The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

Virulence determines beneficial trade‐offs in the response of virus‐infected plants to drought via induction of salicylic acid

It has been hypothesized that plants can get beneficial trade‐offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus‐induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought‐tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus‐infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid‐independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non‐infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.     

Effects of quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested maize on the attraction of the larval parasitoid Cotesia kariyai

Cotesia kariyai Watanabe (Hymenoptera: Braconidae) is a specialist larval parasitoid of Mythimna separata Walker (Lepidoptera: Noctuidae). Cotesia kariyai wasps use herbivore‐induced plant volatiles (HIPVs) to locate hosts. However, complex natural habitats are full of volatiles released by both herbivorous host‐ and non‐host‐infested plants at various levels of intensity. Therefore, the presence of non‐hosts may affect parasitoid decisions while foraging. Here, the host‐finding efficiency of naive C. kariyai from HIPVs influenced by host‐ and non‐host‐infested maize [Zea mays L. (Poaceae)] plants was investigated with a four‐arm olfactometer. Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) was selected as a non‐host species. One unit (1 U) of host‐ or non‐host‐infested plant was prepared by infesting a potted plant with five host or seven non‐host larvae. In two‐choice bioassays, host‐infested plants fed upon by different numbers of larvae, and various units of host‐ and non‐host‐infested plants (infestation units; 1 U, 2 U, and 3 U) were arranged to examine the effects of differences in volatile quantity and quality on the olfactory responses of C. kariyai with the assumption that volatile quantity and quality changes with differences in numbers of insects and plants. Cotesia kariyai was found to perceive quantitative differences in volatiles from host‐infested plants, preferring larger quantities of volatiles from larger numbers of larvae or plants. Also, the parasitoids discriminated between healthy plants, host‐infested plants, and non‐host‐infested plants by recognising volatiles released from those plants. Cotesia kariyai showed a reduced preference for host‐induced volatiles, when larger numbers of non‐host‐infested plants were present. Therefore, quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested plants appear to affect the decision of C. kariyai during host‐habitat searching in multiple tritrophic systems.     

Companion planting – do aromatic plants disrupt host‐plant finding by the cabbage root fly and the onion fly more effectively than non‐aromatic plants?

Brassica and Allium host‐plants were each surrounded by four non‐host plants to determine how background plants affected host‐plant finding by the cabbage root fly (Delia radicum L.) and the onion fly [Delia antiqua (Meig.)] (Diptera: Anthomyiidae), respectively. The 24 non‐host plants tested in field‐cage experiments included garden ‘bedding’ plants, weeds, aromatic plants, companion plants, and one vegetable plant. Of the 20 non‐host plants that disrupted host‐plant finding by the cabbage root fly, fewest eggs (18% of check total) were laid on host plants surrounded by the weed Chenopodium album L., and most (64% of check total) on those surrounded by the weed Fumaria officinalis L. Of the 15 plants that disrupted host‐plant finding in the preliminary tests involving the onion fly, the most disruptive (8% of check total) was a green‐leaved variant of the bedding plant Pelargonium × hortorum L.H. Bail and the least disruptive (57% of check total) was the aromatic plant Mentha piperita × citrata (Ehrh.) Briq. Plant cultivars of Dahlia variabilis (Willd.) Desf. and Pelargonium×hortorum, selected for their reddish foliage, were less disruptive than comparable cultivars with green foliage. The only surrounding plants that did not disrupt oviposition by the cabbage root fly were the low‐growing scrambling plant Sallopia convolvulus L., the grey‐foliage plant Cineraria maritima L., and two plants, Lobularia maritima (L.) Desv. and Lobelia erinus L. which, from their profuse covering of small flowers, appeared to be white and blue, respectively. The leaf on which the fly landed had a considerable effect on subsequent behaviour. Flies that landed on a host plant searched the leaf surface in an excited manner, whereas those that landed on a non‐host plant remained more or less motionless. Before taking off again, the flies stayed 2–5 times as long on the leaf of a non‐host plant as on the leaf of a host plant. Host‐plant finding was affected by the size (weight, leaf area, height) of the surrounding non‐host plants. ‘Companion plants’ and aromatic plants were no more disruptive to either species of fly than the other plants tested. Disruption by all plants resulted from their green leaves, and not from their odours and/or tastes.     

