Induction and accumulation of PR proteins activityduring early stages of root colonizationby the mycoparasite Trichoderma harzianum strain T-203

The biochemical nature of the interaction between the antagonistic fungus Trichoderma harzianum strain T-203 and cucumber roots was studied during the early stages of root colonization by the fungus. Pathogenesis related (PR) proteins of the plant and enzyme activity of the fungus following the penetration and colonization of the roots by T. harzianum were explored up to 72 h post-inoculation. Scanning electron microscopy (SEM) revealed typical fungal structures previously associated with mycoparasitic interactions of T. harzianum strains during biological control. These included hyphal coiling and appressoria formation. Compared to untreated control, cucumber roots treated with T. harzianum T-203 exhibited higher activities of chitinase (EC 3.2.1.14), β-1,3-glucanase (EC 3.2.1.6), cellulase (EC 3.2.1.4) and peroxidase (EC 1.11.1.7), up to 72 h post-inoculation. Plants treated with a chemical inducer of the plant defence response, 2,6-dichloroisonicotinic acid (INA) displayed responses that were similar but not identical to those of plants inoculated with T. harzianum. In vivo staining of chitinase activity in fresh root sections allowed the localization of the activity in roots treated with either T. harzianum T-203 or INA. The formation of fluorescent products mainly in intercellular spaces of the induced roots provided evidence for the involvement of the plant defence system. In addition to its well-recognized mycoparasitic nature, it is suggested that Trichoderma’s association with roots reduce root disease through activation of the plant’s defence response.     

Efficacy of Saccharomyces cerevisiae on controlling the root-knot nematode (Meloidogyne javanica) infection and promoting cucumber growth and yield under laboratory and field conditions

Saccharomyces cerevisiae is a promising plant growth-promoting yeast for different crops. Applicability of S. cerevisiae as a biocontrol agent of the root-knot nematode (Meloidogyne javanica) was investigated on cucumber under growth-room and field conditions. The yeast S. cerevisiae similar to the nematicide, Ethoprophos, when applied as a rhizospheric soil drench treatment led to an obvious reduction of root galling caused by M. javanica and resulted in reducing the nematode reproduction ability on cucumber under growth room and field conditions. The yeast was more effective at 10 than at 5?g/l. Furthermore, the application of S. cerevisiae resulted in improving cucumber plant growth and increasing its fruit yield. High content of total phenolics in cucumber roots of S. cerevisiae-treated plants and hydrogen peroxide-treated plants gives a clue on the ability of the yeast to induced plant resistance in a similar way to exogenous hydrogen peroxide.     

Temperature effects on the reactive oxygen species formation and antioxidant defence in roots of two cucurbit species with contrasting root zone temperature optima

Suboptimal root zone temperature (14°C) was imposed on chilling-sensitive cucumber (Cucumis sativus L.) and chilling-tolerant figleaf gourd (Cucurbita ficifolia Bouché) plants. Exposure of roots to low temperature for up to 10 days caused a strong growth inhibition in cucumber compared with figleaf gourd. Physiological analysis showed that generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion was significantly induced in cucumber plants as fast as 1 day after low root zone temperature treatment. In addition to the significant induction of antioxidant superoxide dismutase activity, low root zone temperature also increased the mitochondrial electron transport allocated to alternative pathway while decreased cytochrome pathway salicylhydroxamic acid-resistant respiration. However, these defense responses could not compensate for the ROS production, resulting in membrane lipid peroxidation and loss of root cell viability in the low root zone temperature treated cucumber roots. In contrast, 14°C root zone temperature had no significant effects on figleaf gourd plant growth, antioxidant enzymes, ROS levels and alternative respiratory pathway. Hence, difference in ROS metabolism would be associated with the remarkable difference in adaptability of cucumber and figleaf gourd plants in response to suboptimal root zone temperature condition.     

