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Hevea brasiliensis is an evergreen woody tree widely used in industrial production in tropical rain forest. However, rubber trees are extremely vulnerable to low temperature stress, which seriously limits the cultivation and use of rubber trees in low temperature areas. NAC transcription factor gene family is common to many plant species, and can participate in a variety of behaviors that are crucial for plants to cope with the challenges from the environment. However, little is known about the evolution history, function and expression patterns of NAC family in rubber trees. Here, 51 HbNAC genes were identified in rubber tree by genome search, and they were divided into 11 subclasses according to phylogenetic relationship. The members of a subfamily share a similar number of introns and exons. We conducted transcriptome analyses to obtain cold-responsive HbNACs, and the transcriptome data showed the expression of HbNAC26 and HbNAC11 was significantly induced by cold stress, suggesting a potential role of these genes in cold stress. The expression pattern of HbNACs in different tissues and different developmental stages was studied by real-time PCR. In addition, the complementary effect of defective yeast mutants indicated that the HbNAC11 gene could be a positive regulator of cold stress in rubber trees. The results of this study can lay a foundation for studying the biological function of NAC and improving cold resistance breeding of rubber trees. [ABSTRACT FROM AUTHOR]

Purpose: Herein, the extraction of lignin from rubber tree residues (Hevea brasiliensis Müll. Arg), particularly from the seed shell, is reported with the aim of providing added value to this plantation. Methods: The protocol employed consisted of an organosolv extraction using formic or acetic acids and p-TsOH as catalyst assisted by ultrasound, avoiding the use of high temperatures and mineral acids. Results: 22% yield of lignin was obtained from the dried rubber seed shell using the formic acid/p-TsOH combination in comparison with 13% yield obtained with acetic acid/p-TsOH. The extracted lignin was oxidized isolating typical fragments derived from syringyl, guaiacyl and p-hydroxyphenyl units. The lignin was characterized by IR, UV, NMR, TGA and DSC. Moreover, total phenol content and antioxidant activity were also evaluated. Conclusion: The results exhibited the presence of typical lignin aromatic composition. The lignin obtained by the seed shell could be effectively employed in numerous future applications and the method could be considered as a low-cost acid catalysed process. [ABSTRACT FROM AUTHOR]

Rubber (Hevea brasiliensis) is a priority tree crop that produces natural rubber (NR), making it an important plantation commodity in the Philippines. However, NR production is confronted with major constraints, including rubber diseases resulting in low latex yield. Twenty-five rubber farms located in five major rubber-producing municipalities (Kidapawan, Antipas, Makilala, Matalam and President Roxas) of Cotabato, Philippines, were surveyed for prevalence of major rubber leaf diseases. Information on farm practices and environmental variables was collected. The majority of rubber farmers were smallholders with hectarage planted ranging between 1 and 15 hectares. The most planted clones are RRIM 600 and PB 260, which are high-yielding yet susceptible to many foliar pathogens. Six leaf diseases, viz. Oidium powdery mildew, Colletotrichum leaf disease, Corynespora leaf fall/spot, Phytophthora leaf blight, bird's eye spot and algal spot, were documented in this study. Powdery mildew was the most prevalent in Cotabato with the highest percentage and severity of infections in all plantations, followed by Colletotrichum leaf disease. Information on disease prevalence in surveyed areas is important for disease management actions. [ABSTRACT FROM AUTHOR]

Large-scale deforestation in the tropics, triggered by logging and subsequent agricultural monoculture has a significant adverse impact on biodiversity due to habitat degradation. Here, we measured the diversity of butterfly species in three agricultural landscapes, agroforestry orchards, oil palm, and rubber tree plantations. Butterfly species were counted at 127 sampling points over the course of a year using the point count method. We found that agroforestry orchards supported a greater number of butterfly species (74 species) compared to rubber tree (61 species) and oil palm plantations (54 species) which were dominated by generalist (73%) followed by forest specialists (27%). We found no significant difference of butterfly species composition between agroforestry orchards and rubber tree plantation, with both habitats associated with more butterfly species compared to oil palm plantations. This indicates butterflies were able to persist better in certain agricultural landscapes. GLMMs suggested that tree height, undergrowth coverage and height, and elevation determined butterfly diversity. Butterfly species richness was also influenced by season and landscape-level variables such as proximity to forest, mean NDVI, and habitat. Understanding the factors that contributed to butterfly species richness in an agroecosystem, stakeholders should consider management practices to improve biodiversity conservation such as ground vegetation management and retaining adjacent forest areas to enhance butterfly species richness. Furthermore, our findings suggest that agroforestry system should be considered to enhance biodiversity in agricultural landscapes. [ABSTRACT FROM AUTHOR]

SGT1 is an essential factor required for R proteins to trigger disease resistance and defense responses in plants. Although its vital role have been thoroughly studied in model plant species, so far no SGT1 gene in rubber tree has been identified. In this study, HbSGT1a and HbSGT1b from rubber trees were cloned. The amino acid sequences identities between HbSGT1a and HbSGT1b is 64.82%, and both of them have conserved TPR, CS and SGS domains, which are the characteristic domains of SGT1 proteins. Expression analysis showed that the transcripts of HbSGT1b were more abundant than HbSGT1a in all tissues. Moreover, transcripts of both HbSGT1a and HbSGT1b were triggered by powdery mildew attack in rubber trees. Additionally, transcripts of HbSGT1a and HbSGT1b were significantly induced by exogenous phytohormones and abiotic stress conditions. Yeast two-hybrid assays demonstrated that both HbSGT1a and HbSGT1b interacted with a heat shock protein 90 (HbHSP90.1). Interestingly, HbSGT1a and HbSGT1b showed a strong interaction. Taken together, these results suggested that HbSGT1 and HbHSP90.1 form a functional complex and carry out essential roles in powdery mildew and stress resistance, as well as hormone signaling in rubber tree. [ABSTRACT FROM AUTHOR]

Powdery mildew, caused by Oidium heveae, is a major threat to rubber plantations worldwide. Population studies are scarce for this pathogen due to the lack of polymorphic molecular markers. In this study, sixteen polymorphic microsatellite loci were identified using DNA extracted from single lesions based on the whole-genome sequences of the pathogen. Primers of these loci were applied to 138 O. heveae samples from five counties in Hainan, China. The number of alleles per locus ranged from 2 to 11 and the gene diversity varied from 0.014 to 0.728. In total, 119 multilocus genotypes (MLGs) were observed for the 138 samples. However, only one sample was significantly distinct from the other samples base on DAPC analysis. Further study indicated that there were three subclusters for the other 137 samples, but no evidence for regional genotypic subdivision was identified. The microsatellite markers developed are very useful to study the genetic structure and the dispersal route of O. heveae, especially as it can use DNA extracted directly from infected leaves. [ABSTRACT FROM AUTHOR]

