Natural variations of TFIIAγ gene and LOB1 promoter contribute to citrus canker disease resistance in Atalantia buxifolia.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease.

Mathematical model studies of the comprehensive generation of major and minor phyllotactic patterns in plants with a predominant focus on orixate phyllotaxis.

Plant leaves are arranged around the stem in a beautiful geometry that is called phyllotaxis. In the majority of plants, phyllotaxis exhibits a distichous, Fibonacci spiral, decussate, or tricussate pattern. To explain the regularity and limited variety of phyllotactic patterns, many theoretical models have been proposed, mostly based on the notion that a repulsive interaction between leaf primordia determines the position of primordium initiation. Among them, particularly notable are the two models of Douady and Couder (alternate-specific form, DC1; more generalized form, DC2), the key assumptions of which are that each leaf primordium emits a constant power that inhibits new primordium formation and that this inhibitory effect decreases with distance. It was previously demonstrated by computer simulations that any major type of phyllotaxis can occur as a self-organizing stable pattern in the framework of DC models. However, several phyllotactic types remain unaddressed. An interesting example is orixate phyllotaxis, which has a tetrastichous alternate pattern with periodic repetition of a sequence of different divergence angles: 180°, 90°, -180°, and -90°. Although the term orixate phyllotaxis was derived from Orixa japonica, this type is observed in several distant taxa, suggesting that it may reflect some aspects of a common mechanism of phyllotactic patterning. Here we examined DC models regarding the ability to produce orixate phyllotaxis and found that model expansion via the introduction of primordial age-dependent changes of the inhibitory power is absolutely necessary for the establishment of orixate phyllotaxis. The orixate patterns generated by the expanded version of DC2 (EDC2) were shown to share morphological details with real orixate phyllotaxis. Furthermore, the simulation results obtained using EDC2 fitted better the natural distribution of phyllotactic patterns than did those obtained using the previous models. Our findings imply that changing the inhibitory power is generally an important component of the phyllotactic patterning mechanism.

Alkaloid diversity in the leaves of Australian Flindersia (Rutaceae) species driven by adaptation to aridity.

The genus Flindersia (Rutaceae) comprises 17 species of mostly Australian endemic trees. Although most species are restricted to rainforests, four have evolved to grow in semi-arid and arid environments. In this study, the leaf alkaloid diversity of rainforest and semi-arid/arid zone adapted Australian Flindersia were compared by LC/MS-MS and NMR spectroscopy. Contrary to expectations, Flindersia alkaloid diversity was strongly correlated with environmental aridity, where species predominating in drier regions produced more alkaloids than their wet rainforest congenerics. Rainforest species were also more chemically similar to each other than were the four semi-arid/arid zone species. There was a significant relationship between the presence of alkaloid structural classes and phylogenetic distance, suggesting that alkaloid profiles are influenced by both genetic and environmental factors. The results suggest that the radiation of Flindersia species out of the rainforest and into drier environments has promoted the evolution of unique alkaloid diversity. Plants growing in arid and semi-arid regions of Australia may represent an untapped source of undescribed specialised metabolites.

Structure and functioning of oil cavities in the shoot apex of Metrodorea nigra A. St.-Hil. (Rutaceae).

This study investigates the histology and subcellular features of secretory cavities during the development of the shoot apex of Metrodorea nigra A. St.-Hil. in order to better understand the functioning of these glands. This Rutaceae species is a very suitable model for studying secretory cavity life span, since the shoot apex exhibits both dormant and growth stages during its annual cycle. Shoot apices were collected during the dormant and growth stages from populations of M. nigra growing under natural conditions. Materials were processed using standard techniques for light and electron microscopy. The secretory cavities originate under the protodermis, and their initiation is restricted to the early developmental stage of shoot organs, which are protected by a hood-shaped structure. Secretory cavities have a multi-seriate epithelium surrounding a lumen that expands schizolysigenously. Oil production begins before lumen formation. When the shoot apex resumes development after the dormant stage, the glands remain active in oil secretion in the developing shoot apex and fully expanded leaves. The mature epithelial cells are flattened and exhibit very thin walls, large oil bodies, leucoplasts surrounded by endoplasmic reticulum, and mitochondria with unusual morphology. The tangential walls of the epithelial cells facing the lumen undergo continuous peeling. The vacuole extrusion appears to be the primary mode of release oil into the lumen, in an exocytotic way. The continuity of oil secretion is ensured by the replacement of the damaged inner epithelial cells by divisions in the parenchyma layer that surround the oil gland, likely a meristematic sheath.