Metabolomic analysis of the interaction between plants and herbivores

Insect herbivores by necessity have to deal with a large arsenal of plant defence metabolites. The levels of defence compounds may be increased by insect damage. These induced plant responses may also affect the metabolism and performance of successive insect herbivores. As the chemical nature of induced responses is largely unknown, global metabolomic analyses are a valuable tool to gain more insight into the metabolites possibly involved in such interactions. This study analyzed the interaction between feral cabbage (Brassica oleracea) and small cabbage white caterpillars (Pieris rapae) and how previous attacks to the plant affect the caterpillar metabolism. Because plants may be induced by shoot and root herbivory, we compared shoot and root induction by treating the plants on either plant part with jasmonic acid. Extracts of the plants and the caterpillars were chemically analysed using Ultra Performance Liquid Chromatography/Time of Flight Mass Spectrometry (UPLCT/MS). The study revealed that the levels of three structurally related coumaroylquinic acids were elevated in plants treated on the shoot. The levels of these compounds in plants and caterpillars were highly correlated: these compounds were defined as the ‘metabolic interface’. The role of these metabolites could only be discovered using simultaneous analysis of the plant and caterpillar metabolomes. We conclude that a metabolomics approach is useful in discovering unexpected bioactive compounds involved in ecological interactions between plants and their herbivores and higher trophic levels.     

Elevated CO2 changes the interactions between nematode and tomato genotypes differing in the JA pathway

Interactions between the root‐knot nematode Meloidogyne incognita and three isogenic tomato (Lycopersicon esculentum) genotypes were examined when plants were grown under ambient (370 ppm) and elevated (750 ppm) CO₂. We tested the hypothesis that, defence‐recessive genotypes tend to allocate ‘extra’ carbon (relative to nitrogen) to growth under elevated CO₂, whereas defence‐dominated genotypes allocate extra carbon to defence, and thereby increases the defence against nematodes. For all three genotypes, elevated CO₂ increased height, biomass, and root and leaf total non‐structural carbohydrates (TNC):N ratio, and decreased amino acids and proteins in leaves. The activity of anti‐oxidant enzymes (superoxide dismutase and catalase) was enhanced by nematode infection in defence‐recessive genotypes. Furthermore, elevated CO₂ and nematode infection did not qualitatively change the volatile organic compounds (VOC) emitted from plants. Elevated CO₂ increased the VOC emission rate only for defence‐dominated genotypes that were not infected with nematodes. Elevated CO₂ increased the number of nematode‐induced galls on defence‐dominated genotypes but not on wild‐types or defence‐recessive genotypes roots. Our results suggest that CO₂ enrichment may not only increase plant C : N ratio but can disrupt the allocation of plant resources between growth and defence in some genetically modified plants and thereby reduce their resistance to nematodes.     

Interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in Catharanthus roseus plants

Aims: We investigated the ultrastructural and molecular interactions between ‘Candidatus Phytoplasma mali’ and the apple endophyte Epicoccum nigrum in the experimental host Catharanthus roseus to determine whether inoculation of endophyte could trigger defence reactions in the host. Methods and Results: Apple proliferation (AP) symptom severity was evaluated in AP‐grafted plants that were treated by E. nigrum and compared with untreated controls. Phytoplasma concentration was quantified by real‐time PCR in treated and untreated plants. Ultrastructural observations revealed that in endophyte‐treated periwinkles, modifications to phytoplasmas, such as irregular shape and cytoplasm confined to the periphery of the cell, and plant cytological changes, such as abundant callose depositions and P‐protein aggregations in the sieve elements, occurred. AP‐grafted plants that were treated by the endophyte (E. nigrum) showed a reduction in symptom severity; in particular, flowers appeared normal in shape and size, when compared with uninfected controls. Real‐time PCR indicated that phytoplasma concentration in AP‐grafted plants treated with E. nigrum was about 2·8 times lower than that in untreated ones. Conclusions: These results demonstrated that the inoculation with E. nigrum influenced phytoplasma infection in C. roseus plants; plant ultrastructural modifications allowed us to hypothesize an enhancing host defence response. Significance and Impact of the Study: At present, curative protocols against this phytoplasma are not available. Alternative approaches are thus required to reduce disease spread. Our study might represent a first step in the clarification of plant–phytoplasma–endophyte relationships to find possible strategies for the control of phytoplasma diseases.     

Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens -transformed roots and Agrobacterium rhizogenes-transformed hairy roots

Medicago truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes.     

Aboveground resource allocation in response to root herbivory as affected by the arbuscular mycorrhizal symbiosis

Aims

Arbuscular mycorrhizal (AM) fungi associate with the majority of terrestrial plants, influencing their growth, nutrient uptake and defence chemistry. Consequently, AM fungi can significantly impact plant-herbivore interactions, yet surprisingly few studies have investigated how AM fungi affect plant responses to root herbivores. This study aimed to investigate how AM fungi affect plant tolerance mechanisms to belowground herbivory.

Methods

We examined how AM fungi affect plant (Saccharum spp. hybrid) growth, nutrient dynamics and secondary chemistry (phenolics) in response to attack from a root-feeding insect (Dermolepida albohirtum).

Results

Root herbivory reduced root mass by almost 27%. In response, plants augmented investment in aboveground biomass by 25%, as well as increasing carbon concentrations. The AM fungi increased aboveground biomass, phosphorus and carbon. Meanwhile, root herbivory increased foliar phenolics by 31% in mycorrhizal plants, and increased arbuscular colonisation of roots by 75% overall. AM fungi also decreased herbivore performance, potentially via increasing root silicon concentrations.

Conclusions

Our results suggest that AM fungi may be able to augment plant tolerance to root herbivory via resource allocation aboveground and, at the same time, enhance plant root resistance by increasing root silicon. The ability of AM fungi to facilitate resource allocation aboveground in this way may be a more widespread strategy for plants to cope with belowground herbivory.

     

Sub‐lethal UV‐C radiation induces callose,hydrogen peroxide and defence‐related gene expression in Arabidopsis thaliana

Exposure of plants to UV‐C irradiation induces gene expression and cellular responses that are commonly associated with wounding and pathogen defence, and in some cases can lead to increased resistance against pathogen infection. We examined, at a physiological, molecular and biochemical level, the effects of and responses to, sub‐lethal UV‐C exposure on Arabidopsis plants when irradiated with increasing dosages of UV‐C radiation. Following UV‐C exposure plants had reduced leaf areas over time, with the severity of reduction increasing with dosage. Severe morphological changes that included leaf glazing, bronzing and curling were found to occur in plants treated with the 1000 J·m^?2 dosage. Extensive damage to the mesophyll was observed, and cell death occurred in both a dosage‐ and time‐dependent manner. Analysis of H₂O₂ activity and the pathogen defence marker genes PR1 and PDF1.2 demonstrated induction of these defence‐related responses at each UV‐C dosage tested. Interestingly, in response to UV‐C irradiation the production of callose (β‐1,3‐glucan) was identified at all dosages examined. Together, these results show plant responses to UV‐C irradiation at much lower doses than have previously been reported, and that there is potential for the use of UV‐C as an inducer of plant defence.     

Insect eggs suppress plant defence against chewing herbivores

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect‐responsive genes after challenge with caterpillars, suggesting that egg‐derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA‐deficient mutant sid2‐1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross‐talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg‐induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.     

Pseudomonas fluorescens mediated suppression of Meloidogyne incognita infection of cowpea and tomato

Plant growth-promoting rhizobacterium, Pseudomonas fluorescens strain BICC602 suppresses root-knot nematode (Meloidogyne incognita) by enhancing defence mechanism leading to induced systemic resistance in cowpea (Vigna unguiculata) cv. L.Walp. and tomato (Solanum lycopersicum) cv. Pusa Ruby. In cowpea, the soil treatment proved more effective than foliar spray on root galling and eggs in roots. However, which factors are necessary in the induction of resistance response in plants against nematodes by BICC602 is not yet known. Salicylic acid (SA) production by some bacteria acts as endogenous signal for the activation of certain plant defence responses. In a split-root trial with tomato as a host plant and M. incognita as challenging parasite, BICC602 induces systemic resistance in tomato plants. Based on the results, it is assumed that P. fluorescens-induced resistance against M. incognita in cowpea and tomato is made either through SA-dependent or SA-independent transduction pathway.