Rubber Tree (Hevea brasiliensis) Bark Necrosis Syndrome I: Still No Evidence of a Biotic Causal Agent.

The main constraint in rubber plantations worldwide is the cessation of latex production because of two syndromes: (i) tapping panel dryness (TPD), a reversible physiological response to overexploitation; and (ii) bark necrosis (BN), an irreversible syndrome spreading from the collar toward the tapping cut. Early BN symptoms develop in the inner phloem tissues but never affect the cambium. Necrotic patches appear in the outer phloem, inducing bark cracking and peeling, but these alterations never lead to tree death. BN spreads gradually to neighboring rubber trees, and evidence of linear disease centers suggest that a pathogen may be involved, possibly transmitted by the tapping knife. Several previous etiological investigations (fungi, phytoplasma, bacteria, and virus) were performed (3) on leaves, bark, and latex using different methods (e.g., isolation, transmission, chemical treatments, and optic and electron microscope studies). Recent works focused on mechanically transmissible pathogens, such as viroid (2) or virus/double strand RNA, using RNA extraction (nonionic cellulose and appropriate ethanol concentrations) and treatment with RNase A, followed with sequential polyacrylamide gel electrophoresis (s-PAGE), reverse transcription-polymerase chain reaction (RT-PCR), degenerate oligonucleotide primer-PCR (DOP-PCR), and cloning and sequence analysis. While numerous viroid-like (between 250 and 400 nucleotides) and double strand virus-like (1,800 bp) low-molecular-weight RNAs were observed, no definite correlation was found with the BN status of trees. Sequencing of the various isolated RNAs only identified plasmids, nonpathogenic bacteria and yeasts, but none of the suspected pathogens. In addition, previous and recent transmission trials (tapping knife disinfection, bud grafting, bark implantation, and etc.) failed to confirm the involvement of a biotic agent. In conclusion, since these etiological investigations were inconclusive, a physiological disease is now suspected that involves exogenous stresses, nonoptimal vascular relations at the rootstock/scion junction and impaired cyanide metabolism (1,4). References: (1) H. Chrestin et al. Plant Dis. 88:1047, 2004. (2) N. Duran-Vila et al. J. Gen. Virol. 69:3069, 1988. (3) D. Nandris et al. Eur. J. For. Pathol. 21:340, 1991. (4) D. Nandris et al. Plant Dis. 88:1047, 2004.

Rubber Tree (Hevea brasiliensis) Bark Necrosis Syndrome II: First Comprehensive Report on Causal Stresses.

Bark necrosis (BN), described and first studied in Côte d'Ivoire in the 1980s (2), affects most modern rubber plantations (i.e., grafted trees with high-yielding clones, intensive exploitation due to tapping frequency, and use of Ethrel as a yield stimulant) worldwide with a wide range of severity across sites. While previous (3) and recent (4) etiological analyses remain inconclusive, environmental factors were shown associated with BN. Numerous epidemiological surveys conducted in various African and Asian plantations on recently tapped blocks (less than 10-year-old trees) revealed the nonrandom location of the earliest single diseased trees. These risky areas are mainly characterized by the proximity of a swamp, plantation road, windrow, old bulldozer track, residual forest stump, or slope break. In BN emergence areas, while no significant correlation was found with chemical soil parameters, physical soil analyses (e.g., penetrometer) revealed higher soil compaction often associated with poorer rhizogenesis in BN trees (comparative root counts made in pits close to healthy or BN trees). Furthermore, initial BN symptoms were preferentially observed near the grafted bud at the rootstock/scion junction (RS/S). Numerous comparative ecophysiological measurements of leaf water potential, stem water potential, and predawn base potential using a plant moisture stress (PMS) pressure chamber indicated water stress in BN trees. These results and preliminary dye transfer studies at the RS/S junction suggested a nonoptimal vascular relation between the root system and the trunk of BN trees. In conclusion, compaction-associated reduced water availability of the soil and poor root capacity to meet the water demand during drier dry seasons combined with disturbed sap flows and recurrent local water drainage (latex flows) are now suspected to jointly act as the main exogenous causal stresses that induce the BN process at the RS/S bud zone before spreading upward to the tapping cut. This multidisciplinary approach gives a new comprehensive scenario for the emergence of this multifactorial physiological disease, now suspected to involve cyanide release (1) into the inner phloem of the rubber tree. References: (1) H. Chrestin. Plant Dis. 88:1047, 2004. (2) D. Nandris et al. Eur. J. For. Pathol. 21:325, 1991. (3) D. Nandris et al. Eur. J. For Pathol. 21:340, 1991. (4) F. Pellegrin et al. Plant Dis. 88:1046, 2004.

