanglongbing (HLB) associated bacterial agent ‘Candidatus Liberibacter asiaticus’ (CLas). The molecular interactions between CLas and D. citri remain unclear. In the present study, protein profiles of mitochondrial, microsomal and cytosolic fractions from uninfected and CLas‐infected adult D. citri are investigated using two‐dimensional gel electrophoresis. The comparative analysis reveals a total of 18, 24 and 20 protein spots that are unique or differentially expressed in mitochondrial, microsomal and cytosolic proteins fractions respectively. These proteins are successfully identified by mass spectrometry. Among the 62 identified proteins, 30 are up‐regulated, whereas 32 are down‐regulated. These proteins include important components in energy metabolism such as ATP synthase, ATPase, ATP/ADP carrier protein, etc.; host stress responses such as heat shock proteins; host detoxification processes (i.e., cytochrome P450 and glutathione S‐transferase); and the cytoskeleton (such as actin, tubulin, myosin and tropomyosin). These data suggest that, after CLas infection, several proteins of D. citri, especially energy metabolism and protein biosynthesis, are altered, and extensive host defence responses are induced. In conclusion, the present study reports proteomic information that is helpful in understanding the vector–pathogen relationship between CLas and D. citri, and could be used to identify potential targets for limiting the spread of CLas, as well as to provide new insights into HLB management.     

Effect of host‐plant and infection with ‘Candidatus Liberibacter asiaticus’ on honeydew chemical composition of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the citrus greening pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) by feeding on citrus phloem sap. Because phloem sap is rich in sugars but low in amino acids, ACP sucks large quantities and excretes most of it as honeydew. We studied the chemical composition of ACP honeydew on various host plants. Honeydew samples were analyzed with gas chromatography–mass spectrometry. Fourteen sugars, 13 amino acids, and six organic acids were detected in the honeydew of ACP. Sugars composed about 95% of the total compounds. Sucrose and trehalose were the predominant sugars, composing about 58 and 23% of the total sugars, respectively. Proline, asparagine, aspartic acid, and glutamic acid were the most abundant amino acids in ACP honeydew. The host plant and its infection with CLas had some effect on the honeydew composition. Glucose, chiro‐inositol, myo‐inositol, inositol, maltose, and turanose were lower in honeydew collected from CLas‐infected citrus compared to that collected from non‐infected trees. In CLas‐infected citrus (pineapple sweet orange, Citrus sinensis L. Osbeck) and Bergera koenigii (L.) Spreng. [curry leaf tree (both Rutaceae)] honeydews, valine, alanine, serine, glutamine, glycine, and the organic acids were lower than in honeydew from healthy citrus. Mannose, galactose, inositol, mannitol, an unknown disaccharide, and proline were higher in the honeydew collected from B. koenigii than in honeydew collected from healthy citrus (pineapple sweet orange), whereas fructose, chiro‐inositol, myo‐inositol, trehalose, and lactic acid were lower. The findings of this study help us understand the metabolism and the nutrient needs of ACP that transmits CLas, the pathogen of huanglongbing in citrus.     

Insect eggs induce a systemic acquired resistance in Arabidopsis

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen‐associated molecular patterns (PAMPs), which are involved in PAMP‐triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg‐induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae‐infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg‐induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.     

Interrelated effects of mycorrhiza and free‐living nitrogen fixers cascade up to aboveground herbivores

