Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to enhanced disease resistance but impaired plant growth

Transgenic tobacco plants expressing a phenylalanine ammonia-lyase cDNA (ShPAL), isolated from Stylosanthes humilis, under the control of the 35S promoter of the cauliflower mosaic virus were produced to test the effect of high level PAL expression on disease resistance. The transgenic plants showed up to eightfold PAL activity and were slowed in growth and flowering relative to non-transgenic controls which have segregated out the transgene. The expression of the ShPAL transgene and elevated PAL levels were correlated and stably inherited. In T-1 and T-2 tobacco plants with increased PAL activity, lesion expansion was significantly reduced by up to 55% on stems inoculated with the Oomycete pathogen Phytophthora parasitica pv. nicotianae, Lesion area was significantly reduced by up to 50% on leaves inoculated with the fungal pathogen Cercospora nicotianae. This study provides further evidence that PAL has a role in plant defence. (C) 2002 Elsevier Science Ltd. All rights reserved.

Turnover of plant lineages shapes herbivore phylogenetic beta diversity along ecological gradients.

Understanding drivers of biodiversity patterns is of prime importance in this era of severe environmental crisis. More diverse plant communities have been postulated to represent a larger functional trait-space, more likely to sustain a diverse assembly of herbivore species. Here, we expand this hypothesis to integrate environmental, functional and phylogenetic variation of plant communities as factors explaining the diversity of lepidopteran assemblages along elevation gradients in the Swiss Western Alps. According to expectations, we found that the association between butterflies and their host plants is highly phylogenetically structured. Multiple regression analyses showed the combined effect of climate, functional traits and phylogenetic diversity in structuring butterfly communities. Furthermore, we provide the first evidence that plant phylogenetic beta diversity is the major driver explaining butterfly phylogenetic beta diversity. Along ecological gradients, the bottom up control of herbivore diversity is thus driven by phylogenetically structured turnover of plant traits as well as environmental variables.

Predicting root defence against herbivores during succession

P>1. Root herbivores and pathogens interfere with basic below-ground plant function, and can thereby affect plant fitness and spatial and temporal patterns in natural plant communities. However, there has been little development of concepts and theories on below-ground plant defence, a deficit that is in contrast to the abundance of theorizing for above-ground plant parts.2. A review of the past 10 years of research on below-ground plant-herbivore interactions has revealed that, similar to above-ground tissues, root defences can be expressed constitutively or induced upon herbivore attack, and can be classified into direct and indirect traits, tolerance, and escape. Indeed, it has been shown that roots tolerate herbivory by outgrowing or re-growing lost tissues, or resist it by producing secondary metabolites that are toxic to herbivores or attract natural enemies of herbivores.3. We propose that, similar to above-ground plant-herbivore theories, the partition of abiotic and biotic factors over ecological succession can serve as the basis for predicting investment in defence strategies below-ground.4. Investigation of herbivore pressure and root responses along primary and secondary successional gradients suggests that: (i) roots are often fast growing, thinner and softer in early compared to later succession. (ii) Insect and nematode herbivore pressure increases until mid-succession and later decreases. (iii) Mycorrhizal abundance increases with succession, and the composition of fungal species changes through succession, often shifting from arbuscular mycorrhizae to ecto-mycorrhizae.5. Based on these findings, and on classical (above-ground) plant defence theory, we suggest the following set of testable hypotheses for below-ground plant defence: (i) During succession, early plants invest most of their resources in growth and less in defences (associated with a general lack of herbivores and pathogens, and with limited availability of resources in the system), therefore relying more on re-growth (tolerance) strategies. (ii) During mid-succession, a buildup of herbivore pressure facilitates replacement by plant species that exhibit greater direct and indirect defence strategies. (iii) Constitutive and inducible levels of defences may trade-off, and early successional plants should rely more on induction of defences after herbivore attack, whereas late successional plants will increasingly rely on constitutively produced levels of physical and chemical defence. (iv) Successional changes in microbial associations have consequences for root defence by improving plant nutrition and defence expression as well as directly competing for root space; however, toxic or impenetrable root defences may also limit association with root symbionts, and so may constrain the expression of root defence.