Rubber Tree (Hevea brasiliensis) Bark Necrosis Syndrome III: A Physiological Disease Linked to Impaired Cyanide Metabolism.

First attempts to discriminate between tapping panel dryness (TPD) and bark necrosis (BN), two Hevea sp. bark diseases leading to the cessation of latex production, showed differences in latex biochemical characteristics (1). Further, contrary to TPD, BN is characterized by inner phloem necrosis starting at the rootstock/scion junction (RS/S) and spreading upward to the tapping cut. Recent etiological (3) and epidemiological studies did not provide evidence of a causative pathogen for BN, but showed that BN is favored by a combination of various stresses (2). Searching for molecular markers of BN using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses highlighted differential expression of some proteins in the latex and bark, especially a 67-kDa protein, which accumulates in the inner phloem of the BN trees. This protein was identified by peptide microsequencing as a linamarase (cyanogenic ß-glucosidase). This led to the suspicion of the involvement of cyanogenesis in the spread of the syndrome inside the inner bark. The cDNAs of enzymes involved in cyanide (CN) metabolism (linamarase, hydroxynitrile lyase, and cyanoalanine synthase) were cloned from our Hevea sp. phloem specific cDNA library. In addition, the most BN-susceptible rubber clones were shown to exhibit higher cyanide potentions in the leaves and bark, together with low cyanoalanine synthase (CAS) gene expression and activity. Furthermore, linamarine (the cyanogene glucoside substrate of linamarase) was shown to accumulate in the phloem at the base of the trunk, especially above the rootstock/scion junction. The results of biochemical and gene expression studies associated with recent ecophysiological advances (2) strongly suggest a possible cell decompartmentalization near the RS/S junction, resulting in a local release of toxic concentration of highly diffusive CN. This, combined with a lethal imbalance between cyanogenic and CN-detoxifying activities (CAS) in the phloem of BN trees, could lead to poisoning of neighboring cells and to the spread of tissue necrosis toward the tapping cut. In conclusion, after providing evidence of exogenous factors favoring BN (2), this report highlights endogenous disorders that may be at the origin of this physiological disease leading to BN. References: (1) D. Nandris et al. Eur. J. For. Pathol. 21:325, 1991. (2) D. Nandris et al. Plant Dis. 88:1047, 2004.

Genetic mapping of a caffeoyl-coenzyme A 3-O-methyltransferase gene in coffee trees. Impact on chlorogenic acid content.

Chlorogenic acids (CGA) are involved in the bitterness of coffee due to their decomposition in phenolic compounds during roasting. CGA mainly include caffeoyl-quinic acids (CQA), dicaffeoyl-quinic acids (diCQA) and feruloyl-quinic acids (FQA), while CQA and diCQA constitute CGA sensu stricto (CGA s.s.). In the two cultivated species Coffea canephora and Coffea arabica, CGA s.s. represents 88% and 95% of total CGA, respectively. Among all enzymes involved in CGA biosynthesis, caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is not directly involved in the CGA s.s. pathway, but rather in an upstream branch leading to FQA through feruloyl-CoA. We describe how a partial cDNA corresponding to a CCoAOMT encoding gene was obtained and sequenced. Specific primers were designed and used for studying polymorphism and locating the corresponding gene on a genetic map obtained from an interspecific backcross between Coffea liberica var. Dewevrei and Coffea pseudozanguebariae. Offspring of this backcross were also evaluated for the chlorogenic acid content in their green beans. A 10% decrease was observed in backcross progenies that possess one C. pseudozanguebariae allele of the CCoAOMT gene. This suggests that CGA s.s. accumulation is dependent on the CCoAMT allele present and consequently on the activity of the encoded isoform, whereby CGA accumulation increases as the isoform activity decreases. Possible implications in coffee breeding are discussed.

Ethylene-Induced Increase in Glutamine Synthetase Activity and mRNA Levels in Hevea brasiliensis Latex Cells.