We investigate the key predictors that influence the soil organic matter (SOM) content in higher altitude plantations. This is to study the potential of carbon sequestration for recapturing CO2 from the atmosphere, which is an important consideration in mitigating global climate change. This research applied distance-based linear models to evaluate and predict the influence of topography and types of land use on soil carbon storage in the tropical mountainous areas of Central Vietnam. Three predictors plantation, altitude, and slope together explained 73.8% of the SOM variation. We focused on four types of plantations including acacia, rubber tree, cassava plantations, and plantations with rotation system between acacia and cassava. We found that they had different carbon accumulation, which depended on various characteristics of the terrain. The acacia plantation forest stored the highest amount of SOM and was less sensitive to changes in altitude. The soil carbon content in cassava plantations was the lowest, and altered considerably with altitude. SOM content in rubber tree and acacia-cassava rotation plantations were similar to each other. Altitude was the most critical driver in affecting the capacity of soil carbon sequestration in each plantation. In higher elevation zones, soil carbon stock was greater, which was probably related to decreasing decomposability by microorganism, and increasing carbon content in foliage and leaf litter. The carbon retention tended to increase gradually over an elevation range of 133–300 m a.s.l. The effects of the terrain slope were observed, when considering certain types of land use, and ones with small altitude disparity. In the slope range of 0° to 20°, the carbon pool showed a tendency to increase its capacity. Higher slope gradients, however, could hinder carbon retention due to organic material loss through erosion. In particular, when the slope was above 30°, the soil organic matter content decreased substantially. [ABSTRACT FROM AUTHOR]

Nutrient management is critical for the development and growth of rubber trees during the immature phase. We present the results of a fertilizer trial during the immature phase of a rubber plantation setup in Cambodia. The main objective of this study was to analyze the response of a specific clone, PB330, in the specific conditions of one of the most important rubber-growing areas of Cambodia. The experiment was arranged in a randomized complete block design with 4 treatments and 4 replications. The four treatments consisted of 3 fertilization treatments with an increasing dose of fertilizers (respectively T1, T2 and T3) compared to a non-fertilized control (T0). The girth of trees was measured every six months from the end of the first year after planting (YAP) to the end of the 6th YAP. Results showed that fertilization had a significant effect on tree girth. At the end of the 6th YAP, fertilization had a significant effect on the number of tappable trees, and stand basal area but not on average tree girth. The most pronounced effect of fertilization was observed from annual increments in girth and stand basal area during the 3rd and 4th YAP. The effect of fertilization on tree growth was evident from all three treatments, although T3 gave the most significant increase compared to T0, increasing doses of fertilizer did not significantly improve the growth parameters. Hence, this study confirmed the importance of mineral nutrition on rubber tree growth during the immature phase even at T1, the lowest level of nutrient inputs. These results can be used to improve the standard fertilizer recommendations for immature rubber trees in Cambodia. We recommend setting up similar trials in different edaphic conditions of the country with the aim to establish nutrient management practices tailored to local biophysical constraints. [ABSTRACT FROM AUTHOR]

Main conclusion: A putative powdery mildew effector can elicit defense responses including reactive oxygen species and callose accumulations in model plants Nicotiana benthamiana and Arabidopsis thaliana and host plant Hevea brasiliensis. Powdery mildew fungi cause severe diseases in many agricultural plants, such as the mildew fungus Erysiphe quercicola infecting the rubber tree (Hevea brasiliensis), causing latex yield losses. However, effectors of E. quercicola were rarely functionally characterized. In this study, we identified a highly specific candidate-secreted effector protein, EqCSEP04187, from E. quercicola. This putative effector is expressed at the late stage but not the early stage during infection. The constitutive expression of EqCSEP04187 in model plants Nicotiana benthamiana and Arabidopsis thaliana elicited defense responses, as did transient expression of EqCSEP04187 in protoplasts of H. brasiliensis. Introducing EqCSEP04187 into another H. brasiliensis-associated fungal pathogen, Colletotrichum gloeosporioides, inhibited H. brasiliensis infection, and infection by E. quercicola was decreased in the A. thaliana eds1 mutant expressing EqCSEP04187. Further analysis suggests that these reductions in infection were the consequences of EqCSEP04187 eliciting defense responses. Our study suggests that this putative effector has elicitor activity that can improve plant resistance. [ABSTRACT FROM AUTHOR]

The rubber trees (Hevea brasiliensis) are exposed to water limitations in the Amazon region that can affect its physiology, making it necessary to select genotypes that are best adapted. This study analyzed the diurnal and seasonal changes in water use and status using traits of gas exchange and water relations in ten rubber tree genotypes in the immaturity phase from large-scale clone trials at three localities in the northwestern Colombian Amazon. In San Vicente del Caguán (semi-humid warm climate), the greatest means for water-use efficiency (WUEe and WUEi) and leaf water potential (ΨL) were observed, in contrast with Belén de los Andaquíes (humid warm) where there was a higher relative water content (RWC). Florencia (humid warm) presented high averages for WUEe and WUEi similar to San Vicente del Caguán. The WUEe and WUEi were greater in the dry season, contrary to those observed in ΨL and RWC. The WUEe was higher in the time range 9:00 to 12:00, while ΨL had a greater mean at 3:00 and a lower average at 12:00. In the dry season, the more efficient genotypes for water use had a higher leaf water potential as well as a lower relative water content, and had an extreme anisohydric behavior. In the wet season, these had a partial isohydric strategy. The FX 4098, MDF 180 and FDR 5597 genotypes were selected because of their greater adaptive capacity for water use and status, as compared to clone IAN 873 (control), which makes them desirable for future climate drought scenarios. [ABSTRACT FROM AUTHOR]

Rooting induction of leafy stem cuttings of Hevea brasiliensis can be used as a relevant tool for the cultivation, conservation of genetic diversity, and rescue of this species in Peru, decimated by decades of extractive practices. In this study, we evaluated the effects of different numbers of leaflets and concentrations of indole-3-butyric acid (IBA) in the rooting capacity of leafy stem cuttings of H. brasiliensis. Cuttings 6–7 cm long were collected from stockplants in a germplasm bank; leaves were pruned to 2 or 5 leaflets, and the cutting base was treated with one of the following IBA concentrations: 0, 1000, 2000, 3000, 4000, or 5000 ppm. Cuttings were placed in trays with a sandy substrate and transferred to plastic mini-tunnels with fogging irrigation. After 29 days, cuttings with 5 leaflets and 2000 ppm of IBA showed the best results in survival (100%), rooting percentage (79%), and roots number (4.1) and length (2.9 cm). The high conversion rate of cuttings to plants indicates the feasibility of this technique as a simple and effective tool, both for the conservation and the commercial propagation of selected rubber tree genotypes. [ABSTRACT FROM AUTHOR]

