Identifying citrus limonoid aglycones by HPLC-EI/MS and HPLC-APCI/MS techniques

HPLC coupled with normal phase electron ionisation (EI) and atmospheric pressure chemical ionisation (APCI)/ mass spectrometry methods has been applied to identify 17 known neutral limonoid aglycones from Citrus sources. The HPLC-MS data from the known limonoids provided chromatographic characteristics, APCI-derived molecular weight data and EI fragmentation data for each limonoid. EI fragmentation patterns for the limonoids were correlated with structural characteristics. The EI fragmentation patterns coupled with APCI-derived molecular weights were utilised as a potential method by which to discern the structural character of unknown citrus limonoids.     

Heterochromatin banding patterns in Rutaceae-Aurantioideae--a case of parallel chromosomal evolution

The heterochromatin banding patterns in the karyotypes of 17 species belonging to 15 genera of Rutaceae subfamily Aurantioideae (= Citroideae) were analyzed with the fluorochromes chromomycin (CMA) and 4'-6-diamidino-2-phenylindole-2HCl (DAPI). All species were diploids, except one tetraploid (Clausena excavata) and two hexaploids [Glycosmis parviflora agg. (aggregate) and G. pentaphylla agg.]. There are only CMA/DAPI bands, including those associated with the nucleolus. Using recent cpDNA (chloroplast DNA) sequence data as a phylogenetic background, it becomes evident that generally more basal genera with rather plesiomorphic traits in their morphology, anatomy, and phytochemistry exhibit very small amounts of heterochromatin (e.g., Glycosmis, Severinia, Swinglea), whereas relatively advanced genera from different clades with more apomorphic characters display numerous large CMA bands (e.g., Merrillia, Feroniella, Fortunella). Heterochromatin increase (from 0.7 to 13.7%) is interpreted as apomorphic. The bands are mostly located in the larger chromosomes and at telomeric regions of larger arms. However, one of the largest chromosome pair has been conserved throughout the subfamily with only very little heterochromatin. The heterochromatin-rich patterns observed in different clades of Aurantioideae appear quite similar, suggesting a kind of parallel chromosomal evolution. In respect to the current classification of the subfamily, it is proposed to divide Murraya s.l. (sensu lato) into Bergera and Murraya s.s. (sensu stricto) and to place the former near Clausena into Clauseneae s.s. and the latter together with Merrillia into Citreae s.l. The subtribes recognized within Clauseneae s.s. and Citreae s.l. appear heterogeneous and should be abandoned. On the other hand, the monophyletic nature of the core group of Citrinae, i.e., the Citrus clade with Eremocitrus, Microcitrus, Clymenia, Poncirus, Fortunella, and Citrus, is well supported.     

Extracellular proteins and early embryo development in Citrus nucellar cell cultures

Highly efficient synchronous embryogenesis was induced in suspension cultures of sour orange ( Citrus aurantium L.) by a change in the carbon source of the growth medium from sucrose to glycerol. In liquid culture the embryos developed into globular structures during a three week period. Embryo development showed an absolute requirement for the continued presence of glycerol. The embryo cell cultures turned green in the light, but light did not affect the course of development. The profiles of soluble cellular protein extracts of embryo and proembryogenic (PEM) cells were very similar as judged by two-dimensional polyacrylamide gel electrophoresis. However, major differences were detected in the profiles of extracellular proteins. PEM cells accumulated extracellular glycoproteins of 53–57 kDa mass. Upon subculture in glycerol containing medium, the accumulation of these proteins ceased within two days. Developing embryos accumulated at least 4 new extracellular polypeptides of 41–42 kDa mass. In addition to these polypeptides, stage specific peroxidases and proteases were found. The relatively extended duration and synchrony in which these early developmental events take place make Citrus cultures an especially useful system for the study of early events in plant embryogenesis.     

Evaluation of Aspergillus aculeatus GC-09 for the biological control of citrus blue mold caused by Penicillium italicum

Blue mold caused by Penicillium italicum is a severe postharvest disease in citrus fruits. In this study, the fermentation product (FP-E) of Aspergillus aculeatus GC-09, an endophytic fungus isolated from a citrus plant, was found to exhibit antifungal activity against P. italicum with a MIC of 0.3125 mg/mL. The fungus A. aculeatus GC-09 was identified based on the studies of morphology and ITS nucleotide sequence. FP-E significantly inhibited the spore germination and mycelial growth of P. italicum. Scanning electron microscopy (SEM) results of P. italicum treated with FP-E showed shrunken, distorted and collapsed hyphae and conidiospores, indicative of the cell membrane damage, which was further confirmed by the propidium iodide (PI) fluorescent staining analysis. Consistent with the microscopy observation, FP-E led to the leakage of cellular constituents from P. italicum, which is evident from the increase in electrical conductivity and nucleic acid contents in the mycelial solution incubated with FP-E. In addition, FP-E treatment considerably increased the intracellular reactive oxygen species (ROS) content, and reduced the enzyme activities of both catalase (CAT) and peroxidase (POD) in P. italicum cells. Furthermore, orange fruits treated with FP-E showed fewer disease symptoms compared to the untreated fruits. These results suggested that the antifungal activity of FP-E might be associated with the disruption of cell membrane integrity, the accumulation of ROS level, and the reduction of the antioxidant enzymes activity of P. italicum. Therefore, A. aculeatus GC-09 might be a potential microbial resource for the biocontrol of citrus postharvest blue mold.     