Host‐plant use by two Orthomeria (Phasmida: Aschiphasmatini) species feeding on Macaranga myrmecophytes

Myrmecophytes depend on symbiotic ants (plant‐ants) to defend against herbivores. Although these defensive mechanisms are highly effective, some herbivorous insects can use myrmecophytes as their host‐plants. The feeding habits of these phytophages on myrmecophytes and the impacts of the plant‐ants on their feeding behavior have been poorly studied. We examined two phasmid species, Orthomeria alexis and O. cuprinus, which are known to feed on Macaranga (Euphorbiaceae) myrmecophytes in a Bornean primary forest. Our observations revealed that: (i) each phasmid species relied on two closely‐related myrmecophytic Macaranga species for its host‐plants in spite of their normal plant‐ant symbioses; and (ii) there was little overlap between their host‐plant preferences. More O. cuprinus adults and nymphs were found on new leaves, which were attended by more plant‐ants than mature leaves, while most adults and nymphs of O. alexis tended to avoid new leaves. In a feeding choice experiment under ant‐excluded conditions, O. alexis adults chose a non‐host Macaranga myrmecophyte that was more intensively defended by plant‐ants and was more palatable than their usual host‐plants almost as frequently as their usual host‐plant, suggesting that the host‐plant range of O. alexis was restricted by the presence of plant‐ants on non‐host‐plants. Phasmid behavior that appeared to minimize plant‐ant attacks is described.     

Cucurbit aphid‐borne yellows virus (CABYV) modifies the alighting,settling and probing behaviour of its vector Aphis gossypii favouring its own spread

Plant pathogens are able to influence the behaviour and fitness of their vectors in such a way that changes in plant–pathogen–vector interactions can affect their transmission. Such influence can be direct or indirect, depending on whether it is mediated by the presence of the pathogen in the vector's body or by host changes as a consequence of pathogen infection. We report the effect that the persistently aphid‐transmitted Cucurbit aphid‐borne yellows virus (CABYV, Polerovirus) can induce on the alighting, settling and probing behaviour activities of its vector, the cotton aphid Aphis gossypii. Only minor direct changes on aphid feeding behaviour were observed when viruliferous aphids fed on non‐infected plants. However, the feeding behaviour of non‐viruliferous aphids was very different on CABYV‐infected than on non‐infected plants. Non‐viruliferous aphids spent longer time feeding from the phloem in CABYV‐infected plants compared to non‐infected plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. Viruliferous aphids showed a clear preference for non‐infected over CABYV‐infected plants at short and long time, while such behaviour was not observed for non‐viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimise the transmission and spread of this phloem‐limited virus.     

Host‐specificity constrains evolutionary host change in the psyllid Prosopidopsylla flava

1. At the higher taxonomic levels Psylloidea have largely co‐evolved with their host plants, and the colonisations of new plant lineages have been relatively few. The mechanisms that have constrained the evolution of host relationships throughout the history of this superfamily are not understood. The host relationships of Prosopidopsylla flava were studied in order to identify possible genetic or ecological constraints to macroevolutionary change in host range, using methodology developed for the host specificity testing of potential biological control agents. 2. The five Prosopis taxa (Leguminosae) tested appeared to be indistinguishable as hosts. Adult feeding required for survival, and for the initiation and continuation of egg production, was specific to Prosopis species. Oviposition occurred on 57 of the 58 non‐Prosopis plant species tested within the Leguminosae and Rosaceae, and was highest on plant species that belonged to the same subfamily. Eggs were inserted into plant tissue by their peduncle but hatched independently of host species. Complete development was restricted to Prosopis, although some early nymphal development was observed on species within the same subfamily as Prosopis (Mimosoideae). 3. Multiple phylogenetic constraints restrict host selection and utilisation by P. flava to Prosopis species, implying a long association between insect and host. Specificity of adult feeding was of special significance, being required for survival, oogenesis, and probably indirectly determining the oviposition host. This supports the hypothesis that genetically set limits in particular aspects of life history are responsible for the inability of some psyllids to readily colonise new plant lineages, rather than stabilising selection.     

Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.     