Aboveground plant performance is strongly influenced by belowground microorganisms, some of which are pathogenic and have negative effects, while others, such as nitrogen‐fixing bacteria and arbuscular mycorrhizal fungi, usually have positive effects. Recent research revealed that belowground interactions between plants and functionally distinct groups of microorganisms cascade up to aboveground plant associates such as herbivores and their natural enemies. However, while functionally distinct belowground microorganisms commonly co‐occur in the rhizosphere, their combined effects, and relative contributions, respectively, on performance of aboveground plant‐associated organisms are virtually unexplored. Here, we scrutinized and disentangled the effects of free‐living nitrogen‐fixing (diazotrophic) bacteria Azotobacter chroococcum (DB) and arbuscular mycorrhizal fungi Glomus mosseae (AMF) on host plant choice and reproduction of the herbivorous two‐spotted spider mite Tetranychus urticae on common bean plants Phaseolus vulgaris. Additionally, we assessed plant growth, and AMF and DB occurrence and density as affected by each other. Both AMF alone and DB alone increased spider mite reproduction to similar levels, as compared to the control, and exerted additive effects under co‐occurrence. These effects were similarly apparent in host plant choice, that is, the mites preferred leaves from plants with both AMF and DB to plants with AMF or DB to plants grown without AMF and DB. DB, which also act as AMF helper bacteria, enhanced root colonization by AMF, whereas AMF did not affect DB abundance. AMF but not DB increased growth of reproductive plant tissue and seed production, respectively. Both AMF and DB increased the biomass of vegetative aboveground plant tissue. Our study breaks new ground in multitrophic belowground–aboveground research by providing first insights into the fitness implications of plant‐mediated interactions between interrelated belowground fungi–bacteria and aboveground herbivores.     

Powdery mildew suppresses herbivore‐induced plant volatiles and interferes with parasitoid attraction in Brassica rapa

The co‐occurrence of different antagonists on a plant can greatly affect infochemicals with ecological consequences for higher trophic levels. Here we investigated how the presence of a plant pathogen, the powdery mildew Erysiphe cruciferarum, on Brassica rapa affects (1) plant volatiles emitted in response to damage by a specialist herbivore, Pieris brassicae; (2) the attraction of the parasitic wasp Cotesia glomerata and (3) the performance of P. brassicae and C. glomerata. Plant volatiles were significantly induced by herbivory in both healthy and mildew‐infected plants, but were quantitatively 41% lower for mildew‐infected plants compared to healthy plants. Parasitoids strongly preferred Pieris‐infested plants to dually‐infested (Pieris + mildew) plants, and preferred dually infested plants over only mildew‐infected plants. The performance of P. brassicae was unaffected by powdery mildew, but C. glomerata cocoon mass was reduced when parasitized caterpillars developed on mildew‐infected plants. Thus, avoidance of mildew‐infested plants may be adaptive for C. glomerata parasitoids, whereas P. brassicae caterpillars may suffer less parasitism on mildew‐infected plants in nature. From a pest management standpoint, the concurrent presence of multiple plant antagonists can affect the efficiency of specific natural enemies, which may in turn have a negative impact on the regulation of pest populations.     

Vector within‐host feeding preference mediates transmission of a heterogeneously distributed pathogen

1. Ecological theory predicts that vector preference for certain host species or discrimination between infected versus uninfected hosts impacts disease incidence. However, little information exists on the extent to which vector within‐host feeding preference mediates transmission. This may be particularly important for plant pathogens, such as sharpshooter transmission of the bacterium Xylella fastidiosa, which are distributed irregularly throughout hosts. 2. We documented the within‐host distribution of two vector species that differ in transmission efficiency, the leafhoppers Draeculacephala minerva and Graphocephala atropunctata, and which are free to move throughout entirely caged alfalfa plants. The more efficient vector D. minerva fed preferentially at the base of the plant near the soil surface, whereas the less efficient G. atropunctata preferred overwhelming the top of the plant. 3. Next we documented X. fastidiosa heterogeneity in mechanically inoculated plants. Infection rates were up to 50% higher and mean bacterial population densities were 100‐fold higher near the plant base than at the top or in the taproot. 4. Finally, we estimated transmission efficiency of the two leafhoppers when they were confined at either the base or top of inoculated alfalfa plants. Both vectors were inefficient when confined at the top of infected plants and were 20–60% more efficient when confined at the plant base. 5. These results show that vector transmission efficiency is determined by the interaction between leafhopper within‐plant feeding behaviour and pathogen within‐plant distribution. Fine‐scale vector and pathogen overlap is likely to be a requirement generally for efficient transmission of vector‐borne pathogens.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.     