Ethylene, used as a stimulant of latex production in Hevea brasiliensis, significantly activates the regenerating metabolism within the laticiferous cells. In this context, attention was focused on glutamine synthetase (GS; EC 6.3.1.2), a key enzyme in nitrogen metabolism. A specific and significant activation of the cytosolic glutamine synthetase (GScyt) in the laticiferous cells after ethylene treatment parallels the increase of latex yield. A marked accumulation of the corresponding mRNA was found, but in contrast, a slight and variable increase of the polypeptide level is at the limit of detection by western blotting. The GS response to ethylene might be mediated by ammonia that increases in latex cytosol following ethylene treatment. The physiological significance for such a regulation by ethylene of the GScyt is discussed in terms of the nitrogen requirement for protein synthesis associated with latex regeneration.

Long-Term Effect of a Pythium Elicitor Treatment on the Growth and Alkaloid Production of Catharanthus roseus Cell Suspensions.

The treatment of a CATHARANTHUS ROSEUS cell suspension culture with a low concentration of PYTHIUM elicitor stimulated the alkaloid production. When these pretreated cells were resuspended in a medium that did not contain the fungal extract, the positive effects of the treatment on alkaloid synthesis and excretion were lost and, moreover, the standard level of production was not recovered. A second treatment of these cells with PYTHIUM elicitor at day 5 of the second culture cycle greatly impaired growth kinetics, but did not stimulate the alkaloid production observed with standard cultures. Repeated treatments with a low concentration of fungal elicitor seemed to have a negative long-term effect on both growth and alkaloid synthesis and did not appear to be a useful process for production purposes.

Hevein, a lectin-like protein from Hevea brasiliensis (rubber tree) is involved in the coagulation of latex.

Hevein, a lectin-like protein is the major protein of vacuolar structures called lutoids in the latex of rubber trees. We have shown both by in planta and ex planta studies that hevein is involved in the coagulation of latex by bringing together rubber particles. This polyvalent bridging between hevein and rubber particles is mediated by N-acetyl-D-glucosamine and involves a receptor glycoprotein of 22 kDa, which is localized on the surface of the rubber particles. The proposed role of hevein helps us to understand the mechanism of coagulation of latex and assigns a physiological intracellular function to one of the smallest lectins.

Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells.

The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the "sink effect" and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast DeltapH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H(+)-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.

Induction of catharanthine synthesis and stimulation of major indole alkaloids production by Catharanthus roseus cells under non-growth-altering treatment with Pythium vexans extracts.

A Catharanthus roseus cell line was selected that synthesised catharanthine exclusively under elicitation.From the first day of culture, treatment with very low concentrations of a Pythium extract did not alter the growth of the suspension but, within 24 hours, induced the synthesis of catharanthine and stimulated the production of ajmalicine. Kinetic analysis showed that serpentine then began to accumulate and that all of these effects lasted more than 7 days. Elicitation also induced changes in the cell/medium distribution of the alkaloids. Higher, although non-lethal, concentrations of the fungal elicitor were shown to impair alkaloid production. This cell line will serve as a model to study the conditions for the expression of catharanthine synthesis at the molecular level.

Early Activation by Ethylene of the Tonoplast H-Pumping ATPase in the Latex from Hevea brasiliensis.

The treatment of Hevea brasiliensis (rubber tree) bark by chloro-2-ethyl phosphonic acid (ethrel), an ethylene-producing compound, induces a significant increase in the tonoplast H(+)-translocating ATPase activity in the latex during the first 24 hours after the application of the stimulating agent. Moreover, the tonoplast-bound ATPase is highly activated when vacuoles (lutoids) are resuspended in ultrafiltrated cytosol. This effect is amplified during ethrel stimulation. Preliminary assays to characterize the endogenous effector(s) suggest that the activator(s) could be a heat-resistant compound with a low molecular weight, most likely an anion. The activation of the tonoplast-bound ATPase and the associated activation of the protons translocation across the lutoid membrane, could explain the cytosolic alkalinization observed in latex following the ethrel treatment of Hevea bark, which results in an enhanced rubber production.

The tonoplast proton-translocating ATPase of higher plants as a third class of proton-pumps.

Taken together, all the data reported recently in the literature suggest that tonoplast ATPase belongs to a new class of proton pumps. To date, the most studied system is the proton-pumping ATPase from the tonoplast of Hevea latex. Its main characteristics are presented. It resembles the mitochondrial ATPase in its specificity, its substrate affinity, and its sensitivity to different inhibitors. However, for some aspects, it resembles the plasma membrane system in its response to other inhibitors tested (quercetin for example). It differs from both ATPases in its sensitivity to nitrate as well as by its molecular structure, i.e. a complex exhibiting a least 4 or 5 polypeptides. These results favor the existence of a third class of proton pumps, intermediate between the F1F0-class and the E1E2-class.