Hevea brasiliensis (rubber tree) is well known as the tree of life for its by-products such as wood and latex. The introduction of super clones with high yield potential such as RRIM 3001 is among the efforts to improve latex and timber yields. However, the RRIM 3001 super clone is prone to a disease known as trunk bulges. The appearing of bulging on the tapping panel causes difficulties during the tapping process for harvesting the latex and affects the quality of wood produced. In this study, we aimed to characterize Bacillus pumilus (B. pumilus) causing trunk bulges disease of RRIM 3001 super clone rubber tree isolated from the infected rubber trees in Malaysia. During October to December 2017, samples with trunk bulges symptoms were collected from four rubber plantations with reported outbreaks. Ten bacterial isolates from vascular tissues of rubber tree with trunk bulges symptom were confirmed as Bacillus pumilus species by the production of yellow pigmentation, circular, raised and entire form colonies on Bacillus differentiation agar medium. All of the isolates are Gram-positive, rod-shaped, motile, as well as endospores formers. They were positive for catalase activities and negative in both potassium hydroxide (KOH) and sulfide productions. All bacterial isolates utilized glucose, inositol, mannitol, sucrose, xylose and trehalose, but did not utilize lactose and arabinose. Application of 16S rRNA, species-specific primers; Bsub5F/Bsub3R and UP-1S/UP-2Sr (GyrB gene) successfully identified all isolates as B. pumilus. Phylogenetic analysis of both genes further clustered these isolates into the same cluster to a B. pumilus reference strain in the GenBank database (GenBank Accession Nos. KF158227 and MF968898). The results from this study are useful for developing management strategies of B. pumilus in rubber plantations, since rubber tree has been recognized as important commodity crop with high economic value in Malaysia. [ABSTRACT FROM AUTHOR]

The multigene family WRKY is plant-specific transcription factors, which is involved in numerous aspects of plant growth and development. Here, we report a genome-wide search for the rubber tree WRKY (HbWRKY) genes and their expression profiles at various tissues including latexes. We also present a comprehensive overview of whole HbWRKY gene family, including gene structure, chromosomal locations, conserved protein domains, gene duplications and phylogenetic inferences. We identify a total of 108 full-length HbWRKY genes, which were classified into the three major subgroups. The genome-scale syntenic analysis showed that WGDs might have played a major role in the evolution of the HbWRKY gene family. Our further real-time reverse transcription-PCR (qRT-PCR) experiments validated that the HbWRKY genes are divergently expressed and gene expression divergence seemingly existed among the duplicated WRKY genes, suggesting a fundamentally functional divergence of the duplicated WRKY paralogs in rubber tree. We also investigated the gene expression profiling of the HbWRKYs under salt stress, to which the majority of genes differentially responded. This study acquires a new insight into the evolution of the HbWRKYs, which will help further functional characterization of theses candidate genes in rubber tree. [ABSTRACT FROM AUTHOR]

The rubber tree (Hevea brasiliensis)–cassava (Manihot esculenta) intercropping system is one of the most popular intercropping patterns for small-holder rubber planters. However, this practice is not recommended in some rubber-producing countries, since it is believed that the rubber tree and cassava share some identical pathogens and that the intercropping system will deplete soil fertility. The effects of cassava allelochemicals on rubber tree pathogens have never been reported. Using controlled plate tests, pot tests, and field experiments, this study aimed to assess the possible adverse effects that cassavas may impart to rubber plantations. The results showed: (1) although the leaf extract, decomposed leaf extract and root exudates of cassava inhibited or had no effect on the mycelial growth of the rubber tree pathogen Corynespora cassiicola, 10 mg mL−1 and 100 mg mL−1 cassava leaf water extracts and testing concentrations of cassava root extraction significantly promoted the mycelial growth of the rubber tree pathogen Rigidoporus lignosus, which increased the risk of rubber tree white root disease; (2) the overall soil fertility was increased significantly in the rubber tree–cassava intercropping system. However, the soil phosphorus content was reduced significantly in the field experiment; (3) the average girth of rubber trees was significantly reduced, and the girth uniformity was decreased in the intercropping system. Based on the experiment results, we suggested that an optimised fertiliser recommendation, particularly on the phosphorus amount, should be developed and implemented for the rubber tree–cassava intercropping system. In addition, since the intercropping of cassava will increase the risk of R. lignosus-induced rubber tree white root disease, which is very hard to prevent and eradicate once rubber trees are infected, the intercropping of cassava in rubber plantation rows should not be recommended in the R. lignosus prevailing areas. [ABSTRACT FROM AUTHOR]

Key message: The proteomic analysis of vascular tissues in rubber tree reveals differentially expressed proteins related to laticifer differentiation in mature phloem and secondary cell wall formation in mature xylem for latex/wood-yield improvement. Rubber tree (Hevea brasiliensis) is a latex-producing plant that has worldwide economic importance for the rubber and wood industries. Natural rubber latex is produced from laticiferous vessels (laticifers) located in the secondary phloem tissue of rubber trees. Improving latex production from rubber tree clones by studying the molecular genetics of vascular development has received much attention, but less information has been obtained from proteomic approaches. This study performed a comparative proteomic analysis of the vascular tissues in high latex-yield and high wood-yield rubber tree clones. One primary vascular tissue (newly developed stem) and two secondary vascular tissues (mature phloem/laticifers and mature xylem/wood) were investigated by qualitative/quantitative proteomic analysis using GeLC-MS/MS. The differentially expressed proteins (DEPs) in the specific vascular tissues were analyzed, and the protein functions in the biological processes related to vascular development and to the specific characteristics of each clone were clarified. The predicted protein–protein interaction network and GO annotation revealed DEPs related to photosynthesis, carbohydrate metabolism, energy production and jasmonic acid-responsive proteins involved in positive regulation of laticifer differentiation in mature phloem of the high latex-yield clone, while DEPs related to cell cytoskeleton maintenance, secondary cell wall components and auxin-, brassinosteroid-, abscisic acid-responsive proteins involved in xylem differentiation were abundant in mature xylem of the high wood-yield clone. This is the first report to demonstrate the correlation and functions of DEPs in phloem/laticifers and xylem/wood differentiation in rubber trees. The regulation of vascular development could be useful in improvement of latex and wood yields from rubber trees. [ABSTRACT FROM AUTHOR]

Rubber [Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg.] plantations are the largest cultivated forest type in tropical China. Returning organic materials to the soil will help to maintain the quality and growth of rubber trees. Although many studies have demonstrated that organic waste materials can be used to improve soil fertility and structure to promote root growth, few studies have studied the effects of organic amendments on soil fertility and root growth in rubber tree plantations. Here, bagasse, coconut husk or biochar were applied with a chemical fertilizer to test their effects on soil properties after 6 months and compared with the effects of only the chemical fertilizer. Results showed that the soil organic matter content, total nitrogen, available phosphorus and available potassium after the chemical fertilizer (F) treatment were all significantly lower than after the chemical fertilizer + bagasse (Fba), chemical fertilizer + coconut husk (Fco) or chemical fertilizer + biochar (Fbi) (p < 0.05). Soil pH in all organic amendments was higher than in the F treatment, but was only significantly higher in the Fbi treatment. In contrast, soil bulk density in the F treatment was significantly higher than in treatments with the organic amendments (p < 0.05). When compared with the F treatment, soil root dry mass increased significantly by 190%, 176% and 33% in Fba, Fco and Fbi treatments, respectively (p < 0.05). Similar results were found for root activity, number of root tips, root length, root surface area and root volume. Conclusively, the application of bagasse, coconut husk and biochar increased soil fertility and promoted root growth of rubber trees in the short term. However, bagasse and coconut husk were more effective than biochar in improving root growth of rubber trees. [ABSTRACT FROM AUTHOR]