Host‐plant genotypic diversity and community genetic interactions mediate aphid spatial distribution

Genetic variation in plants can influence the community structure of associated species, through both direct and indirect interactions. Herbivorous insects are known to feed on a restricted range of plants, and herbivore preference and performance can vary among host plants within a species due to genetically based traits of the plant (e.g., defensive compounds). In a natural system, we expect to find genetic variation within both plant and herbivore communities and we expect this variation to influence species interactions. Using a three‐species plant‐aphid model system, we investigated the effect of genetic diversity on genetic interactions among the community members. Our system involved a host plant (Hordeum vulgare) that was shared by an aphid (Sitobion avenae) and a hemi‐parasitic plant (Rhinanthus minor). We showed that aphids cluster more tightly in a genetically diverse host‐plant community than in a genetic monoculture, with host‐plant genetic diversity explaining up to 24% of the variation in aphid distribution. This is driven by differing preferences of the aphids to the different plant genotypes and their resulting performance on these plants. Within the two host‐plant diversity levels, aphid spatial distribution was influenced by an interaction among the aphid's own genotype, the genotype of a competing aphid, the origin of the parasitic plant population, and the host‐plant genotype. Thus, the overall outcome involves both direct (i.e., host plant to aphid) and indirect (i.e., parasitic plant to aphid) interactions across all these species. These results show that a complex genetic environment influences the distribution of herbivores among host plants. Thus, in genetically diverse systems, interspecific genetic interactions between the host plant and herbivore can influence the population dynamics of the system and could also structure local communities. We suggest that direct and indirect genotypic interactions among species can influence community structure and processes.     

Arbuscular mycorrhizal fungi reduce growth and infect roots of the non‐host plant Arabidopsis thaliana

The arbuscular mycorrhizal (AM) symbiosis is widespread throughout the plant kingdom and important for plant nutrition and ecosystem functioning. Nonetheless, most terrestrial ecosystems also contain a considerable number of non‐mycorrhizal plants. The interaction of such non‐host plants with AM fungi (AMF) is still poorly understood. Here, in three complementary experiments, we investigated whether the non‐mycorrhizal plant Arabidopsis thaliana, the model organism for plant molecular biology and genetics, interacts with AMF. We grew A. thaliana alone or together with a mycorrhizal host species (either Trifolium pratense or Lolium multiflorum) in the presence or absence of the AMF Rhizophagus irregularis. Plants were grown in a dual‐compartment system with a hyphal mesh separating roots of A. thaliana from roots of the host species, avoiding direct root competition. The host plants in the system ensured the presence of an active AM fungal network. AM fungal networks caused growth depressions in A. thaliana of more than 50% which were not observed in the absence of host plants. Microscopy analyses revealed that R. irregularis supported by a host plant was capable of infecting A. thaliana root tissues (up to 43% of root length colonized), but no arbuscules were observed. The results reveal high susceptibility of A. thaliana to R. irregularis, suggesting that A. thaliana is a suitable model plant to study non‐host/AMF interactions and the biological basis of AM incompatibility.     

Herbivore species identity rather than diversity of the non‐host community determines foraging behaviour of the parasitoid wasp Cotesia glomerata

Extensive research has been conducted to reveal how species diversity affects ecosystem functions and services. Yet, consequences of diversity loss for ecosystems as a whole as well as for single community members are still difficult to predict. Arthropod communities typically are species‐rich, and their species interactions, such as those between herbivores and their predators or parasitoids, may be particularly sensitive to changes in community composition. Parasitoids forage for herbivorous hosts by using herbivore‐induced plant volatiles (indirect cues) and cues produced by their host (direct cues). However, in addition to hosts, non‐suitable herbivores are present in a parasitoid's environment which may complicate the foraging process for the parasitoid. Therefore, ecosystem changes in the diversity of herbivores may affect the foraging efficiency of parasitoids. The effect of herbivore diversity may be mediated by either species numbers per se, by specific species traits, or by both. To investigate how diversity and identity of non‐host herbivores influence the behaviour of parasitoids, we created environments with different levels of non‐host diversity. On individual plants in these environments, we complemented host herbivores with 1–4 non‐host herbivore species. We subsequently studied the behaviour of the gregarious endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) while foraging for its gregarious host Pieris brassicae L. (Lepidoptera: Pieridae). Neither non‐host species diversity nor non‐host identity influenced the preference of the parasitoid for herbivore‐infested plants. However, after landing on the plant, non‐host species identity did affect parasitoid behaviour, whereas non‐host diversity did not. One of the non‐host species, Trichoplusia ni Hübner (Lepidoptera: Noctuidae), reduced the time the parasitoid spent on the plant as well as the number of hosts it parasitized. We conclude that non‐host herbivore species identity has a larger influence on C. glomerata foraging behaviour than non‐host species diversity. Our study shows the importance of species identity over species diversity in a multitrophic interaction of plants, herbivores, and parasitoids.     