The interactions of tropical soda apple mosaic tobamovirus and Gratiana boliviana (Coleoptera: Chrysomelidae), an introduced biological control agent of tropical soda apple (Solanum viarum)

Tropical soda apple (Solanum viarum Dunal (Solanaceae) is a South American invasive plant of rangelands, pastures and natural areas in Florida. A chrysomelid beetle from South America, Gratiana boliviana Spaeth, has been released at >300 locations in Florida for biological control of tropical soda apple since 2003. Tropical soda apple is a host of several plant viruses, including the newly described tropical soda apple mosaic virus (TSAMV). We investigated the influence of TSAMV infection of tropical soda apple plants on developmental time, leaf tissue consumption, longevity, fecundity, and feeding preference of G. boliviana, and also tested transmission of the virus by the beetle. Developmental time was approximately 10% slower, and adults consumed only about 50% as much leaf tissue, for beetles fed on infected plants compared to uninfected plants. Longevity did not differ between females reared on infected and uninfected plants, but females fed on uninfected plants produced 71% more eggs than those fed on infected plants. Adult G. boliviana preferentially fed on uninfected plants when given a choice. There was no evidence of TSAMV transmission by G. boliviana. The potential impacts of TSAMV infection on the effectiveness of G. boliviana as a biological control agent are discussed.     

Influence of the forest caterpillar hunter Calosoma sycophanta on the transmission of microsporidia in larvae of the gypsy moth Lymantria dispar

• 1 The behaviour of predators can be an important factor in the transmission success of an insect pathogen. We studied how Calosoma sycophanta influences the interaction between its prey [Lymantria dispar (L.) (Lepidoptera, Lymantriidae)] and two microsporidian pathogens [Nosema lymantriae (Microsporidia, Nosematidae) and Vairimorpha disparis (Microsporidia, Burellenidae)] infecting the prey.
• 2 Using laboratory experiments, C. sycophanta was allowed to forage on infected and uninfected L. dispar larvae and to disseminate microsporidian spores when preying or afterwards with faeces.
• 3 The beetle disseminated spores of N. lymantriae and V. disparis when preying upon infected larvae, as well as after feeding on such prey. Between 45% and 69% of test larvae became infected when C. sycophanta was allowed to disseminate spores of either microsporidium.
• 4 Laboratory choice experiments showed that C. sycophanta did not discriminate between Nosema‐infected and uninfected gypsy moth larvae. Calosoma sycophanta preferred Vairimorpha‐infected over uninfected gypsy moth larvae and significantly influenced transmission.
• 5 When C. sycophanta was allowed to forage during the latent period on infected and uninfected larvae reared together on caged, potted oak saplings, the percentage of V. disparis infection among test larvae increased by more than 70%. The transmission of N. lymantriae was not affected significantly in these experiments.
• 6 Beetles never became infected with either microsporidian species after feeding on infected prey.
• 7 We conclude that the transmission of N. lymantriae is not affected. Because no V. disparis spores are released from living larvae, feeding on infected larvae might enhance transmission by reducing the time to death and therefore the latent period.

     

Extended larval development time for aphid parasitoids in the presence of plant endosymbionts

Abstract 1. Variation in plant chemistry does not only mediate interactions between plants and herbivores but also those between herbivores and their natural enemies, and plants and natural enemies. 2. Endophytic fungi complete their whole life cycle within the host plant’s tissue and are associated with a large diversity of plant species. Endophytes of the genus Neotyphodium alter the chemistry of the host plant by producing herbivore toxic alkaloids. 3. Here we asked whether the endophyte‐tolerant aphid species Metopolophium festucae could be defended against its parasitoid Aphidius ervi when feeding on endophyte‐infected plants. In a laboratory experiment, we compared life‐history traits of A. ervi when exposed to hosts on endophyte‐infected or endophyte‐free Lolium perenne. 4. The presence of endophytes significantly increased larval and pupal development times, but did not affect the mortality of immature parasitoids or the longevity of the adults. Although the number of parasitoid mummies tended to be reduced on endophyte‐infected plants, the number of emerging parasitoids did not differ significantly between the two treatments. 5. This shows that the metabolism of individual aphids feeding on infected plants may be changed and help in the defence against parasitoids. An increase in parasitoid development time should ultimately reduce the population growth of A. ervi. Therefore, endophyte presence may represent an advantage for endophyte‐tolerant aphid species through extended parasitoid development and its effect on parasitoid population dynamics.     