Colletotrichum leaf disease (CLD), mainly caused by the Colletotrichum gloeosporioides species complex (CGSC), is a serious disease of rubber tree in most rubber-growing countries. Few fungicides are available for rubber farmers to manage this disease. To control CLD effectively, it is important to test the baseline sensitivity and efficacy of fungicides against CGSC isolates. Penthiopyrad is a new succinate dehydrogenase inhibitor (SDHI) that has been registered for use in many countries. In this study, baseline sensitivity to penthiopyrad was established by mycelial growth methods using 91 CGSC isolates collected from rubber trees in China. The EC50 values of penthiopyrad ranged from 0.243 to 3.377 mg/L with a mean EC50 value of 1.685 mg/L. Penthiopyrad greatly decreased the mycelial biomass and increased cell membrane permeability of CGSC isolates. The mycelia were severely distorted, and the number of top branches increased with penthiopyrad treatment. Penthiopyrad inhibited succinate dehydrogenase activity, causing a decrease in the ATP content of CGSC isolates, and significantly inhibited conidia germination and appressorium formation on rubber tree leaves. The protective and curative efficacy of 100 mg/L penthiopyrad reached up to 98.53% and 66.79% against CLD, respectively. Our results demonstrated the high inhibitory activity of penthiopyrad against CGSC isolates and its potential for CLD management on rubber tree. [ABSTRACT FROM AUTHOR]

The genus Hevea is native to the Brazilian Amazon forest, and H. brasiliensis, popularly known as the rubber tree, is the main source of raw material for the production of natural rubber in the world. To characterize the fungal communities that inhabit the tissues of this species, a total of 550 endophytic fungi were isolated, of which 212 were from native trees located in the state of Acre and 338 in the state of Amazonas. These fungi were grouped into 115 OTUs. The phylum Ascomycota was dominant (93%) and the most abundant isolates belonged to the families Glomerallaceae (genus Colletotrichum), Diaporthaceae (genus Diaporthe), Nectriaceae (genus Fusarium), Hypocreaceae (genus Trichoderma), and Trichocomaceae (genus Penicillium). The diversity and richness of fungi in the state of Acre did not differ significantly between leaves, stems, and roots; however, in the state of Amazonas, the diversity was greater in the stems and roots than in the leaves. In general, the diversity of endophytic fungi was greater in the state of Acre. Comparisons between communities of fungi showed more differences between those that inhabit different tissues than in different states. Furthermore, the fungal communities of roots and stems were similar, and both these were dissimilar from leaves. Isolates identified in this study will be tested for their potential to produce metabolites of interest for industry and for the sustainable development of agribusiness. [ABSTRACT FROM AUTHOR]

South American Leaf Blight (SALB) is a fungal disease caused by the pathogen Pseudocercospora ulei. It is the main disease affecting rubber trees in Latin America. This study was designed to investigate the changes in the spectral reflectance of rubber leaflets inoculated with P. ulei under controlled conditions. Young leaflets of two clones of Hevea brasiliensis with different degrees of resistance to SALB were tested. The results demonstrated that SALB severity affected the spectral properties of the rubber leaflets, increasing the spectral reflectance in the visible range, decreasing the near-infrared range, and causing variating in the spectral vegetation indices. The changes were more obvious in the most susceptible clone (FX 3864) than in the most resistant one (FX 4098). The SALB severity classes were correlated with the relative chlorophyll content, the leaflet phenology, and the appearance of disease symptoms after the pathogen inoculation. New mathematical models for predicting the SALB severity in the leaflets B (10–18 days old) and C (22–30 days old) in the two rubber tree clones were designed. [ABSTRACT FROM AUTHOR]

In this study, 2n gametes were induced in the rubber tree RRIM 600 by high-temperature exposure and used for the production of triploid offspring. A relationship between megasporogenesis and the external morphology of male and female flower buds was established to guide the treatments. The relationship between megasporogenesis and microsporogenesis of adjacent male flower buds was also estimated. The developmental stage of megasporogenesis was estimated indirectly based on these relationships. Finally, 5 open-pollinated triploid offspring were obtained; the mean efficiency of triploid production was approximately 1.2%. The induction efficiency was approximately 9.1% when meiotic stages of megasporogenesis from the diplotene stage to the diakinesis stage were exposed to a high temperature of 44 °C for 4 h. Simple sequence repeat (SSR) markers revealed that the 2n gametes were derived from the RRIM 600 female parent. This study was the first to induce 2n female gametes in rubber trees and will serve as the basis for the germplasm creation of triploids and breeding of new varieties of rubber trees. [ABSTRACT FROM AUTHOR]

Two Flower locus T-like genes were isolated and characterized in rubber tree. Both of them were found to have conserved genomic characteristics of the phosphatidylethanolamine-binding protein family and their encoding proteins possess conserved key amino acid residues of FT. RT-qPCR results showed that HbFT1 was restricted in mature leaves, male and female flowers whereas HbFT2 showed the main expression in reproductive organs. Both of them can be induced by short-day conditions but with different expression patterns and regulated by temperature. Transgenic wild-type Arabidopsis can reduce the flowering time and change plant morphogenesis, with fewer rosette leaves and terminal inflorescence to some degrees, as well as the phenotype of ft-10 can also be successfully rescued by ectopic expressing each of them, respectively, suggesting that both of them can exert the function of AtFT. However, they displayed different seasonal-change expression patterns, with HbFT1 mainly accumulating in September for adult trees and in October for juvenile trees and HbFT2 transcript accumulating in March, which was the main floral initiation period, even though both of them showed higher transcript levels in adult trees than 2-year-old trees, implying their different biological functions during growth and development as well as flowering transition in rubber tree. [ABSTRACT FROM AUTHOR]

During growth of woody plant-trunk, the secondary meristem functions in giving rise the xylem and phloem. Rubber tree (Hevea brasiliensis Muell. Arg.), in addition, contains laticifers (latex producing vessels) in the vicinity of phloem. Insights into regulatory mechanisms of gene networks underlying laticifer proliferation in rubber tree has remained very limited. The candidate vascular development-related genes were selected to investigate for expression profile in phloem and xylem tissues of high latex yield- and high wood yield-clones of rubber tree. The differential gene expression between the mature branch-xylem and -phloem tissues was clearly observed. The cis-regulatory motif analysis revealed the existent of putative jasmonic acid (JA)- and brassinosteroid (BR)-responsive regulatory motifs in promoter regions of these genes, and consequently the effect of exogenous application of JA, BR or their respective signaling inhibitors, on the formation of laticifers in rubber tree was demonstrated. Interestingly, the laticifer numbers were significantly increased in JA-treatment, correlated with up-regulation of phloem development-related genes in both rubber tree clones. On the contrary, the laticifers were decreased in BR-treatment accompanying by up-regulation of xylem development-related genes, especially in high wood yield-rubber tree clone. BR-inhibitor treatment also enhanced laticifer numbers, while JA-inhibitor suppressed laticifer differentiation. Taken together, this study unveils the molecular interplay between JA/BR on vascular development in rubber tree and how this impacts the appearance of laticifers in this plant. This process is vital for a better understanding on laticifer differentiation and its impact in the manipulation of wood and latex yield in rubber tree improvement program. [ABSTRACT FROM AUTHOR]

