Positive feedback with mycorrhizal fungi alleviates negative effects of intercropping the energy crop <Emphasis Type="Italic">Jatropha curcas</Emphasis> with <Emphasis Type="Italic">Crotalaria retusa</Emphasis>

Little is known about the arbuscular mycorrhizal status of the ligneous plant Jatropha curcas, an energy crop that raises high expectations worldwide. We hypothesized that its early mycorrhization and growth could be improved by co-culturing it with Crotalaria retusa, a mycotrophic legume species. Soil samples collected from a 15-year-old J. curcas hedgerow were transferred to the greenhouse, along with soil sampled from the contiguous fallow field. Three pot-culture modalities were studied for 3 months: jatropha alone, jatropha sowed after the clipping of 2-month-old C. retusa, and jatropha sowed next to 2-month-old C. retusa. J. curcas biomass was significantly lower when it was co-cultured with C. retusa in both soil types as compared to when it was grown individually, while its biomass following the cut of C. retusa was not impacted. J. curcas shoot P content was significantly improved only when both plant species grew in the hedgerow soil, and so was mycorrhization intensity. Additionally, the composition of the J. curcas root mycorrhizal community was closer to that of C. retusa when using this hedgerow soil. Overall, J. curcas development was not improved by its association with C. retusa, but the soil cropping history appeared essential to their mycorrhizal interactions. These were favored by a soil mycorrhizal community shaped by multiple years of J. curcas monoculture. Improved knowledge about these preferential association patterns with J. curcas is needed to improve its co-culture with compatible mycotrophic legumes.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Leaf-residing <Emphasis Type="Italic">Methylobacterium</Emphasis> species fix nitrogen and promote biomass and seed production in <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Background

Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant–microbe interactions.

Results

An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to α, β, γ-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female–male flower ratio.

Conclusion

The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria–plant interaction may significantly contribute to Jatropha’s tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant’s nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.

     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Insufficient Evidence of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Invasiveness: Experimental Observations in Burkina Faso,West Africa

Biofuel plants such as Jatropha curcas L. have potential to support the livelihoods of rural communities and contribute to sustainable rural development in Africa, if risks and uncertainties are minimized. Yet, recent papers have warned of the risk of biological invasions in such tropical regions as a consequence of the introduction of exotic biofuel crops. We investigated the seed dispersal risk and invasiveness potential of both J. curcas monoculture plantations and live fences into adjacent cultivated and uncultivated land use systems in Sissili province, Burkina Faso. Invasiveness potential was assessed through (i) detecting evidence of natural regeneration in perimeters around J. curcas plantations and live fences, (ii) assessing seed dispersal mechanisms, and (iii) assessing seedling establishment potential through in situ direct seed sowing. Spontaneous regeneration around the plantation perimeters of the three sites was very low. Individual seedling density around J. curcas live fences was less than 0.01 m^?2 in all sites. Seventy percent of the seedlings were found close to the live fence and most of them derived from the same year (96 %), which indicates low seed-bank longevity and seedling survival. J. curcas can be dispersed by small mammals and arthropods, particularly rodents and ants. In some sites, such as in Onliassan, high secondary seed dispersal by animals (up to 98 %) was recorded. There were highly significant differences in germination rates between seeds at the soil surface (11 %) and those buried artificially at 1–2-cm depth (64 %). In conclusion, we failed to find convincing evidence of the spreading of J. curcas or any significant impact on the surrounding environment.     

Overexpression of the ABC transporter gene <Emphasis Type="Italic">TsABCG11</Emphasis> increases cuticle lipids and abiotic stress tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The cuticle, composed primarily of wax and cutin, covers most plant aerial surfaces and plays a vital role in interactions between plants and their environment. Some ATP-binding cassette G subfamily (ABCG) members are involved in cuticular lipid molecule exportation to outside in the plant surface. Thellungiella salsugineum, a relative of Arabidopsis thaliana with a heavy cuticle, has extreme stress tolerance. TsABCG11, an ABCG transporter was cloned (GenBank accession number JQ389853), and its structure was studied. qRT-PCR showed that TsABCG11 expression varied in different organs of T. salsugineum and was upregulated under ABA, NaCl, drought and cold conditions. The rosette leaves from 4-week-old TsABCG11 overexpressed (OE) Arabidopsis plants displayed lower rates of water loss and decreased chlorophyll-extracted rates compared to wild-type plants. TsABCG11-OE plants also exhibited significantly increased total cuticular wax and cutin monomer amounts, mainly due to prominent changes in the C₂₉, C₃₁, and C₃₃ alkanes in the wax and C18:2 dioic in cutin monomers, respectively. TsABCG11-OE seedlings exhibit lower root growth inhibition under 100 mM of NaCl or 1 µM of ABA than the wild type. Four-week-old TsABCG11-OE plants exhibited higher photosynthetic rates and water-use efficiency under cold stress (4 °C) than control plants. These results indicate that TsABCG11 plays an important role in cuticle lipid exportation and is involved in abiotic stresses, probably having a close relationship with extreme stress tolerance in T. salsugineum.     