Spatial dispersion and sampling of citrus whitefly,Dialeurodes citri,for control decisions in a citrus orchard

1 Monitoring the citrus whitefly Dialeurodes citri (Ashmead) in citrus orchards in Israel is necessary for control decisions in IPM in the period before the establishment of natural enemies. The main damage caused by D. citri is due to sooty mould, which colonizes the sweet secretions of this pest. 2 Using Taylor’s Power Law to describe the variance–mean relationship, the dispersion of the citrus whitefly was found to be aggregated at all developmental stages. The action threshold was estimated using regression of sooty mould level on egg or larval counts. 3 Two-stage sampling plans were constructed in different pest generations, for eggs and larval stages, on the basis of the variance between leaves on a tree and the variance between trees in a plot. 4 Fixed sample size plans were prepared, with possible numbers of trees and the numbers of leaves per tree to be sampled, in order to attain a given precision level. 5 Sequential sampling plans were also drawn up, on the basis of four leaves per tree, where trees are sampled until a decision can be reached. The maximum number of trees to be sampled was set according to the corresponding fixed sample size plan. 6 Simulation of the sequential sampling plans showed them to be highly reliable in terms of the decision reached with economy in sample size for high and low infestation levels.     

Reclassification of citrus pseudo greasy spot causal yeasts, and a proposal of two new species, Sporobolomyces productus sp. nov. and S. corallinus sp. nov.

Three basidiomycetous yeast strains, No. 3 (=MAFF 654001), No. 4 (=MAFF 654002), and No. 6 (=MAFF 654003), isolated from diseased leaves of Citrus unshiu and formerly identified as Sporobolomyces roseus, were reported to cause citrus pseudo greasy spot (PGS). In the course of investigating the causal agent of citrus PGS, we confirmed, using conventional and molecular phylogenetic methods, that these yeasts caused citrus PGS but did not belong to S. roseus. No. 3 (=MAFF 654001) and No. 4 (=MAFF 654002) were closely related to S. subbrunneus, and No. 6 (=MAFF 654003) to S. dimmenae, but each of them was distinctive from these species. We have proposed two new species, S. productus and S. corallinus, as the causal pathogens of citrus PGS.     

Ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in the presence of orange-peel oil

Summary Two ethanologenic yeasts, Saccharomyces cerevisiae and Kluyveromyces marxianus, were used to ferment sugar solutions modeling hydrolyzed Valencia orange peel waste at 37°C. Orange stripper oil produced from orange peel was added in various amounts to determine its effect on ethanol production. The minimum peel oil concentration that inhibited ethanol production was determined after 24, 48 and 72 h and the two yeasts were compared to one another in terms of ethanol yield. Minimum inhibitory peel oil concentrations for ethanol production were 0.05% at 24 h, 0.10% at 48 h, and 0.15% at 72 h for both yeasts. S. cerevisiae produced more ethanol than K. marxianus at each time point.     

柑桔叶点霉侵染琯溪蜜柚的超微结构观察及致病机理分析

为探明黑斑病对琯溪蜜柚(Citrus grandis‘Guanximiyou’)果皮细胞的侵染破坏特点及作用机理,人工接种柑橘叶点霉亚洲种(Phyllosticta citriasiana)后,在扫描电镜与透射电镜下观察蜜柚真皮细胞的变化。结果表明,受到侵染后,真皮细胞的液泡膜首先发生褶皱、断裂,最终消失;叶绿体的淀粉粒消失、膜出现褶皱;质膜内陷、局部断裂;类囊体发生膨胀并解体;线粒体数量明显增加,基粒清晰可见,之后线粒体形态改变,双层膜消失,内部空胞化。未观察到细胞间隙及细胞内有菌丝分布,且细胞壁结构始终稳定。P.citriasiana危害蜜柚果皮细胞与毒素作用有关,侵染位点可能是细胞膜,信息通过胞间连丝穿过细胞壁,进入细胞内部。     

长白山木贼山柰素含量对反射光谱参数的响应

对长白山地区木贼山柰素含量和反射光谱参数的关系进行了研究,探索了判别木贼药材质量优劣的快捷方法。根据反射光谱曲线（Unispec-Sc光谱分析仪）导出的若干参数,对木贼样品进行聚类分析,采用HPLC法分别测定木贼山柰素的含量,分析了反射光谱参数与山柰素含量的相关性。结果表明：蓝边面积（SDb）、绿峰反射率（Rg）等参数与山柰素含量呈显著负相关（r分别为-0.991,-0.982）,530～570 nm波段的反射率与山柰素含量亦呈显著的负相关(︱r︱>0.95)。通过比较SDb、Rg,可简捷地判断长白山地区木贼质量的优劣。增强530～570 nm波段的入射光线,可刺激山柰素含量的增加,但需要大田实验进一步验证.     

两类柑桔杂种砧木对脐橙(Citrus sinensis Osbeck)幼树生长和叶片糖营养含量年变化的影响

以体细胞杂种红桔＋枳和红桔＋粗柠檬、有性杂种Troyer枳橙和Swingle枳柚作砧木的耐湿脐橙(Citrus sinensis Osbeck)2年生嫁接苗为试材,通过盆栽试验研究了砧木对苗木生长和叶片糖营养含量年变化动态的影响。结果表明：红桔＋枳的生长势和花量明显地强于和大于其它砧木,红桔＋粗柠檬的生长和花量表现介于2种有性杂种之间。叶绿素a／b和类胡萝卜素含量在不同砧木间无显著差异,而不同砧木显著或极显著地影响叶绿素含量各指标。砧木影响叶片可溶性糖和淀粉含量年变化动态的基本特征。在休眠期,红桔＋枳的可溶性糖和淀粉含量都高于2种有性杂种砧木,而红桔＋粗柠檬则反之;在生长期,红桔＋粗柠檬的可溶性糖和淀粉含量表现出高于有性杂种砧木的趋势,而红桔＋枳则介于2种有性杂种砧木之间。同时还重点讨论了2种体细胞杂种砧木的利用价值。