SEED DESICCATION TOLERANCE AND CRYOPRESERVATION OF PHILIPPINE CALAMANSI [x Citrofortunella microcarpa (BUNGE) WIJNANDS].

BACKGROUND: The traditional on farm conservation of Calamansi [x Citrofortunella microcarpa (Bunge) Wijnands], an important indigenous Citrus species in the Philippines, is now being threatened by shifting agricultural crop production, climate change, and increasing biotic and abiotic stresses. OBJECTIVE: The study aimed to characterize the desiccation and cryopreservation tolerance of seeds as the basis for complementary long term ex situ conservation. MATERIALS AND METHODS: Intact seeds were desiccated in an airtight container filled with activated silica gel for 0-96 h. Seeds placed in cryotubes were subjected to rapid freezing in liquid nitrogen, rapid thawing in a water bath at 50 degree C for 3 min, and cultured on MS basal medium for seedling recovery and growth. Recovered seedlings were potted out in plastic bags filled with coir dust: garden soil mixture (1:1 v/v) and maintained in the nursery. RESULTS: Significant reduction in percentage germination was obtained at in a moisture content (MC) window between 24.3% and 4.2% and complete loss of viability at below 3.2%. The number of germinated embryos per seed was significantly reduced following desiccation from a mean of 4.2 embryos per seed for the untreated control to 1.2 to 1.02 embryos per seed at 33.3-4.2% MC, respectively. Recovery and germination of seeds after cryopreservation were obtained in a MC window between 24.3% and 4.2% with the maximum seed germination (27%) obtained at 13.4%. Germination abnormalities such as incomplete germination, greening and or enlargement of cotyledon without shoot emergence were observed in both desiccated and cryopreserved seeds. Variations in response to seed desiccation and cryopreservation were observed among Calamansi accessions tested. Maximum seedling recovery after liquid nitrogen storage varied between 12.5% and 61.5%. Recovered seedlings from desiccation and cryopreservation treatments survived ex vitro establishment and showed normal growth and similar morphology with the non-treated control seedlings. CONCLUSION: The partial tolerance of Calamansi seeds to desiccation and subsequent recovery after cryopreservation provides the basis for long term ex situ preservation of this valuable germplasm, although further optimization is needed.

Fragmentation effects and genetic diversity of the key semidecidual forest species Metrodorea nigra in Southwestern Brazil.

Studies of genetic diversity in plant species present in the remaining fragments of the Atlantic Forest are very important for understanding their resilience to such a degraded ecosystem. We analyzed the genetic diversity of 3 populations of the high-density understory species Metrodorea nigra St. Hill. (Rutaceae) located in forest remnants in the region of Ribeirão Preto, SP, Brazil (M13-Rib, BSQ-Rib, and FAC-Crav), by using simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) molecular markers for conservation purposes. A total of 133 polymorphic loci were observed in 136 inter-simple sequence repeat loci (average of 17 per primer). The Nei genetic diversity (HE) was relatively high considering all populations (0.31). The BSQ-Rib population exhibited the highest value (0.27), followed by the M13-Rib (0.26) and FAC-Crav (0.24) populations. The simple sequence repeat markers analyzed showed a high number of alleles (K = 104), with an average of 14.85 alleles per locus. The average observed heterozygosity was 0.516 and the average expected heterozygosity was 0.771, ranging from 0.688 (FAC-Crav) to 0.765 (BSQ-Rib). The fixation indexes showed positive and significant differences from zero for all sample sets, indicating inbreeding, which may have resulted from the species' mating patterns and the barochoric seed dispersal system of M. nigra. Both markers indicated differentiation among populations, with higher values observed for inter-simple sequence repeat markers. No significant differences between juvenile and adult generations in any of the fragments were observed, indicating the resilience of M. nigra to the effects of fragmentation and reduced habitat.

Fusion within and between whorls of floral organs in Galipeinae (Rutaceae): structural features and evolutionary implications.