Key message: An ICE-like transcription factor mediates jasmonate-regulated cold tolerance in the rubber tree (Hevea brasiliensis), and confers cold tolerance in transgenic Arabidopsis. The rubber tree (Hevea brasiliensis) is susceptible to low temperatures, and understanding the mechanisms regulating cold stress is of great potential value for enhancing tolerance to this environmental variable. In this study, we find that treatment with exogenous methyl jasmonate (MeJA) could significantly enhance Hevea brasiliensis cold tolerance. In addition, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments show that JASMONATE ZIM-DOMAIN(JAZ) proteins, HbJAZ1 and HbJAZ12, key repressors of JA signaling pathway, interact with HbICE2, a novel ICE (Inducer of CBF Expression)-like protein. HbICE2 was nuclear-localised and bound to the MYC recognition (MYCR) sequence. The transcriptional activation activity of HbICE2 in yeast cells was dependent on the N-terminus, and overexpression of HbICE2 in Arabidopsis resulted in elevated tolerance to chilling stress. Furthermore, dual-luciferase transient assay reveals that HbJAZ1 and HbJAZ12 proteins inhibit the transcriptional function of HbICE2. The expression of C-repeat-binding factor (CBF) signalling pathway genes including HbCBF1, HbCBF2 and HbCOR47 were up-regulated by MeJA. Taken together, our data suggest that the new ICE-like transcription factor HbICE2 is involved in jasmonate-regulated cold tolerance in Hevea brasiliensis. [ABSTRACT FROM AUTHOR]

Tropical plant rubber tree (Hevea brasiliensis) is the sole source of commercial natural rubber and low-temperature stress is the most important limiting factor for its cultivation. To characterize the gene expression profiles of H. brasiliensis under the cold stress and discover the key cold stress-induced genes. Three cDNA libraries, CT (control), LT2 (cold treatment at 4 °C for 2 h) and LT24 (cold treatment at 4 °C for 24 h) were constructed for RNA sequencing (RNA-Seq) and gene expression profiling. Quantitative real time PCR (qRT-PCR) was conducted to validate the RNA-Seq and gene differentially expression results. A total of 1457 and 2328 differentially expressed genes (DEGs) in LT2 and LT24 compared with CT were respectively detected. Most significantly enriched KEGG pathways included flavonoid biosynthesis, phenylpropanoid biosynthesis, plant hormone signal transduction, cutin, suberine and wax biosynthesis, Pentose and glucuronate interconversions, phenylalanine metabolism and starch and sucrose metabolism. A total of 239 transcription factors (TFs) were differentially expressed following 2 h or/and 24 h of cold treatment. Cold-response transcription factor families included ARR-B, B3, BES1, bHLH, C2H, CO-like, Dof, ERF, FAR1, G2-like, GRAS, GRF, HD-ZIP, HSF, LBD, MIKC-MADS, M-type MADS, MYB, MYB-related, NAC, RAV, SRS, TALE, TCP, Trihelix, WOX, WRKY, YABBY and ZF-HD. The genome-wide transcriptional response of rubber tree to the cold treatments were determined and a large number of DEGs were characterized including 239 transcription factors, providing important clues for further elucidation of the mechanisms of cold stress responses in rubber tree. [ABSTRACT FROM AUTHOR]

The phytohomorne methyl jasmonate (MeJA) is known to trigger extensive reprogramming of gene expression leading to transcriptional activation of many secondary metabolic pathways. However, natural rubber is a commercially important secondary metabolite and little is known about the genetic and genomic basis of jasmonate-elicited rubber biosynthesis in rubber tree (Hevea brasiliensis). RNA sequencing (RNA-seq) of H. brasiliensis bark treated with 1 g lanolin paste containing 0.02% w/w MeJA for 24 h (M2) and 0.04% w/w MeJA for 24 h (M4) was performed. A total of 2950 and 2850 differentially expressed genes in M2 and M4 compared with control (C) were respectively detected. Key genes involved in 2-C-methyl-D-erythritol 4-phosphate, rubber biosynthesis, glycolysis and carbon fixation (Calvin cycle) pathway were found to be up-regulated by MeJA treatment. Particularly, the expression of 3-hydroxy-3-metylglutaryl coenzyme A reductase in MVA pathway was down-regulated by MeJA treatment, but the expression of farnesyl diphosphate synthase (FPS) and cis-prenyltransferase (CPT, or rubber transferase) in rubber biosynthesis pathway were up-regulated by MeJA treatment. Up-regulation of critical genes in JA biosynthesis in response to MeJA treatment exhibited the self-activation of JA biosynthesis. In addition, up-regulated genes of great regulatory importance in cross-talk between JA and other hormone signaling, and of transcriptional regulation were identified. The increased expression levels of FPS and CPT in rubber biosynthesis pathway possibly resulted in an increased latex production in rubber tree treated with MeJA. The present results provide insights into the mechanism by which MeJA activates the rubber biosynthesis and the transcriptome data can also serve as the foundation for future research into the molecular basis for MeJA regulation of other cellular processes. [ABSTRACT FROM AUTHOR]

Foliar nitrogen (N) is an important index to evaluate the content of nitrogen in rubber trees. Hence, a fast way utilizing the spectroscopy to estimate the nitrogen content level of the rubber tree under natural environment has been preliminarily investigated in this paper. The combined features strategy is adopted in this study. The evaluation model is developed by back propagation (BP) algorithm and Adaboost algorithm. The results of simulation experiment show that satisfactory classification performance has been achieved using the proposed model and the percentage of samples correctly classified by the proposed model in the validation set is 92.98%. [ABSTRACT FROM AUTHOR]

Little is known about the phosphorus (P) fractions and P lability of agroforest soils in tropical regions, particularly those of cacao ( Theobroma cacao L.) agroforests. We hypothesized that the effect of P fertilization on the distribution of P fractions in the soil based on the source-sink relationship differs for different cacao agroforestry systems. The cacao agroforestry systems studied were the following: open cacao-cabruca, closed cacao-cabruca, cacao + erythrina, mixed cacao + rubber tree, and cacao + rubber tree intercropping. A natural forest and an unfertilized pasture were used as reference systems. The P fractions were determined using the Hedley sequential extraction method, and the P transformation processes were evaluated via structural equation modeling. The impact of low P fertilizer input on the P fractions varied according to the specific environmental conditions of each cacao production system. Consequently, there was high dissimilarity among all of the cacao sites. In all of the cacao agroforestry systems, there was an increase in inorganic P (Pi), especially the labile fraction (resin-Pi and NaHCO-Pi), but organic P (Po) increased only in the cacao + erythrina system and in the rubber tree planting row of the cacao + rubber tree intercropping system. As a result, the fitted structural models indicated that the inter-relationships of the geochemical processes were more important for determining the P availability than the biological processes. However, the Po concentrations and relative proportion were high in all of the cacao agroforestry systems, thereby revealing the high potential of supplying P to plants via the mineralization process in the eventual removal of mineral fertilization. [ABSTRACT FROM AUTHOR]