Assessment of Genetic Stability in Three Generations of In Vitro Propagated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Plantlets Using ISSR Markers

Tissue culture has been widely employed in Jatropha curcas L. for the clonal multiplication of superior genotypes. However, the evaluation of genetic stability is necessary to detect somaclonal variants. In this context, the present aim was to evaluate the genetic stability of J. curcas plantlets, obtained via indirect organogenesis, by means of ISSR markers. To supply the explant sources for in vitro propagation, the first generation of plants was produced from in vitro germination of J. curcas seeds. Fragments of cotyledonary leaves were inoculated into medium supplemented with 1.5 mg L^?1 BAP and 0.05 mg L^?1 of IBA for induction of callogenesis. The resulting calli were transferred to bud induction medium. Subsequently, the buds were cultured in medium for elongation, giving rise to the second generation of plants. These plants provided new buds, which were excised and subcultured in elongation medium, yielding a third generation of plants. To evaluate genetic stability in three plant generations, twelve ISSR primers were used, resulting in 124 bands showing 41.93 % of polymorphism. Increase was observed in the level of somaclonal variation (SV) over the generations. The present study reports, for the first time, the analysis of genetic stability in J. curcas plantlets regenerated via indirect organogenesis by means of ISSR markers. The results suggest that the indirect route is associated to higher levels of genetic instability, which also increased with successive subcultures. The ISSR markers were efficient in detecting SV, and the generated genetic variability may be useful for breeding programs.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Genetic variability,character association and divergence studies in <Emphasis Type="Italic">Jatropha curcas</Emphasis> for improvement in oil yield

Key message

Variability in traits of 15, diverse 6-year-old candidate plus trees of Jatropha curcas was determined to identify the best gain heritable traits correlating with oil yield for Jatropha improvement.

Abstract

Study was carried out on 15 6-year-old candidate plus trees of Jatropha curcas adapted on semi-arid wasteland with an objective to assess variation in morphological, physiological and oil quality characters. Heritable and non-heritable components of the total variability of the characters were determined by genotypic (GCV) and phenotypic (PCV) co-efficient of variation, heritability and genetic advance (GA) and the best gain traits for Jatropha improvement through selection and breeding were assessed. Further, association among the traits were assessed and germplasm were separated into different clusters. Significant variation was found among the different genotypes for all the characters. The photosynthetic and transpiration rate correlated with oil content, seed and oil yield. The chlorophyll pigments correlated positively with the photosynthetic rate and oil content. The seed oil content varied considerably from 27.68 % (JCN01) to 37.49 % (JCN14) and had high heritability, but it had low PCV and GCV and moderate GA. The oil yield plant^?1 had high genetic variability and varied significantly from 0.07 (JCN15) to 0.47 kg plant^?1 (JCN09/IC 565733). Though the different fatty acids differed significantly with different germplasm and also had high heritability, they had low PCV, GCV and GA. Seed weight, fruit weight, seed weight fruit^?1 and seed yield plant^?1 strongly correlated with oil yield and had moderate to high GCV, PCV, coupled with high heritability and GA. Germplasm were separated into four distinct clusters with a maximum inter distance found between cluster II and IV, and minimum between cluster I and III. The study helped to identify the superior germplasm among diverse genotypes of J. curcas that can serve as parents with desirable characters like high oil yield, low stomatal conductance and high water use efficiency for further breeding purposes.

     

Effects of Calcium and Calmodulin Antagonists on Chilling Stress-Induced Proline Accumulation in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Regulation of proline accumulation in plants under chilling stress remains unclear. In this paper, we treated Jatropha curcas seedlings under chilling stress with exogenous calcium chloride (CaCl₂), the plasma membrane Ca²⁺-channel blocker lanthanum chloride (LaCl₃), calmodulin antagonists, chlorpromazine (CPZ), and trifluoperazine (TFP) and investigated the effects of calcium and calmodulin (CaM) on proline accumulation and chilling tolerance. The results showed that CaCl₂ treatment significantly enhanced chilling stress-induced proline accumulation. CaCl₂ also induced an almost immediate and rapid increase of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and glutamate dehydrogenase activities, the key enzymes in the glutamate pathway of proline biosynthesis, and up-regulated P5CS expression, but it decreased the activity of proline dehydrogenase (ProDH), a key enzyme of proline degradation, and inhibited ProDH expression. Treatment with LaCl₃, CPZ, and TFP exhibited the opposite effects to those by CaCl₂ treatment. Moreover, CaCl₂, LaCl₃, CPZ, and TFP had little effect on the activities of ornithine aminotransferase and arginase, the key enzymes in the ornithine pathway of proline biosynthesis. These results indicated that Ca²⁺-CaM might be involved in signal transduction events, leading to proline accumulation in J. curcas seedlings under chilling stress, and that Ca²⁺-induced proline accumulation is a combined result of the activation of the glutamate pathways of proline biosynthesis and the simultaneous inhibition of the proline degradation pathway. In addition, CaCl₂ treatment increased tissue vitality, decreased the content of the lipid peroxidation product malondialdehyde (MDA), and alleviated electrolyte leakage in J. curcas seedlings under chilling stress, indicating that exogenous Ca²⁺ can enhance chilling tolerance, and proline might be a key factor in this increased chilling tolerance.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Expression of plant antimicrobial peptide <Emphasis Type="Italic">pro-SmAMP2</Emphasis> gene increases resistance of transgenic potato plants to <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> pathogens

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.     

A scientific note on the evidence for the production and use of wax by a captive female <Emphasis Type="Italic">Bombus</Emphasis><Emphasis Type="Italic">sylvestris</Emphasis>

The presence of wax glands in the socially parasitic subgenus Bombus (Psithyrus) was established only recently (Sramkova and Ayasse, 2008), but the uses for the produced wax are unknown. In this investigation nest-searching Bombus sylvestris females were collected and kept in darkness, in conditions of controlled temperature and relative humidity, using rearing conditions for non-parasitic bumblebees. The results show that in Bombus sylvestris not only wax glands are present but they also can produce wax which can be used as a building material.     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.