BACKGROUND AND AIMS: Most genera of the neotropical Galipeinae (tribe Galipeeae, Rutoideae) exhibit several forms and degrees of fusion between the floral organs, including the union of petals into an apparently sympetalous corolla, the joining of the stamens among themselves and to the corolla, and the partial to complete connation of carpels. Though these and others floral traits are currently used in the circumscription of species in Galipeinae, few studies have shown in detail in which way (postgenital or congenital) and to what extent these fusions occur. To elucidate these anatomical conditions, a structural study of the flowers of the Galipeinae species was carried out. METHODS: Flowers of six species from three genera of Galipeinae were studied in their morphology, anatomy and development with stereomicroscopy, light microscopy and scanning electron microscopy (SEM). KEY RESULTS: The floral tube is formed by synorganization of stamens with petals in all species, and exhibits three main patterns: (1) Conchocarpus heterophyllus and C. minutiflorus have a floral tube formed by marginal coherence/adherence of petals and filaments due to interwining trichomes (postgenital connection); (2) Erythrochiton brasiliensis has a tube formed by congenital fusion of petals and filaments; and (3) Galipea jasminiflora and Conchocarpus macrophyllus have a tube formed distally with the first pattern, and proximally with the second pattern. Although floral tubes seem to be homologous within Galipeinae, this is not true at the level of the family: the floral tube of Correa (from an only distantly related clade of the family) is formed by postgenital union of the petals representing a convergent structure. The gynoecium of the studied species of Galipeinae shows a great variability in the extent of fusion of carpel flanks. Even though different structures for the mature gynoecium were found in each genus, all genera show postgenitally fused carpel apices, which is related to the formation of a compitum, as described earlier for other members of Rutaceae. CONCLUSIONS: The degree and diversity of fusions of floral organs in Galipeinae is unique within the order Sapindales. A study of the amount of diversification of Galipeinae in South America and comparison with other clades of Rutaceae would be of interest.

Long-term demographic consequences of a seed dispersal disruption.

The loss or decline of vertebrate frugivores can limit the regeneration of plants that depend on them. However, empirical evidence is showing that this is still very scarce, as functionally equivalent species may contribute to maintain the mutualistic interaction. Here, we investigated the long-term consequences of the extinction of frugivorous lizards on the population persistence of a Mediterranean relict shrub Cneorum tricoccon (Cneoraceae). We examined the demographic parameters among 26 insular and mainland populations, which encompass the entire plant distributional range, comparing populations with lizards with those in which these are extinct, but in which alien mammals currently act as seed dispersers. Plant recruitment was found to be higher on island populations with lizards than on those with mammals, and the long-term effects of the native disperser's loss were found in all vital phases of plant regeneration. The study thus gives evidence of the cascading effects of human-induced changes in ecosystems, showing how the disruption of native ecological processes can lead to species regression and, in the long term, even to local extinctions.

Floral ontogeny of Ruteae (Rutaceae) and its systematic implications.

Floral development was investigated in Ruta graveolens and Psilopeganum sinense, representing two genera in the tribe Ruteae. Special attention was paid to the sequence of initiation of organ whorls in the androecium and gynoecium. The antepetalous stamens arise at the same level as the antesepalous stamens in both species. The carpels are antepetalous in both taxa, indicating the androecium in both genera is obdiplostemonous. Compared with floral ontogeny of the ancestral genus Phellodendron (Toddalioideae), the obdiplostemonous androecium is a derived condition. The floral apex in P. sinense is quadrangular before initiation of the two carpels. Additionally, there are four dorsal and four ventral traces in the ovary. Integrated morphological and anatomical evidence indicates that the bicarpellate gynoecium in Psilopeganum most likely evolved from a tetracarpellate ancestor. Considering the similarities in morphological, geographical and chromosomal features, the ancestor may be Ruta-like. Further molecular phylogenetic and genetic studies are needed to verify this assumption.

Resprouting from roots in four Brazilian tree species.

Previous studies pointed out that species richness and high density values within the Leguminosae in Brazilian forest fragments affected by fire could be due, at least partially, to the high incidence of root sprouting in this family. However, there are few studies of the factors that induce root sprouting in woody plants after disturbance. We investigated the bud formation on root cuttings, and considered a man-made disturbance that isolates the root from the shoot apical dominance of three Leguminosae (Bauhinia forficata Link., Centrolobium tomentosum Guill. ex Benth, and Inga laurina (Sw.) Willd) and one Rutaceae (Esenbeckia febrifuga (St. Hil.) Juss. ex Mart.). All these species resprout frequently after fire. We also attempted to induce bud formation on root systems by removing the main trunk, girdling or sectioning the shallow lateral roots from forest tree species Esenbeckia febrifuga and Hymenaea courbaril L. We identified the origin of shoot primordia and their early development by fixing the samples in Karnovsky solution, dehydrating in ethyl alcohol series and embedding in plastic resin. Serial sections were cut on a rotary microtome and stained with toluidine blue O. Permanent slides were mounted in synthetic resin. We observed different modes of bud origin on root cuttings: close to the vascular cambium (C. tomentosum), from the callus (B. forficata and E. febrifuga) and from the phloematic parenchyma proliferation (I. laurina). Fragments of B. forficata root bark were also capable of forming reparative buds from healing phellogen formed in callus in the bark's inner side. In the attempt of bud induction on root systems, Hymenaea courbaril did not respond to any of the induction tests, probably because of plant age. However, Esenbeckia febrifuga roots formed suckers when the main trunk was removed or their roots were sectioned and isolated from the original plant. We experimentally demonstrated the ability of four tree species to resprout from roots after disturbance. Our results suggest that the release of apical dominance enables root resprouting in the studied species.