S-Adenosylmethionine decarboxylase (SAMDC) is a key rate-limiting enzyme involved in polyamines biosynthesis, and it plays important roles in plant growth, development and stresses response. However, no SAMDC gene was reported in rubber tree. Here we report characteristics of an SAMDC gene ( HbSAMDC1) in rubber tree. HbSAMDC1 contains a 1080 bp open reading frame (ORF) encoding 359 amino acids. Quantitative real-time PCR analyses revealed that HbSAMDC1 exhibited distinct expression patterns in different tissues and was regulated by various stresses, including drought, cold, salt, wounding, and HO treatments. HbSAMDC1 5′ untranslated region (UTR) contains a highly conserved overlapping tiny and small upstream ORFs (uORFs), encoding 2 and 52 amino acid residues, respectively. No introns were located in the main ORF of HbSAMDC1, whereas two introns were found in the 5′ UTR. In transgenic tobaccos, the highly conserved small uORF of HbSAMDC1 is found to be responsible for translational repression of downstream β-glucuronidase reporter. To our knowledge, this is the first report on molecular cloning, expression profiles, and 5′ UTR characteristics of HbSAMDC1. These results lay solid foundation for further elucidating HbSAMDC1 function in rubber tree. [ABSTRACT FROM AUTHOR]

Key message : Ethephon (an ethylene releaser) is commonly used to enhance latex productivity of rubber tree ( Hevea brasiliensis ), but the underlying mechanism has been elusive. The review presents the recent advances in understanding the molecular mechanism of increasing latex production in rubber tree by ethylene application. Abstract: Rubber tree ( Hevea brasiliensis) is currently the only commercial sources of natural rubber which is an important industrial material. Application of ethephon (an ethylene generator) has been a measure extensively adopted in worldwide rubber plantations to increase the productivity of rubber tree since the 1960s. In the recent years, adoption of genomic, transcriptomic, and proteomic approaches has brought great progress in understanding the mechanism of ethylene stimulation of latex yield. In this review, an update on the molecular mechanism of ethylene stimulation of latex production in H. brasiliensis is presented. Ethylene stimulation can be roughly ascribed to the acceleration of latex regeneration and the prolongation of the flow of latex. The relevant biochemical pathways and processes are comprehensively reviewed. The possibility that the limiting factors of the biosynthesis of rubber identified in bark glycolysis and photosynthetic carbon fixation can be utilized in molecular diagnostic and genetic engineering for rubber high-yielding variety improvement is discussed. Unsolved problems of the ethylene stimulation and a major challenge to understand the regulatory mechanism of the ethylene stimulation are also indicated. [ABSTRACT FROM AUTHOR]

Rubber tree ( Hevea brasiliensis) is a pivotal source for natural rubber production. Polyisoprene is synthesized in laticifer vessels, which are developed from vascular cambium in the phloem area of the inner bark tissue. The transcription factors and phytohormones were reported to be involved in network regulation of plant vascular tissue differentiation. Methyl jasmonate (MeJA) was reported to act as a stimulator for laticifer vessel formation in rubber tree, but the regulatory mechanism remains largely unknown. In this study, the correlation of laticifer vessel proliferation in phloem tissue upon MeJA treatment and the MeJA-responsive gene expression was investigated. Histochemical study of the laticifer revealed the circle and diffused structure around the secondary phloem of the stem. The number of laticifer vessels was increased from the top shoot to the lower stem part. The higher average number of laticifer vessels in stems of high latex-producing clones than in low latex/high wood yield clones suggested the correlation of laticifer vessel number and latex yield potential. This study demonstrated the temporal differential expression of MeJA-responsive genes upon MeJA treatment and adjusted back to a normal level after 3 months in two high latex-producing clones. The increasing number of laticifer vessels in MeJA-treated plants confirmed the consequent effect of MeJA treatment, and the possible roles of these genes in relation with laticifer vessel proliferation are discussed. A better understanding of gene function in laticifer development would be beneficial in rubber tree improvement and exploitation. [ABSTRACT FROM AUTHOR]

Rubber tree ( Hevea brasiliensis) is of major economic importance in Southeast Asia and for small land holders in Thailand in particular. Due to the high value of latex, plantations are expanding into unsuitable areas, such as the northeast province of Thailand where soil fertility is very low and therefore appropriate management practices are of primary importance. Arbuscular mycorrhizal fungi (AMF) contribute to plant growth through a range of mechanisms and could play a key role in a more sustainable management of the rubber plantations. We described the diversity of AMF associated with rubber tree roots in Northeast Thailand in relation to tree age and soil parameters along a chronosequence of rubber tree plantations. Cassava fields were included for comparison. Rubber tree and cassava roots harbored high diversity of AMF (111 Virtual Taxa, VT), including 20 novel VT. AMF VT richness per sample was consistently high (per site mean 16 to 21 VT per sample) along the chronosequence and was not related to soil properties. The composition of AMF communities differed between cassava and rubber tree plantations and was influenced by soil texture and nutrient content (sand, K, P, Ca). AMF community composition gradually shifted with the age of the trees. Our results suggest that the high diversity of AMF in this region is potentially significant for maintaining high functionality of AMF communities. [ABSTRACT FROM AUTHOR]

Background: Forward genetics approaches are not popularly applied in non-model plants due to their complex genomes, long life cycles, backward genetic studies etc. Researchers have to adopt reverse genetic methods to characterize gene functions in non-model plants individually, the efficiency of which is usually low. Results: In this study, we report a gain-of-function in Arabidopsis (GAINA) strategy which can be used for batch identification of functional genes in a plant species. This strategy aims to obtain the gain-of-function of rubber tree genes through overexpressing transformation ready full-length cDNA libraries in Arabidopsis. An initial transformation test produced about two thousand independent transgenic Arabidopsis lines, in which multiple obvious aberrant phenotypes were observed, suggesting the gain-of-function of rubber tree genes. The transferred genes were further isolated and identified. One gene identified to be metallothionein-like protein type 3 gene was further transferred into Arabidopsis and reproduced a similar aberrant phenotype. Conclusion: The GAINA system proves to be an efficient tool for batch identification of functional genes in Hevea brasiliensis, and also applicable in other non-model plants. [ABSTRACT FROM AUTHOR]