Arbuscular mycorrhizal fungi mediate below-ground plant-herbivore interactions: a phylogenetic study

Ecological interactions are complex networks, but have typically been studied in a pairwise fashion. Examining how third-party species can modify the outcome of pairwise interactions may allow us to better predict their outcomes in realistic systems. For instance, arbuscular mycorrhizal fungi (AMF) can affect plant interactions with other organisms, including below-ground herbivores, but the mechanisms underlying these effects remain unclear. Here, we use a comparative, phylogenetically controlled approach to test the relative importance of mycorrhizal colonization and plant chemical defences (cardenolides) in predicting plant survival and the abundance of a generalist below-ground herbivore across 14 species of milkweeds (Asclepias spp.). Plants were inoculated with a mixture of four generalist AMF species or left uninoculated. After 1month, larvae of Bradysia sp. (Diptera: Sciaridae), a generalist below-ground herbivore, colonized plant roots. We performed phylogenetically controlled analyses to assess the influence of AMF colonization and toxic cardenolides on plant growth, mortality and infestation by fungus gnats. Overall, plants inoculated with AMF exhibited greater survival than did uninoculated plants. Additionally, surviving inoculated plants had lower numbers of larvae in their roots and fewer non-AM fungi than surviving uninoculated plants. In phylogenetic controlled regressions, gnat density in roots was better predicted by the extent of root colonized by AMF than by root cardenolide concentration. Taken as a whole, AMF modify the effect of below-ground herbivores on plants in a species-specific manner, independent of changes in chemical defence. This study adds to the growing body of literature demonstrating that mycorrhizal fungi may improve plant fitness by conferring protection against antagonists, rather than growth benefits. In addition, we advocate using comparative analyses to disentangle the roles of shared history and ecology in shaping trait expression and to better predict the outcomes of complex multitrophic interactions.

Perception and modification of plant flavonoid signals by rhizosphere microorganisms

Flavonoids are a diverse class of polyphenolic compounds that are produced as a result of plant secondary metabolism. They are known to play a multifunctional role in rhizospheric plant-microbe and plant-plant communication. Most familiar is their function as a signal in initiation of the legume-rhizobia symbiosis, but, flavonoids may also be signals in the establishment of arbuscular mycorrhizal symbiosis and are known agents in plant defence and in allelopathic interactions. Flavonoid perception by, and impact on, their microbial targets (e.g. rhizobia, plant pathogens) is relatively well characterized. However, potential impacts on 'non-target' rhizosphere inhabitants ('non-target' is used to distinguish those microorganisms not conventionally known as targets) have not been thoroughly investigated. Thus, this review first summarizes the conventional roles of flavonoids as nod gene inducers, phytoalexins and allelochemicals before exploring questions concerning 'non-target' impacts. We hypothesize that flavonoids act to shape rhizosphere microbial community structure because they represent a potential source of carbon and toxicity and that they impact on rhizosphere function, for example, by accelerating the biodegradation of xenobiotics. We also examine the reverse question, 'how do rhizosphere microbial communities impact on flavonoid signals?' The presence of microorganisms undoubtedly influences the quality and quantity of flavonoids present in the rhizosphere, both through modification of root exudation patterns and microbial catabolism of exudates. Microbial alteration and attenuation of flavonoid signals may have ecological consequences for below-ground plant-microbe and plant-plant interaction. We have a lack of knowledge concerning the composition, concentration and bioavailability of flavonoids actually experienced by microbes in an intact rhizosphere, but this may be addressed through advances in microspectroscopic and biosensor techniques. Through the use of plant mutants defective in flavonoid biosynthesis, we may also start to address the question of the significance of flavonoids in shaping rhizosphere community structure and function.

Contrasting effects of necrotrophic and biotrophic plant pathogens on the aphid Aphis fabae

Phytophagous insects have to contend with a wide variation in food quality brought about by a variety of factors intrinsic and extrinsic to the plant. One of the most important factors is infection by plant pathogenic fungi. Necrotrophic and biotrophic plant pathogenic fungi may have contrasting effects on insect herbivores due to their different infection mechanisms and induction of different resistance pathways, although this has been little studied and there has been no study of their combined effect. We studied the effect of the biotrophic rust fungus Uromyces viciae-fabae (Pers.) Schroet (Basidiomycota: Uredinales: Pucciniaceae) and the necrotrophic fungus Botrytis cinerea Pers. (Ascomycota: Helotiales: Sclerotiniaceae) singly and together on the performance of the aphid Aphis fabae Scop. (Hemiptera: Aphididae) on Vicia faba (L.) (Fabaceae). Alone, botrytis had an inhibitory effect on individual A. fabae development, survival and fecundity, while rust infection consistently enhanced individual aphids’ performance. These effects varied in linear relation to lesion or pustule density. However, whole-plant infection by either pathogen resulted in a smaller aphid population of smaller aphids than on uninfected plants, indicating a lowering of aphid carrying capacity with infection. When both fungi were applied simultaneously to a leaf they generally cancelled the effect of each other out, resulting in most performance parameters being similar to the controls, although fecundity was reduced. However, sequential plant infection (pathogens applied five days apart) led to a 70% decrease in fecundity and 50% reduction in intrinsic rate of increase. The application of rust before botrytis had a greater inhibitory effect on aphids than applying botrytis before rust. Rust infection increased leaf total nitrogen concentration by 30% while infection by botrytis with or without rust led to a 38% decrease. The aphids’ responses to the two plant pathogens individually is consistent with the alteration in plant nutrient content by infection and also the induction of different plant defence pathways and the possible cross-talk between them. This is the first demonstration of the complex effects of the dual infection of a plant by contrasting pathogens on insect herbivores. Key words: Vicia faba, Botrytis cinerea, Uromyces viciae-fabae, tripartite interactions, induced resistance

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae pv. phaseolicola

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of Pseudomonas syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K+, that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca2+, Fe2/3+ Mg2+, sucrose, β-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000.