Induced carbon reallocation and compensatory growth as root herbivore tolerance mechanisms

Upon attack by leaf herbivores, many plants reallocate photoassimilates below ground. However, little is known about how plants respond when the roots themselves come under attack. We investigated induced resource allocation in maize plants that are infested by the larvae Western corn rootworm Diabrotica virgifera virgifera. Using radioactive ¹¹CO₂, we demonstrate that root‐attacked maize plants allocate more new ¹¹C carbon from source leaves to stems, but not to roots. Reduced meristematic activity and reduced invertase activity in attacked maize root systems are identified as possible drivers of this shoot reallocation response. The increased allocation of photoassimilates to stems is shown to be associated with a marked thickening of these tissues and increased growth of stem‐borne crown roots. A strong quantitative correlation between stem thickness and root regrowth across different watering levels suggests that retaining photoassimilates in the shoots may help root‐attacked plants to compensate for the loss of belowground tissues. Taken together, our results indicate that induced tolerance may be an important strategy of plants to withstand belowground attack. Furthermore, root herbivore‐induced carbon reallocation needs to be taken into account when studying plant‐mediated interactions between herbivores.     

Growth substances in roots of tomato (Lycopersicon esculentum Mill.) infected with root-knot nematodes (Meloidogyne spp.)

Roots of tomato plants with galls caused by larvae of Meloidogyne spp. contained a similar concentration of auxin as uninfected roots, but a larger total amount because the roots of infected plants were heavier. The body contents and saliva or excretions of M. incognita larvae contained too little auxin to account for the increased amounts in infected roots. Roots with galls contained more bound auxin, released by alkaline hydrolysis or incubation after maceration, and more tryptophan and other amino acids, than uninfected roots. The larvae may hydrolyse the plant proteins to yield tryptophan, which may then react with the endogenous phenolic acids to produce auxin.     

Effects of potato plants expressing a barley cystatin on the predatory bug Podisus maculiventris via herbivorous prey feeding on the plant

The aim of this study was to assess the effects of potato plants expressing a barley cystatin on a potentially cystatin-susceptible natural enemy by predation on susceptible and non-susceptible preys feeding on the plant. We have focussed on the impact of the variant HvCPI-1 C⁶⁸ → G, in which the only cysteine residue was changed by a glycine, on the growth and digestive physiology of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the Egyptian cotton leafworm (ECW), Spodoptera littoralis. Moreover, we have studied the prey-mediated effects of the barley cystatin at the third trophic level, using the predatory spined soldier bug (SSB), Podisus maculiventris, as a model. Feeding trials conducted with CPB larvae reared on transgenic potato plants expressing the C⁶⁸ → G variant resulted in significantly lower weight gains compared to those fed on non-transformed (NT) plants. On the contrary, larger weight gains were obtained when ECW larvae, that lack digestive cysteine proteases, were reared on transgenic potato expressing the cystatin, as compared to larvae fed on NT plants. No negative effects on survival and growth were observed when SSB nymphs were exposed to HvCPI-1 C⁶⁸ → G by predation on either CPB or ECW larvae reared on transgenic potato plants expressing the barley cystatin, despite the fact that the inhibitor suppressed in vitro gut proteolysis of the predatory bug. To investigate the physiological background, biochemical analysis were carried out on guts of insects dissected at the end of the feeding assays. Fernando álvarez-Alfageme and Manuel Martínez are contributed equally to this work.