The transient mesophyll protoplast expression system has become a powerful tool for rapid gene functional analysis, and it has been used successfully in several plant species, including model plants Arabidopsis and rice, but not in rubber tree. In this study, we describe an improved method to isolate high quality rubber tree mesophyll protoplasts. The transient expression of exogenous DNA constructs in this mesophyll protoplast system was observed by using green fluorescent protein, and was detected by immunoblot. Furthermore, we used two typical pathogen-associated molecular patterns flg22 and chitin from bacteria and fungi, respectively, as inducers of plant innate immunity responses to detect the immunity response of rubber tree. Our data showed that flg22 and chitin induced activation of mitogen-activated protein kinases, generation and accumulation of reactive oxygen species, and transcription of defense genes. Our study demonstrated that the rubber tree mesophyll protoplast system can be used for functional analysis of immune responses of rubber tree and the dissection of cell signaling pathways. This transient mesophyll protoplast system may be applied to analyse gene function of rubber tree, a perennial plant for which the applicable transformation system was limited. [ABSTRACT FROM AUTHOR]

Yeast abundance and species diversity in the latex of rubber tree Hevea brasiliensis (Willd. ex Juss.) Müll. Arg., on its green leaves, and in soil below the plant were studied. The yeasts present in the fresh latex in numbers of up to 5.5 log(CFU/g) were almost exclusively represented by the species Candida heveicola. This species was previously isolated from Hevea latex in China. In the course of natural modification of the latex (turned from liquid to solid form), yeast diversity increased, while yeast abundance decreased. The yeasts in thickened and solidified latex were represented by typical epiphytic and ubiquitous species: Kodamea ohmeri, Debaryomyces hansenii, Rhodotorula mucilaginosa, and synanthropic species Candida parapsilosis and Cutaneotrichosporon arboriformis. The role of yeasts in latex modification at the initial stages of succession and their probable role in development of antifungal activity in the latex are discussed. [ABSTRACT FROM AUTHOR]

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest. [ABSTRACT FROM AUTHOR]

The Ascomycota fungus Microcyclus ulei is a major threat for natural rubber production. The ascospores play a major role in the survival and dissemination of the fungus. The present study indicates that the ascospores are present on the leaves shortly after infection. Although their abundance is higher on leaflets 50-70 days after they reach maturity, ascospores are still found on 6 months-old leaves, ensuring the survival of the fungus during the dry season in the case of no defoliation of the rubber trees. Indeed, their viability does not depend on the age of the leaf. The pycnidiospores, whose role in the lifecycle of the fungus is not established, are also present on the leaves several months after their maturity. The ascospores are disseminated only if the leaves are wetted and exposed to an airflow. The discharge of ascospores starts a few minutes after a single wetting event but the number of ascospores discharged decreases rapidly and stops 90 min after the wetting event. However, the perithecia are rapidly able to discharge ascospores if wetted again but at a lower level. Compared to other Ascomycota, the discharge of ascospores is quick but brief. [ABSTRACT FROM AUTHOR]

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs. [ABSTRACT FROM AUTHOR]

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding. [ABSTRACT FROM AUTHOR]

To construct a linkage map enriched with tapping panel dryness (TPD)-related markers, we firstly utilized rubber tree ESTs associated with TPD to develop intron length polymorphism (ILP) markers. In this study, 52 new ILP markers were further developed. Together with the ILP markers previously reported, 102 ILP markers developed from TPD-related ESTs were analyzed within 39 Hevea germplasm in detail. The PCR success rate and polymorphism rate of ILP markers was 97.06 and 61.62 %, respectively. The results based on PCR amplification and sequence analyses provided the evidences on cross-species/genera transferability of rubber tree ILP markers. The average polymorphic information content (PIC) values of 39 Hevea germplasm were about 0.1719, indicating that the genetic base of Hevea germplasm selected in this study was very narrow. Among 39 Hevea germplasm, the PIC value of wild rubber tree accessions was the highest, followed by Hevea species and cultivated rubber tree clones. Based on the similarity coefficient of ILP markers, 39 Hevea germplasm were divided into three groups including cultivated clones, wild accessions and Hevea species, suggesting that the classification was generally related to the characterization of Hevea germplasm. The ILP markers developed in this study further enriches the number of molecular marker in rubber tree, and the ILP markers will have a wide application in DNA fingerprinting, genetic diversity, marker-assisted selection and genetic mapping, etc. Moreover, the ILP markers transferred cross-euphorbiaceae family might be utilized in cassava, castor bean and physic nut. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is an essential macronutrient for plant growth and development. P deficiency could affect rubber tree productivity seriously, and understanding the mechanism responses of the rubber tree under the P deficiency will be helpful to improving rubber tree productivity. The molecular mechanism by which the rubber trees respond to a P-deficiency is a complex network involving many processes. To identify the genes differentially expressed in that response, we constructed subtractive suppression hybridization libraries for roots of plants growing under deficient or sufficient conditions. We identified 94 up-regulated genes from the forward library and 45 down-regulated from the reverse library. These differentially expressed genes were categorized into eight groups representing functions in metabolism, transcription, signal transduction, protein synthesis, transport, stress responses, photosynthesis, and development. We also performed quantitative real-time PCR to investigate the expression profiles of eight randomly selected clones. Our results provide useful information for further study of the molecular mechanism for adaptations to a P-deficiency in this species. Further characterization and functional analysis of these differentially expressed genes will help us improve its phosphorus utilization and overall productivity. [ABSTRACT FROM AUTHOR]

Plant microRNAs (miRNAs) have been demonstrated to play an important regulatory role in a variety of biological processes ranging from plant growth and development, defensive pathways, as well as biotic and abiotic stress responses. MiRNAs have been extensively studied in plant models with available genome sequence data. The Euphorbiaceae family is a large flowering plant family, which is of considerable economic importance, but relatively limited genome sequence data. The four Euphorbiaceous species; rubber tree, cassava, castor bean and leafy spurge; were selected for this study. Using conserved plant miRNAs sequences aligned to the GenBank expressed sequenced tag database of each target plants, and further potential precursor miRNA-secondary structure analysis, the 526 potential miRNAs belonging to 17 miRNA families and their targets were identified. The majority of miRNA families was able to be identified in cassava. The minimal folding free energy index of precursor miRNAs was significantly higher than other non-coding or coding RNAs. The largest group of miRNA target genes consists of regulatory genes predicted to encode transcription factors, while non-regulatory genes involving in various functions. Some miRNAs contain different targets, suggesting the diverse functions of miRNAs depending on plant species and environmental conditions. The 6 expressed Hbr-miRNAs were verified in rubber tree. Gaining insight into the miRNA targets will help us to understand the range of miRNA expression regulation and to more coherently describe the functional importance of miRNAs. These findings considerably facilitate the basis for broaden future investigation on the role of these miRNAs in the economic important Euphorbiaceous species. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree ( Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high-throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 115 miRNAs belonging to 56 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly and predicted 20 novel miRNAs. Further, computational analysis uncovered potential targets of the known and novel miRNAs. Predicted target genes included not only transcription factors but also genes involved in various biological processes including stress responses, primary and secondary metabolism, and signal transduction. In particular, genes with roles in rubber biosynthesis are predicted targets of miRNAs. This study provides a basic catalog of miRNAs and their targets in rubber tree to facilitate future improvement and exploitation of rubber tree. [ABSTRACT FROM AUTHOR]