Resprouting from roots in four Brazilian tree species

Previous studies pointed out that species richness and high density values within the Leguminosae in Brazilian forest fragments affected by fire could be due, at least partially, to the high incidence of root sprouting in this family. However, there are few studies of the factors that induce root sprouting in woody plants after disturbance. We investigated the bud formation on root cuttings, and considered a man-made disturbance that isolates the root from the shoot apical dominance of three Leguminosae (Bauhinia forficata Link., Centrolobium tomentosum Guill. ex Benth, and Inga laurina (Sw.) Willd) and one Rutaceae (Esenbeckia febrifuga (St. Hil.) Juss. ex Mart.). All these species resprout frequently after fire. We also attempted to induce bud formation on root systems by removing the main trunk, girdling or sectioning the shallow lateral roots from forest tree species Esenbeckia febrifuga and Hymenaea courbaril L. We identified the origin of shoot primordia and their early development by fixing the samples in Karnovsky solution, dehydrating in ethyl alcohol series and embedding in plastic resin. Serial sections were cut on a rotary microtome and stained with toluidine blue O. Permanent slides were mounted in synthetic resin. We observed different modes of bud origin on root cuttings: close to the vascular cambium (C. tomentosum), from the callus (B. forficata and E. febrifuga) and from the phloematic parenchyma proliferation (I. laurina). Fragments of B. forficata root bark were also capable of forming reparative buds from healing phellogen formed in callus in the bark’s inner side. In the attempt of bud induction on root systems, Hymenaea courbaril did not respond to any of the induction tests, probably because of plant age. However, Esenbeckia febrifuga roots formed suckers when the main trunk was removed or their roots were sectioned and isolated from the original plant. We experimentally demonstrated the ability of four tree species to resprout from roots after disturbance. Our results suggest that the release of apical dominance enables root resprouting in the studied species. Rev. Biol. Trop. 57 (3): 789-800. Epub 2009 September 30.

Estudios anteriores señalaron que la alta riqueza de especies de Leguminosae en fragmentos de bosques afectadas por el fuego podría deberse, al menos parcialmente, a la alta incidencia de brotes de raíz en esta familia. Sin embargo, existen pocos estudios. Investigamos la formación de brotes en tres Leguminosae (Bauhinia forficata Link., Centrolobium tomentosum Guill. ex Benth, e Inga laurina (Sw.) Willd) y una Rutaceae (Esenbeckia febrifuga (St. Hil.) Juss. ex Mart.). Todas estas especies suelen rebrotar después de los incendios. También hicimos cortes experimentales en árboles forestales: Esenbeckia febrifuga e Hymenaea courbaril L. Observamos tres orígenes: cerca del cambium vascular (C. tomentosum), desde el callo (B. forficata y E. febrifuga) y de parénquima (I. laurina). Hymenaea courbaril no respondió a ninguna de las pruebas de inducción, probablemente debido a la edad de la planta. Sin embargo, las raíces de Esenbeckia febrifuga forman retoños tras retirar el tronco o separar sus raíces. Hemos demostrado experimentalmente la capacidad de las cuatro especies de árbol de rebrotar a partir de sus raíces tras una perturbación grave.

Sphingobium rhizovicinum sp. nov., isolated from rhizosphere soil of Fortunella hindsii (Champ. ex Benth.) Swingle.

The taxonomic status of a pale-yellow-coloured bacterial isolate from rhizosphere soil of Fortunella hindsii (Champ. ex Benth.) Swingle was characterized using a polyphasic taxonomic approach. Comparative analysis of the 16S rRNA gene sequence showed that the isolate constituted a distinct branch within the genus Sphingobium. The generic assignment was confirmed by chemotaxonomic data, which revealed the presence of a fatty acid profile that was characteristic for the genus Sphingobium, consisting of straight-chain saturated and unsaturated as well as 2-OH fatty acids and the lack of 3-OH fatty acids, ubiquinone with ten isoprene units (Q-10) as the predominant respiratory quinone, and a polar lipid pattern that consisted of the predominant compounds phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, diphosphatidylglycerol, sphingoglycolipid and an unknown glycolipid. Spermidine was the major polyamine component. The genotypic and phenotypic data (physiology and fatty acid and polar lipid profiles) showed that the isolate merits classification as representing a novel species of the genus Sphingobium, for which the name Sphingobium rhizovicinum sp. nov. is proposed. The type strain is CC-FH12-1T (=CCM 7491(T)=BCRC 17770T) [corrected]

Acridone and furoquinoline alkaloids from Teclea gerrardii (Rutaceae: Toddalioideae) of southern Africa.

