Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Fatty acid profiles of esterified sterol glucosides from juice vesicles of citrus fruits

Vesicular lipids of 24 citrus cultivars were extracted and purified, and the esterified sterol glucosides (ESG) separated from other vesicular lipids by chromatography. Fatty acids of the ESG were analysed by gas-liquid chromatography as their methyl esters. Statistical analysis showed that five out of six citrus species or biotypes could be differentiated from each other on the basis of profiles of the five major ESG fatty acids. Differences between midand late-season sweet orange cultivers were detected. Small differences were observed between white- and red-fleshed grapefruit cultivars. Profiles of the minor acids showed higher relative percentages of iso-branched fatty acids in lemons and limes, and higher relative percentages of 12: 0 and 14: 0 fatty acids in mandarin and mandarin hybrids than in other cultivars.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

鲜活饵料和人工配合饲料对鳜肌肉营养成分和质构特性的影响

研究以饲喂鲜活饵料为对照组,比较分析人工饲料对鳜(Siniperca chuatsi)常规营养成分、氨基酸组成及营养价值评价、脂肪酸组成和肌肉质构特性的影响。实验结果显示,摄食人工饲料组鳜肌肉蛋白质水平与鲜活饵料组无显著性差异(P>0.05),粗脂肪含量显著较高(P<0.05)。在两种饲喂模式下鳜肌肉中氨基酸评分(AAS)、必需氨基酸指数(EAAI)、化学评分(CS)和F值均无显著差异(P>0.05),但第一限制性氨基酸均为蛋氨酸+半胱氨酸(Met+Cys),该结果可为鳜人工饲料配方优化提供指导。人工饲料组鳜肌肉中饱和脂肪酸(SFA)、单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)含量均极显著高(P<0.01),其中C20﹕5(EPA)和C22﹕6(DHA)含量极显著高于鲜活饵料组,表明人工饲料可通过营养素的均衡配比以提供更优质的脂肪酸营养。人工饲料组鳜肌肉硬度、咀嚼性、胶黏性及回复性均极显著高于鲜活饵料组(P<0.01),而黏性极显著低于鲜活饵料组(P<0.01),表明人工饲料饲喂提升了鳜的肌肉质构特性。综上所述,相比于鲜活饵料组,人工饲料...     

ISSR-PCR technique: a useful method for characterizing new allotetraploid somatic hybrids of mandarin

Somatic hybrids between Redblush and Duncan grapefruit (Citrus paradisi Macfadyen) with Avana, Tardivo di Ciaculli mandarin (C. deliciosa Tenore) and Fortune mandarin (C. reticulata Blanco) were obtained by symmetric protoplast fusion. Isozyme banding pattern analysis and flow cytometry were used for early screening. DNA of the hybrids was then extracted and subjected to inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) using different primers anchored at the 5' and 3' ends. PCR products were resolved on 1.5% agarose gels and stained with ethidium bromide. All the bands from the parental cultivars were present in the somatic hybrids. The usefulness and efficiency of the ISSR-PCR method as a screening technique for Citrus somatic hybrids are discussed.     

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root-tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

Effect of Photoperiod on Isoenzymic Composition of Citrus and Poncirus

Long day photoperiod treatments given to Dancy and Clementine tangerines (Citrus reticulata Blanco.), the Mineola tangelo (C. parodisi Macf. × C. reticulata Blanco.) and the Rubideaux trifoliate (Poncirus trifoliata (L.) Raf.) produced 1.5 to 2.5 times the stem area, total linear growth and number of branches compared to short day treatments. Growing shoot tips were snbjected lo etectrophoretic analyses of tbe isoenzymic composition of esterases, teucine aminopeptidases, peroxidases and amylases. Differences were observed in the number and activity of the isoenzymes between the sbort and the long day treated plants of the three Citrus species aiid Hnbideaux trifoliate orange. The possible significance of tbese differences is discnssed in relation to Ihe growth in response to the photoperiodic treatments.     