Pseudomonas syringae pv tomato DC3000 (Pto) is the causal agent of the bacterial speck of tomato, which leads to significant economic losses in this crop. Pto inhabits the tomato phyllosphere, where the pathogen is highly exposed to light, among other environmental factors. Light represents a stressful condition and acts as a source of information associated with different plant defence levels. Here, we analysed the presence of both blue and red light photoreceptors in a group of Pseudomonas. In addition, we studied the effect of white, blue and red light on Pto features related to epiphytic fitness. While white and blue light inhibit motility, bacterial attachment to plant leaves is promoted. Moreover, these phenotypes are altered in a blue-light receptor mutant. These light-controlled changes during the epiphytic stage cause a reduction in virulence, highlighting the relevance of motility during the entry process to the plant apoplast. This study demonstrated the key role of light perception in the Pto phenotype switching and its effect on virulence.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H+-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

It's the first bites that count: Survival of first-instar monarchs on milkweeds

Mortality of first instars is generally very high, but variable, and is caused by many factors, including physical and chemical plant characters, weather and natural enemies. Here, a summary of detailed field-based studies of the early-stage survival of a specialist lepidopteran herbivore is presented. First-instar larvae of the monarch butterfly, Danaus plexippus, a milkweed specialist, generally grew faster and survived better on leaves when latex flow was reduced by partial severance of the leaf petiole. The outcome depended on milkweed species, and was related to the amount of latex produced, as well as other plant characters, such as leaf hairs, microclimate and concentration of secondary metabolites. Even for a so-called 'milkweed specialist', larval performance and survival appears to be related to the concentration of cardenolides produced by the plants (a potential chemical defence against herbivory). This case study of monarchs and milkweeds highlights the need for field-based experiments to assess the effect of plant characters on the usually poor survival of early instar phytophagous insects. Few similar studies concerning the performance and survival of first-instar, eucalypt-specific herbivores have been conducted, but this type of study is considered essential based on the findings obtained using D. plexippus.

Genotypic variation in proanthocyanidin status in the Leucaena genus

The proanthocyanidin (PA) status of 116 accessions from the Leucaena genus representing 21 species, 6 subspecies, 3 varieties and 4 interspecific hybrids was evaluated under uniform environmental and experimental conditions at Redland Bay, Queensland, Australia in October 1997. The PA content of lyophilized youngest fully expanded leaves was measured spectrophotometrically by the butanol/HCl assay referenced to L. leucocephala ssp. glabrata standard PA and expressed as L. leucocephala ssp. glabrata PA equivalents (LLPAE). Considerable interspecific variation in PA concentration existed within the genus, ranging from 0-339 g LLPAE/kg dry matter (DM). Taxa including L. confertiflora, L. cuspidata, L. esculenta and L. greggii contained very high (> 180 g LLPAE/kg DM) PA concentrations. Similarly, many agronomically superior accessions from L. diversifolia, L. pallida and L. trichandra contained extremely high (up to 250 g LLPAE/kg DM) PA concentrations, although these taxa exhibited wide intraspecific variation in PA content offering the potential to select accessions with lower (120-160 g LLPAE/kg DM) PA content. Commercial cultivars of L. leucocephala ssp. glabrata, known to produce forage of superior quality, contained low amounts of PA (33-39 g LLPAE/kg DM). Artificial interspecific hybrids had PA contents intermediate to those of both parents, Lesser-known taxa. including L. collinsii, L. lanceolata, L. lempirana, L. macrophylla, L. magnifica, L. multicapitula, L. salvadorensis and L. trichodes, contained undetectable to low (0-36 g LLPAE/kg DM) quantities of PA and have potential as parents to breed interspecific hybrids of low PA status and superior forage quality. Extractable PA was the dominant PA component, accounting for 91% of total PA within the genus. Regression analysis of accession ranks from different experiments compared to these results indicated that genetic regulation of Leucaena spp. PA content was consistent (P < 0.01) under different edapho-climatic environments. The distribution of PA within the Leucaena genus did not concur with the predictions of various evolutionary and phylogenetic plant defence theories.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores, and indirect defences in ant-attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high and low elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high elevation ecotypes. We observed an elevation-driven trade-off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade-offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.