The combined hexane/CH(2)Cl(2) extract of the stem bark of Teclea gerrardii (Rutaceae: Toddalioideae) has yielded two acridone alkaloids, 3-hydroxy-1-methoxy-N-methylacridone (tegerrardin A) (1) and 3-hydroxy-N-methyl-1-(gamma,gamma-dimethylallyloxy)acridone (tegerrardin B) (2), three known acridones (3-5), two known furoquinolines (6,7), and the acridone precursor tecleanone (8). Arborinine (3) and evoxine (6) displayed moderate antiplasmodial activity against the CQS D10 strain of Plasmodium falciparum, with IC(50) values of 12.3 and 24.5 microM, respectively.

Allelochemicals from Stauranthus perforatus, a Rutaceous tree of the Yucatan Peninsula, Mexico.

Aqueous leachates and a CHCl3-MeOH (1:1) extract of roots of Stauranthus perforatus showed a significant phytotoxic effect on Amaranthus hypochondriacus and Echinochloa crus-galli. Bioassay-directed fractionation of the active organic extract led to the isolation and characterization of ten secondary metabolites, which included two pyranocoumarins [xanthyletin (1) and 3-(1',1'-dimethylallyl)-xanthyletin (2)], four furanocoumarins [chalepensin (3), ammirin (4), chalepin (5) and 2'-isopropyl-psoralene (6)], two lignans [asarinin (7) and fargesin (8)], one sesquiterpene [4,5-epoxi-beta-caryophyllene (9)], and one alkamide [pellitorine (10)]. From these compounds, 2'-isopropyl-psoralene (6) or anhydromarmesin, is reported for the first time as a natural product, whereas compounds 4-10 are now reported as being present in S. perforatus. Metabolites 1, 3-5 and 10 caused significant inhibition of radicle growth of A. hypochondriacus and E. crus-galli. Furthermore, in a greenhouse experiment the decomposition of the leaves and roots in the soil had a significant inhibitory effect on the growth of weeds. The allelopathic action of the decomposition of roots was evident up to the sixth week of the experiment. The effect of leaves was comparable to that of DPCA (dimethyl tetrachloroterephthalate), a commercial herbicide. Finally different concentrations of Stauranthus root powder were combined with maize kernels and used to feed corn weevil. The treatments resulted in high mortality of this insect.

The dissipation of tebuconazole and propiconazole in boronia (Boronia megastigma Nees).

The broad spectrum, systemic fungicides tebuconazole and propiconazole are used to control rust in boronia (Boronia megastigma Nees). Gas chromatography combined with either a benchtop quadrupole mass spectrometer or a high-resolution mass spectrometer allowed for the monitoring of both pesticides in boronia leaves, flowers, and concrete. Field trials were established at two sites to determine the rate of dissipation of tebuconazole and propiconazole in boronia. At site 1, two application rates of 125 and 250 g active ingredient/hectare (ai/ha) tebuconazole were employed. Treatments were repeated 17 days later. At harvest, 286 days after the final application, tebuconazole was detected at levels of 0.06 +/- 0.05 and 0.5 +/- 0.1 [mg/kg +/- standard error, on a dry matter basis (DMB)] in the leaves collected from plots treated with 125 and 250 g ai/ha of tebuconazole, respectively. The oil produced from the flowers collected at the final harvest had residues of tebuconazole at levels of 0.06 +/- 0.03 and 0.10 +/- 0.08 mg/kg for the 125 and 250 g ai/ha application rates, respectively. Two repeat applications of 125 g ai/ha propiconazole were also used at site 1. Residues of propiconazole were detected at 0.09 +/- 0.03 mg/kg (DMB) 286 days after the final application. At site 2, treatments of 125 g ai/ha of tebuconazole were applied twice. At harvest, 279 days after the final application of tebuconazole, residues were recorded at 0.30 +/- 0.09 mg/kg in the leaves (DMB) while the oil produced had 0.20 +/- 0.07 mg/kg.