Associative learning of host presence in non‐host environments influences parasitoid foraging

1. Parasitoids are known to utilise learning of herbivore‐induced plant volatiles (HIPVs) when foraging for their herbivorous host. In natural situations these hosts share food plants with other, non‐suitable herbivores (non‐hosts). Simultaneous infestation of plants by hosts and non‐hosts has been found to result in induction of HIPVs that differ from host‐infested plants. Each non‐host herbivore may have different effects on HIPVs when sharing the food plant with hosts, and thus parasitoids may learn that plants with a specific non‐host herbivore also contain the host. 2. This study investigated the adaptive nature of learning by a foraging parasitoid that had acquired oviposition experience on a plant infested with both hosts and different non‐hosts in the laboratory and in semi‐field experiments. 3. In two‐choice preference tests, the parasitoid Cotesia glomerata shifted its preference towards HIPVs of a plant–host–non‐host complex previously associated with an oviposition experience. It could, indeed, learn that the presence of its host is associated with HIPVs induced by simultaneous feeding of its host Pieris brassicae and either the non‐host caterpillar Mamestra brassicae or the non‐host aphid Myzus persicae. However, the learned preference found in the laboratory did not translate into parasitisation preferences for hosts accompanying non‐host caterpillars or aphids in a semi‐field situation. 4. This paper discusses the importance of learning in parasitoid foraging, and debates why observed learned preferences for HIPVs in the laboratory may cancel out under some field experimental conditions.     

Effects of single and multiple herbivory by host and non‐host caterpillars on the attractiveness of herbivore‐induced volatiles of sugarcane to the generalist parasitoid Cotesia flavipes

It is well known that parasitoids are attracted to volatiles emitted by host‐damaged plants; however, this tritrophic interaction may change if plants are attacked by more than one herbivore species. The larval parasitoid Cotesia flavipesCameron (Hymenoptera: Braconidae) has been used intensively in Brazil to control the sugarcane borer, Diatraea saccharalisFabricius (Lepidoptera: Pyralidae) in sugarcane crops, where Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), a non‐stemborer lepidopteran, is also a pest. Here, we investigated the ability of C. flavipes to discriminate between an unsuitable host (S. frugiperda) and a suitable host (D. saccharalis) based on herbivore‐induced plant volatiles (HIPVs) emitted by sugarcane, and whether multiple herbivory (D. saccharalis feeding on stalk + S. frugiperda feeding on leaves) in sugarcane affected the attractiveness of HIPVs to C. flavipes. Olfactometer assays indicated that volatiles of host and non‐host‐damaged plants were attractive to C. flavipes. Even though host‐ and non‐host‐damaged plants emitted considerably different volatile blends, neither naïve nor experienced wasps discriminated suitable and unsuitable hosts by means of HIPVs emitted by sugarcane. With regard to multiple herbivory, wasps innately preferred the odor blend emitted by sugarcane upon non‐host + host herbivory over host‐only damaged plants. Multiple herbivory caused a suppression of some volatiles relative to non‐host‐damaged sugarcane that may have resulted from the unaltered levels of jasmonic acid in host‐damaged plants, or from reduced palatability of host‐damaged plants to S. frugiperda. In conclusion, our study showed that C. flavipes responds to a wide range of plant volatile blends, and does not discriminate host from non‐host and non‐stemborer caterpillars based on HIPVs emitted from sugarcane. Moreover, we showed that multiple herbivory by the sugarcane borer and fall armyworm increases the attractiveness of sugarcane plants to the parasitoids.     