With a long-term goal of constructing a linkage map enriched with gene-specific markers in rubber tree ( Hevea brasiliensis Muell. Arg.), we utilized rubber tree ESTs associated with tapping panel dryness (TPD) to develop intron-flanking PCR markers. After downloading and assembling the rubber tree ESTs associated with TPD, we predicted the exon/exon junction sites (E/E) by aligning rubber tree transcripts with the genomic sequences of castor bean ( Ricinus communis L.). Based on the predicted E/E, the primers flanking intron(s) and no intron were designed. Compared with the markers designed by conventional method, the PCR success rate of the markers designed with the predicted E/E increased 28-30%, whereas the polymorphism rate of intron-flanking EST-PCR markers was approximately 3.43-fold increase. Therefore, the intron-flanking marker was more polymorphism-generating efficient than the markers designed by conventional methods. In addition, analyzing the polymorphic information content (PIC) among Hevea germplasm showed that the polymorphism of wild rubber tree accessions was higher than one of cultivated rubber tree clones and Hevea species. This study enriches the categories and numbers of molecular markers in rubber tree, and the markers developed in this research will have a wide application in DNA fingerprinting, marker-assisted selection and genetic mapping in rubber tree. This research also indicates that it is possible to develop intron-flanking EST-PCR markers of rubber tree with castor bean genome as reference sequences, which provides new insights into developing intron-flanking EST-PCR markers for rubber tree or other plant species without genomic information. [ABSTRACT FROM AUTHOR]

Corynespora cassiicola is the causal agent of Corynespora Leaf Fall (CLF) disease. CLF is one of the most important fungal diseases of rubber trees in Asia and Africa but disease outbreaks have not been reported in South America. Cassiicolin, a small cysteine-rich glycoprotein secreted by the pathogenic C. cassiicola isolate CCP, was previously identified as a potential disease effector in rubber tree. Recently, the cassiicolin-encoding gene ( Cas1) was characterized and shown to be expressed in the early phase of infection. In this study, we investigated whether previously undetected strains of C. cassiicola are present in South American rubber plantations by examining the fungal endophyte population found in asymptomatic rubber tree leaves. Four isolates were identified as C. cassiicola. Genes encoding new forms of the cassiicolin precursor protein (Cas3 and Cas4) were identified from these isolates. Three of four isolates were able to induce symptoms on the cultivar they were isolated from in a detached leaf assay, with different kinetics and intensities. One isolate had the same pathogenicity profile as the pathogenic isolate CCP; the other two isolates developed symptoms late during the course of infection, suggesting saprotrophic capabilities. However, no Cas3 or Cas4 transcripts could be detected upon inoculation with the endophytic isolates, whereas the reference gene Cas1 was expressed upon inoculation with the CCP isolate. This work demonstrated that C. cassiicola is present in South America in an endophytic form and that it may evolve from an endophytic to a saprophytic or even potentially pathogenic life style. [ABSTRACT FROM AUTHOR]

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers. [ABSTRACT FROM AUTHOR]

Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression in budded plants should be verified for functional analysis and further development. [ABSTRACT FROM AUTHOR]

The components of genetic resistance from the Hevea brasiliensis cultivar MDF 180 against South American Leaf Blight (SALB) caused by Microcyclus ulei were investigated by QTL mapping. MDF 180 has already been described as a cultivar with a high level of partial and long-lasting resistance. The resistance of progeny individuals from a cross between a susceptible cultivar and MDF 180 was assessed both under controlled conditions of inoculation by three M. ulei isolates and under natural infection in a field trial. Genetic maps of the two parents of this progeny were mainly established based on microsatellites and AFLP markers. No resistance QTL were found in the susceptible parent. In the resistant parent, we identified a qualitative gene responsible for the resistance against isolates from French Guiana and a major quantitative resistance factor determining the resistance against isolates from the state of Bahia (Brazil). The qualitative resistance gene was denominated M15md and was located in the linkage group g15. Four minor resistance QTLs were also identified, two of which showed an epistatic interaction with M15md. The durability of the resistance of MDF 180 is discussed in light of these data. [ABSTRACT FROM AUTHOR]

Endophytic fungi live inside plants as symbionts for at least one period of their life cycle. They play an important role in plant defence and could be promising sources of valuable natural bioactive products. The present study aimed to select endophytic fungi antagonistic to Microcyclus ulei, the agent of South American Leaf Blight (SALB) responsible for the weak development of rubber plantations in Latin America. Endophytic fungi were isolated from leaves of three cultivars (FX3864, CDC312, MDF180) of Hevea brasiliensis (the rubber tree) with distinct resistance levels to the attack by Microcyclus ulei. Leaf samples were collected from a 5,000 ha area of the Michelin plantations (Igrapiúna, Bahia, Brazil). A total of 435 colonies of endophytic fungi were isolated, purified, and preserved in sterilized distilled water. Lyophilized culture filtrates obtained from 88 fungal isolates, grown in liquid malt extract medium, were tested in vitro at concentrations of 50% and 12.5% for their inhibitory potential against the germination of M. ulei PMB26 conidia. Thirteen filtered cultures of endophytic fungi that presented inhibitory activity above 80% (at the concentration 12.5%) were characterized taxonomically by sequencing the ITS regions of rDNA, revealing the following taxa: Fusarium sp., Gibberella sp., Glomerella cingulata, Microsphaeropsis sp., Myrothecium sp., Pestalotiopsis sp. and Phomopsis sp. Three taxa were detected only in the resistant cultivars: Microsphaeropsis sp ., and Pestalotiopsis sp. in MDF180, and Myrothecium sp. in CDC312. The present work constitutes the first study showing inhibitory activity of rubber tree endophytic fungi on Microcyclus ulei. These results provide an encouraging basis for the identification of new bioactive molecules for an effective control of South American leaf blight. [ABSTRACT FROM AUTHOR]

An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing antibiotic resistance marker-free transgenic plants. [ABSTRACT FROM AUTHOR]

Life cycle and behaviour of Cunaxatricha tarsospinosa Castro & Den Heyer from rubber trees in Brazil were studied, with Tenuipalpus heveae Baker offered as prey. The study was conducted at 25.4 ± 0.2°C, 83 ± 5% RH and 12:12 h L:D photophase. The egg stage was the longest immature stage, lasting 17.1 ± 1.3 days (mean ± SE); total juvenile development was completed in 33.2 ± 2.8 days. Lifetime fecundity was 12.0 ± 2.2 eggs. Intrinsic rate of population increase was low, suggesting that T. heveae may not be a good prey for the predator. All specimens of C. tarsospinosa collected in the field for this study were females, no males were found. Concurrently, only females were obtained in the laboratory. This seems to be the first report of thelytokous parthenogenesis for cunaxids. Similar to earlier reports for some Cunaxinae and Coleoscirinae, prey were captured when predators were actively searching for them. [ABSTRACT FROM AUTHOR]