Contrasts between Citrus species in response to salinisation: An analysis of photosynthesis and water relations for different rootstock-scion combinations

Leaf gas exchange, water relations and ion content were measured on two-year-old Valencia orange (Citrus sinensis [L.] Osbeck), Washington Navel orange (C. sinensis) and Marsh grapefruit (C. parodisi Macfad) scions budded to either Trifoliata (Poncirus infoliata [L] Raf) or Cleopatra mandarin (C. reticuLua Blanco) rootstoeks. Trees were watered with dülute nutrient solution containing either 0 or 50 _mM NaCl for 77 days. Leaf chloride concentrations (cell sap basis) were higher in all scions budded on “Trifoliata but sodium levels were lower than in equivalent foliage budded on Cleopatra mandarin rootstock. Foliar salt levels also varied according to scion. Leaves of Marsh grapefruit had higher levels of both sodium and chloride than leaves of either Valencia orange or Washington Navel orange on both rootstocks. Accumulation of sodium and chloride in salinised leaves caused a reduction in leaf osmotic potential of 0.2–1.4 MPa. and leaf water potential declined by as much as 0.5 MPa. Turgor pressure in salinised leaves was thus maintained at or above the control level. Osmotic potentials determined by psychrometry compared with pressure-volume curves were taken to imply that some accumulation of sodium or chloride in the apoplast of salinised leaves may have occurred. Despite turgor maintenance both _co2 assimilation and stomatal conductance were reduced by salinity. Following onset of leaf response to salinisation, gas exchange was impaired to a greater extent in scions budded to Cleopatra mandarin compared to those on Trifoliata. Amongst those scions. leaves of salt-treated Marsh grapefruit showed greater reductions in gas exchange than Valencia orange or Washington Navel orange budded on either rootstock. Increased sensitivity of 1Marsh grapefruit was correlated with a higher foliar sodium and chloride content in this scion. Scion differences in sensitivity of leaf gas exchange to solute concentration were independent of rootstock and appeared unrelated to leaf prolinebetaine concentrations. This implies an inherent difference between scion species with respect to salt tolerance, rather than variation in their capacity to acquire that type of compatible solute. In terms of rootstock effects, all scions proved more sensitive to salinity when budded to Cleopatra mandarin compared with Trifoliata. That response was attributed to a disproportionately higher concentration of leaf sodium in scions on Cleopatra mandarin.     

Fatty acids of triglycerides from Citrus juice sacs

The fatty acid composition of trigylcerides from oranges, grapefruit, lemons and limes, was determined by GLC. Each species possessed its own intrinsic fatty acid pattern which might be used to differentiate it from the other species. The five major acids in all species were palmitic, palmitoleic, oleic, linoleic and linolenic. Collectively these acids comprised greater than 92% of the total acid content. Lemons were distinguished from all other species by their higher 16/16:1 ratios while grapefruit showed the highest total percentage of 16 and 16:1 acids. Lemons and limes contained higher percentages of branched-chain acids than oranges and grapefruit.     

Fatty acid composition of sterol esters from Citrus sinensis,C. paradisi,C. limon aurantifolia and C. limettioides sacs

The fatty acid compositions of sterol esters from 4 citrus species, viz, orange. grapefruit, lemon and lime, were determined by GLC. Each species possessed its own intrinsic fatty acid pattern which could be used to differentiate it from the other species. In most cases varieties within a species had fatty acid patterns which could be used for varietal differentiation. In all citrus tested except Columbia lime, the major acid was linoleic acid; this acid varied from 10 to 56% of the total acid content. The ratios of 16/16:1 were distinct for each citrus species. The C₂₂-C₂₉ fatty acids were prevalent in citrus sterol esters ranging from 6·5% for some orange and grapefruit varieties to over 41% for two lime varieties. In all varieties C₂₄ was the most prominent of these longer chain fatty acids. Argentation TLC indicated that these longer chain fatty acids primarily were esterified to dimethyl sterols. ft*|One of the laboratories of the Southern Region, Agricultural Research Service, U.S. Department of Agriculture.     

Cellular distribution and linkage of D-(-)-3-hydroxy fatty acids in Bacteroides species.

Two strains of Bacteroides asaccharolyticus and two strains of Bacteroides fragilis were analyzed for total fatty acid, total lipid fatty acid, and total bound fatty acid profiles. Extracted lipids and defatted cell residues were subjected to sequential alkaline and acid methanolyses to distinguish ester- and amide-linked fatty acids in each fraction. In the lipid fractions, all the ester-linked fatty acids were nonhydroxylated, whereas all of the amide-linked fatty acids were hydroxylated. In the nonextractable fractions, both hydroxy and nonhydroxy fatty acids were found in both ester and amide linkage, although hydroxy acids predominated. The fatty acid profiles of the bound fractions differed widely from those of the lipid fractions. Bound fatty acid represented approximately 10% of the total cellular fatty acids.     