Geographic pattern of host‐associated differentiation in the cotton fleahopper,Pseudatomoscelis seriatus

Host‐associated differentiation (HAD) is the occurrence of genetically distinct, host‐associated lineages. Most of the cases of HAD in phytophagous insects have been documented in specialist insects inhabiting feral ecosystems or in generalist parthenogens in agroecosystems. Herein we report HAD in the cotton fleahopper, Pseudatomoscelis seriatus (Reuter) (Hemiptera: Miridae), a native, generalist, non‐parthenogenetic insect feeding on native wild hosts [horsemint, Monarda punctata L. (Lamiaceae) and woolly croton, Croton capitatus Michx. (Euphorbiaceae)] and on cotton [Gossypium hirsutum L. (Malvaceae)] in the USA. Examination of genome‐wide genetic variation with AFLP markers and Bayesian analyses of P. seriatus associated with three different host plant species at five locations in Texas revealed a geographic pattern of HAD. The geographic pattern of HAD corresponded with differences in precipitation among the locations studied. In three locations, two distinct lineages of P. seriatus were found in association with horsemint and cotton/woolly croton, whereas in two other locations, populations associated with the different host plants studied were panmictic. We suggest that precipitation differences among locations translate into heterogeneity in vegetation distribution, composition, and phenology, which altogether may contribute to the observed geographic pattern of HAD.     

Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing‐mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA‐dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus‐derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non‐transgenic N. benthamiana plants as well as transgene‐derived siRNAs (t‐siRNAs) derived from the immune‐transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t‐siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t‐siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t‐siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24‐nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24‐nucleotide t‐siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24‐nucleotide t‐siRNAs is associated with crinivirus immunity in transgenic plants.     

Do virus‐resistant plants pose a threat to non‐target ecosystems? II. Risk assessment of an Australian pathosystem using multi‐scale field experiments

It has been widely argued that the acquisition of novel disease resistance genes by wild host populations following the release of novel pathogen‐resistant plants into agricultural systems could pose a significant threat to non‐target plant communities. However, predicting the magnitude of ecological release in wild plant populations following the removal of disease remains a major challenge. In this paper we report on the second phase of a tiered risk assessment designed to investigate the role of disease on host growth, survival, fecundity and fitness in a model pathosystem (the pasture species Trifolium repens infected with Clover yellow vein virus, ClYVV) and to assess the level of risk posed to at‐risk native plant communities in southeast Australia by newly developed genetically modified and conventionally bred virus‐resistant T. repens genotypes. Multi‐year field experiments conducted in woodland and grassland environments using host‐pathogen arrays derived from 14 ClYVV isolates and 21 T. repens genotypes indicate that viral infection reduces fecundity, growth and survival of wild T. repens plants but that the severity of these effects depends on host tolerance to infection, isolate aggressiveness and specific spatial and temporal environmental conditions. Demographic modelling showed that by reducing host survival and growth, ClYVV also limits the intrinsic population growth rate and niche size of wild T. repens populations. Given the significant fitness cost associated with viral infection we conclude that virus‐resistant T. repens genotypes may pose a threat to some high conservation‐value non‐target ecosystems in SE Australia. We also argue that long‐term, multi‐tiered experiments conducted in a range of controlled and non‐controlled environments are necessary to detect and accurately quantify risks associated with the release of disease‐resistant plants in general.     

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.     

Host‐associated differentiation in a highly polyphagous,sexually reproducing insect herbivore

Insect herbivores may undergo genetic divergence on their host plants through host‐associated differentiation (HAD). Much of what we know about HAD involves insect species with narrow host ranges (i.e., specialists) that spend part or all their life cycle inside their hosts, and/or reproduce asexually (e.g., parthenogenetic insects), all of which are thought to facilitate HAD. However, sexually reproducing polyphagous insects can also exhibit HAD. Few sexually reproducing insects have been tested for HAD, and when they have insects from only a handful of potential host‐plant populations have been tested, making it difficult to predict how common HAD is when one considers the entire species' host range. This question is particularly relevant when considering insect pests, as host‐associated populations may differ in traits relevant to their control. Here, we tested for HAD in a cotton (Gossypium hirsutum) pest, the cotton fleahopper (CFH) (Pseudatomoscelis seriatus), a sexually reproducing, highly polyphagous hemipteran insect. A previous study detected one incidence of HAD among three of its host plants. We used Amplified fragment length polymorphism (AFLP) markers to assess HAD in CFH collected from an expanded array of 13 host‐plant species belonging to seven families. Overall, four genetically distinct populations were found. One genetically distinct genotype was exclusively associated with one of the host‐plant species while the other three were observed across more than one host‐plant species. The relatively low degree of HAD in CFH compared to the pea aphid, another hemipteran insect, stresses the likely importance of sexual recombination as a factor increasing the likelihood of HAD.