Delay expression of limonoid UDP-glucosyltransferase makes delayed bitterness in citrus

Genes encoding limonoid UDP-glucosyltransferase from albedo of six Citrus species with different levels of delayed bitterness are isolated and cloned in vector pTZ57R/T. Our results indicate that gene sequence of sweet lime (with intense juice delayed bitterness) have complete identity with Satsuma mandarin (without distinctive juice delayed bitterness). Also gene sequence of Marsh seedless grapefruit, local orange and Thompson navel orange (with mild juice delayed bitterness) have very similarity with Satsuma mandarin. On the other hand, this gene started to express 60, 120, and 210 days after full blooming in albedo of Satsuma mandarin, sweet oranges and sour orange, and both grapefruit and sweet lime, respectively. Expression pattern of limonoid glucosyltransferase gene in leaves was quite different with albedo. Thus, we supposed the delayed bitterness in this species was related to delay in expression of limonoid glucosyltransferase gene in albedo and lower limonoid glucoside accumulation in fruits.     

Differentiation of Gram-Negative, Nonfermentative Bacteria Isolated from Biofilters on the Basis of Fatty Acid Composition, Quinone System, and Physiological Reaction Profiles

Gram-negative, nonfermentative bacteria isolated from biofilters for off-gas treatment of animal-rendering-plant emissions were differentiated by whole-cell fatty acid analysis, quinone analysis, and numerical taxonomy based on their physiological reaction profiles. The last system consisted of 60 physiological tests and was arranged as a microtest system on microtitration plates. Based on fatty acid analyses, 31 isolates were separated into six clusters and five single-member clusters. The isolates of two clusters were identified as Alcaligenes faecalis and Pseudomonas diminuta. The remaining nine clusters were characterized by their fatty acid profiles, quinone systems, and physiological reaction profiles. Clusters resulting from fatty acid analyses were compared with those resulting from physiological reaction profiles. Six clusters could be confirmed this way. The efficiency of the physiological test system was increased by the prearrangement of the isolates according to their quinone type.     

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.     

Experimental study on the effect of diet on fatty acid and stable isotope profiles of the squid Lolliguncula brevis

Fatty acid and stable isotope analyses have previously been used to investigate foraging patterns of fish, birds, marine mammals and most recently cephalopod species. To evaluate the application of these methods for dietary studies in squid, it is important to understand the degree to which fatty acid and stable isotope signatures of prey species are reflected in the squids' tissue. Four groups of Lolliguncula brevis were fed on prey species with distinctly different fatty acid and stable isotope profiles over 30 consecutive days. One group of squid were fed fish for fifteen days, followed by crustaceans for a further fifteen days. A second and third group were fed exclusively on fish or crustaceans for thirty days. And a fourth group was fed on a mixture of fish and crustaceans for thirty days. Analysis of squid tissue showed that, after 10 days of feeding, fatty acid profiles of squid tended to reflect those of their prey. Squid that fed on a single prey type, i.e. fish or crustacean, showed only minor modifications in fatty acid proportions after the initial change and fatty acid profiles were clearly distinguishable between the two feeding groups. Shifts in fatty acid proportions towards respective prey profiles could clearly be observed in squid the diet of which was swapped after 15 days. Clear differences could also be seen in fatty acid profiles of squid feeding on a mixed diet with trends towards either fish or crustacean fatty acid signatures. Stable isotope signatures of squid tissues clearly distinguished between animals feeding on different diets and supported findings from fatty acid analysis, thus indicating both methods to be viable tools in feeding studies on squid species.     

Formation of Adventitious Buds in Decapitated Citrus Seedlings and the Effect of Some Growth Regulators

Young seedlings of various citrus species give rise, after decapitation,to adventitious buds at the cut end of the epicotyl. With suchtreatment, more buds formed on seedlings of sweet orange, sourorange, and grapefruit than of lemon, mandarin, and calamondin.Anatomical observations at the cut stem end show formation ofbuds occurring from tissues close to the cambium. Growth regulatorsapplied at the cut epicotyl end modified the patterns of budformation. 6-Benzylaminopurine (BAP) promoted bud formation;indole-3-butyric acid (IBA), abscisic acid (ABA), and gibberellicacid (GA₃) inhibited it. BAP reversed the inhibitory effectof GA₃. Gibberellic acid caused a typical elongation of thesprout axis. The use of decapitated seedlings as a tool forstudying stem differentiation in citrus is suggested.     

Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis

Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids.