Severinia Buxifolia Research Articles

Severinia buxifolia Leaves: Isolation, Characterization of Major Metabolites from the Bioactive Fractions and their Antiprotozoal Activity

In an effort to explore herbal drugs as treatment for some neglected tropical diseases (NTDs), Severinia buxifolia (Poir) Ten, was selected and investigated for its antiprotozoal activity. Based on this objective, the ethyl acetate (EA), n-hexane (HE), methylene chloride (MC), n-butanol (BU) fractions of the leaves of S. buxifolia were screened for the in vitro antiprotozoal activity against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Hexane and MC fractions showed good antiprozoal activity (IC50 for HE8.56, 8.64, 3.37, 10.26 µg/mL and MC 8.20, 12.7, 32.69, 2.63 µg/mL) against T. cruzi, L. infantum,T. brucei,and P. falciparum respectively. However, the EA and BU were inactive. Accordingly, two major compounds were isolated from HE identified as and friedelin (1) and β-sitosterol (2) while two coumarins were isolated from MC and identified as seselin (3) and (+)-ulopterol (4). Identification of the compounds was carried out based on mass spectrometry, proton and carbon 1D NMR, as well as 1H-13C HSQC and HMBC spectral analysis. Among isolated compounds, only seselin showed antiprotozoal activity with IC50 of 38.47 and 38.5 mg/mL against T. cruzi and P. falciparum, respectively and no cytotoxicity. Furthermore, an HPLC fingerprint for each fraction was achieved with the aim of authenticating the plant chemical profile and identified seslin and ulopterol as major constituents of the plant extract.

Severinia buxifolia

This datasheet on Severinia buxifolia covers Identity.

Effects of solvent-solvent fractionation on the total terpenoid content and in vitro anti-inflammatory activity of Serevenia buxifolia bark extract.

Severinia buxifolia (Rutaceae) is often used as a traditional medical plant. The present study was carried out to estimate the effects of solvents (petroleum ether and hexane: ethyl acetate) used in liquid–liquid extraction to total terpenoid content (TTC) and in vitro anti‐inflammatory activity of the extracts obtained from S. buxifolia bark. The results showed that solvent fractionation increased the TTC compared with crude extracts. The hexane: ethyl acetate bark extract fraction (HEF) had the highest TTC (731.48 µg/ml) in comparison with the petroleum ether bark extract fraction (PEF) (564.81 µg/ml) and the crude extract (CE) (184.26 µg/ml). In addition, one of composition of terpenoid of S. buxifolia, namely ursolic acid, was determined by HPLC method from the crude CE and the fractions PEF and HEF: 2.44 μg/g DW, 3.56 μg/g DW and 5.04 μg/g DW, respectively. The samples had an in vitro anti‐inflammatory activity comparable with that of two reference standards (aspirin and indomethacin). Particularly, the HEF fraction had the highest in vitro anti‐inflammatory activity (i.e., albumin denaturation: IC50 = 147.91 μg/mL, heat‐induced hemolysis: IC50 = 159.91 μg/mL, proteinase inhibition: IC50 = 117.72 μg/mL, and lipoxygenase activity: IC50 = 90.45 μg/mL). Besides, the preliminary experiments of this study were conducted to determine the influences of maceration factors (solvent type, temperature, and time) for S. buxifolia bark extract. The TTC ranged from 453.70 to 842.59 mg linalool/g DW, and the extraction yield from 2.40% to 5.120% in all extracts. Based on TTC and EY, the hexane: acetone mixture is recommended as the optimal solvent to obtain the crude bark extract (CE) at 46°C for 24 hr of maceration. Extracts of S. buxifolia bark are a promising source for the treatment of inflammatory diseases.

Evaluation of the Use of Different Solvents for Phytochemical Constituents, Antioxidants, and In Vitro Anti-Inflammatory Activities of Severinia buxifolia

Severinia buxifolia (Rutaceae) is a promising source of bioactive compounds since it has been traditionally used for the treatment of various diseases. The present study aimed at evaluating the impact of different solvents on extraction yields, phytochemical constituents and antioxidants, and in vitro anti-inflammatory activities of S. buxifolia. The results showed that the used solvents took an important role in the yield of extraction, the content of chemical components, and the tested biological activities. Methanol was identified as the most effective solvent for the extraction, resulting in the highest extraction yield (33.2%) as well as the highest content of phenolic (13.36 mg GAE/g DW), flavonoid (1.92 mg QE/g DW), alkaloid (1.40 mg AE/g DW), and terpenoids (1.25%, w/w). The extract obtained from methanol exhibited high capacity of antioxidant (IC50 value of 16.99 μg/mL) and in vitro anti-inflammatory activity (i.e., albumin denaturation: IC50 = 28.86 μg/mL; antiproteinase activity: IC50 = 414.29 μg/mL; and membrane stabilization: IC50 = 319 μg/mL). The antioxidant activity of the S. buxifolia extract was found to be 3-fold higher than ascorbic acid, and the anti-inflammatory activity of S. buxifolia extract was comparable to aspirin. Therefore, methanol is recommended as the optimal solvent to obtain high content of phytochemical constituents as well as high antioxidants and in vitro anti-inflammatory constituents from the branches of S. buxifolia for utilization in pharmacognosy.

Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq

The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian “true citrus fruit trees” (genera Citrus, Poncirus, and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus, and Clymenia) was inconsistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae.

DNA Barcoding of Highly Threatened Sacred Plant - Aegle marmelos L.

DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, the matK chloroplast gene was tested for species identification of highly threatened sacred plant Aegle marmelos L., to document their reliable identification, discrimination, similarities and evolutionary trend with their related taxa for future use. DNA isolation from leaf sample of Aegle marmelos was carried out by using a modified Cetyl Trimethyl Ammonium Bromide method (CTAB). Gradient PCR amplification was performed for the isolated DNA using matK gene and the primers, matK472F and matK1248R. Pairwise sequence alignments were made with BLAST and multiple sequence alignments were also made with ClustalW. Based on the sequence alignments, dendrograms were constructed using the software, Mega 5 and Neighbor Joining tree method was used to study the phylogenetic aspects of the species studied and with their related taxa. The success rate of PCR amplification was 90-95% and the success rate of DNA sequence was 95%. The maximum identity of the present study sample was 100% with Aegle marmelos and the similarity of other related species, Severinia buxifolia, Citrus trifoliata and Citrus reticulata was 97% respectively.

Possible role of plant volatiles in tolerance against huanglongbing in citrus

abstractVolatile organic compounds (VOC) play an important role in protecting plants from insect and pathogen attack. In this study, we investigated the leaf volatile profiles of 14 citrus varieties. The VOC in citrus leaves were extracted with n-hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Overall, 4six volatile compounds were identified in the n-hexane extract from citrus leaves. Most of the detected compounds belonged to 3 main groups (monoterpenes, sesquiterpenes, and aliphatic aldehydes). Principle component analysis was used to examine the relative distribution of the studied varieties to each other. Interestingly, volatile profiles of varieties that are tolerant to Candidatus Liberibacter asiaticus (CLas) were different from those of the susceptible ones. Tolerant and moderately-tolerant cultivars contained relatively higher amounts of volatiles than susceptible varieties. In addition, tolerant varieties were also higher in specific compounds which are known for their antimicrobial activities. These compounds include Aldehydes (undecanal, neral, geranial, and citronellal) and some monoterpenes such as linalool, d-limonene, myrcene, α- and β- phellandrene. In addition, some sesquiterpene compounds including t-caryophellene, γ-elemene, β-elemene, germacrene D, and geranyl acetate were higher in tolerant and moderately tolerant cultivars. Severinia buxifolia which is known for its tolerance to CLas and many other pathogens contained higher levels of santalenes and coumarins. Our results indicated that citrus leaf volatiles might play a role in citrus tolerance to CLas. The results of this study may help in understanding of the mechanism of citrus tolerance against CLas.

Freeze Response of Citrus and Citrus-related Genotypes in a Florida Field Planting

A test population consisting of progenies of 92 seed-source genotypes (hereafter called “parent genotypes”) of Citrus and Citrus relatives in the field in east–central Florida was assessed after natural freeze events in the winters of 2010 and 2011. Eight seedlings per parent genotype were planted in a randomized complete block design; however, as a result of mortality, the number of plants assessed in some genotype groups was reduced at some or all sampling dates. The citrus diseases huanglongbing and citrus canker were endemic in the planting and may have influenced tree response to cold temperatures. Unusually low temperatures (near –4 °C each winter) for east–central Florida were experienced during the trial period. Defoliation and dieback were significantly greater in the winter of 2011 than in the winter of 2010. The winter in 2011 was preceded by a period of extraordinarily low temperatures in mid-December with no period of cool temperatures to allow trees to acclimate. In 2010 the average defoliation was 53% ± 28% and less than 13% of the trees exhibited any noticeable dieback, whereas in 2011, the average defoliation and dieback were 93% ± 17% and 51% ± 35%, respectively. Within the genus Citrus, several progenies were identified that had 16% to 24% dieback in 2011 and these were from parent genotypes C. reticulata (CRC 2590) (23%), C. sinensis (CRC 3858) (24%), C. maxima (CRC 3945) (16%), C. hassaku (CRC 3907 and 3942) (16% and 17%), C. aurantium (CRC 628 and 2717) (18% and 7%), C. taiwanica (CRC 2588) (21%), and C. neo-aurantium (C. obovoidea + C. unshiu graft chimera) (CRC 3816) (23%). Within other genera in the Aurantiodeae, Poncirus trifoliata (CRCs 301, 3957, 3549, and 4007), Severinia buxifolia (CRC 1497), Bergera koenigii (CRC 3165), and Glycosmis pentaphylla (CRC 3285) had the least amount of dieback, all at less than 23%. The two species within the Toddalioideae subfamily of the Rutaceae (Casimiroa edulis and Zanthoxylum ailanthoides) had among the least amount of dieback (1% and 8%, respectively). When considered by groups, the Citrons and Australian natives had the greatest amount of dieback in 2011, 68% and 65%, respectively. The trifoliates (Poncirus and hybrids) had the least dieback, ranging from 4% to 40%. The information from this study may be useful in germplasm enhancement and Citrus breeding targeting greater cold tolerance.

Live Population Dynamics of 'Candidatus Liberibacter asiaticus', the Bacterial Agent Associated with Citrus Huanglongbing, in Citrus and Non-Citrus Hosts.

Citrus huanglongbing (HLB) is a century-old destructive disease which presents an unprecedented challenge to citrus industries worldwide. In Florida, HLB is associated with the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' and is mainly transmitted by Asian citrus psyllid (Diaphorina citri). Quantification of the pathogen population in a host aids in investigation of virulence mechanisms and disease management. Recently a procedure was developed to detect live bacterial populations using a novel DNA-binding dye, propidium monoazide, in conjunction with real-time polymerase chain reaction (PMA-qPCR). Chinese box orange (Severinia buxifolia) is a common ornamental present in Florida which could host D. citri and 'Ca. L. asiaticus'. For 20 months, the change of the live 'Ca. L. asiaticus' populations in graft- and psyllid-transmitted Valencia sweet orange (Citrus sinensis 'Valencia') and S. buxifolia plants was monitored by PMA-qPCR. Our results showed that the live 'Ca. L. asiaticus' population was significantly lower in the months of December, January, and February than the rest of the year in both hosts. No statistically significant pattern in the total bacterial population was observed in either host. This pattern may indicate a seasonal growth of 'Ca. L. asiaticus' along with the growth of both plants. These new findings should provide useful information on HLB management.

CALIDAD DE NARANJA ‘VALENCIA’ INJERTADA EN 20 PORTAINJERTOS

The objective of the present study was to determine the rootstocks among a group of 20 that confer equal or better quality to the fruit of ‘Valencia’ orange with respect to that achieved with sour orange. The rootstocks evaluated were Troyer, Carrizo, Swingle, Sour orange (regional control), Cleopatra, Morton, Rubidoux, Rough Lemon, C-35, Sunki, Volkameriana, Macrophylla, Amblicarpa, Yuma, Taiwanica, Pomeroy, Shekwasha, Flying Dragon, Severinia buxifolia and Sacaton. The response variables were fruit size, skin color, roughness and thickness of skin, weight of the fruit parts (juice, skin, pulp, seeds) and internal quality. There were differences among rootstocks in equatorial and polar diameter, percentage of skin color of the fruit rind and weight of the fruit, seeds and skin, as well as degrees Brix, soluble solids and acidity. The rootstocks that matched the quality of fruit produced with Sour orange were: Flying Dragon, Cleopatra, Sacaton, Yuma, Morton, Rubidoux and Troyer.

Nuclear and maternal phylogeny within Citrus and four related genera based on nuclear genes sequence SNPs and mitochondrial InDels

Despite considerable morphological differentiation Citrus, Fortunella, Poncirus, Microcitrus and Eremocitrus genera are sexually compatible. Species of these genera are mainly diploid (2n=18). If the origin of cultivated Citrus from four basic taxa (C. maxima, C. medica, C. reticulata and C. micrantha) is now well documented, their phylogenetic relationships with Citrus wild species and related genera is still unclear. In the present work we analyse their nuclear and maternal phylogeny by using respectively SNPs on gene sequences and mitochondrial InDels. A total of 7.15 kb were amplified by PCR from 11 genes (Table1) and sequenced (Sanger) for 33 genotypes. The varietal sample was composed of 7 C. reticulata, 5 C. maxima, 5 C. medica, 4 papeda, 5 Fortunella, 3 Poncirus, 2 Microcitrus, 1 Eremocitrus. Severinia buxifolia was used as outgroup. SNPs were mined using BioEdit and SeqMan softwares and phylogenetic analysis done in http://phylemon.bioinfo.cipf.es with different approaches (Phylip (v. 3.68), Phyml Best AIC Tree (v. 1.02b), PhyML (v. 3.00). For maternal phylogeny, 4 InDel markers developed by (Froelicher et al. 2011) have been used. The average frequency per Kb of SNPs and InDels were respectively 59.88 and 1.33 in coding region and 110.99 and 16.31 in non-coding ones. A total of 506 SNP and 23 InDels were identified (Table1). Within Citrus, the papeda group was the most polymorphic species, with 185 polymorphisms, followed by C. reticulata (125), C. maxima (48), and C. medica (27). A newmitotype was observed for Microcitrus australasica while two different mitotypes were identified for Fortunella. Nuclear and mitochondrial phylogenetic analysis reveal that C. reticulata and Fortunella form a consistent clade clearly differentiated from the clade including the other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha).. Inclusion of more genes sequences is undergoing and will improve the resolution of the phylogenetic analysis. (Resume d'auteur)

Phylogenetic relationships of Citrus and its relatives based on rbcL gene sequences

We sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. Both trees supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, the trees did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. Furthermore, Atalantia species showed polytomy. The classification of Swingle and Reece should be reviewed.

The evolution of CMA bands in Citrus and related genera

Most species of Citrus and related genera display a similar karyotype with 2n = 18 and a variable number of terminal heterochromatic blocks positively stained with chromomycin A(3) (CMA(+) bands). Some of these blocks are 45S rDNA sites, whereas others may correspond to the main GC-rich satellite DNA found in several Citrus species. In the present work, the distribution of the 45S rDNA and the main satellite DNA isolated from C. sinensis (CsSat) were investigated by in situ hybridization in seven species of Citrus, two species of closely related genera (Fortunella obovata and Poncirus trifoliata) and four species of the subfamily Aurantioideae, which were less related to Citrus (Atalantia monophylla, Murraya paniculata, Severinia buxifolia, and Triphasia trifolia). In Citrus, Fortunella, and Poncirus, most CMA(+) bands colocalized only with CsSat sites, whereas others colocalized only with rDNA sites. However, some of these species displayed a few CMA(+) bands that colocalized with sites of both probes and other CMA(+) bands that did not colocalized with any of the probes. On the other hand, in the four species less related to Citrus, no CsSat signal was found on chromosomes. On Southern blot, the CsSat probe hybridized with genomic DNA from Citrus, Fortunella, and Poncirus at high stringency only, while under the less stringent conditions, it also hybridized with distantly related species. Therefore, CsSat sequences are the principal component of the heterochromatic blocks of Citrus, Poncirus, and Fortunella, whereas CsSat-like sequences seem to be widespread in the subfamily Aurantioideae. These data further suggest that the variable number of terminal CMA(+) bands observed on chromosomes of Citrus and related genera are probably the consequence of amplification or reduction in the number of CsSat-like sequences distributed on chromosome termini, paralleled by mutation and homogenization events, as proposed by the library hypothesis.

Complete 1H and 13C NMR data assignment of new constituents from Severinia buxifolia

Phytochemical analysis of different organs of the rutaceaeous plant Severinia buxifolia led to the isolation of a new limonoid, a new acridone alkaloid, and a new flavone. Structure elucidation and signal assignment were achieved by the extensive use of 1D and 2D NMR experiments (selective 1D NOE, COSY, NOESY, HSQC, HMBC).

CMA staining analysis of chromosomes in several species of Aurantioideae

Fluorochrome staining with chromomycin A3 (CMA) was used to characterize and compare the CMA banding patterns of chromosomes of 17 species from 13 genera of Aurantioideae, which is one of the seven subfamilies of Rutaceae. All species used in this study had 2n = 18 chromosomes. These chromosomes were classified into five types based on the number and position of CMA-positive bands; B: one telomeric and one proximal band, C: two telomeric bands, D: one telomeric band, E: without bands and Dst: type D with a satellite chromosome. Each species possessed two or four types of chromosomes and unique CMA banding patterns. The CMA banding patterns were 6D + 12E in Clausena anisata (Willd.) Hook. f.,1C + 3D + 14E in Murraya koenigii (L.) Spreng., 1C + 11D + 6E in Murraya paniculata (L.) Jack, 1B + 1C + 10D + 6E in Merrillia caloxylon (Ridl.) Swing., 1C + 9D + 7E + 1Dst in Paramignya lobata Burkill, 2C + 5D + 11E in Severinia buxifolia (Poir.) Tenore, 5D + 13E in Hesperethusa crenulata (Roxb.) Roem., 16D + 2E in Citropsis gabunensis (Engl.) Swing. et M. Kell, 14D + 4E in Citropsis schweinfurthii (Engl.) Swing. et M. Kell, 8D + 10E in Atalantia monophylla DC., 1C + 3D + 14E in Atalantia ceylanica (Arn.) Oliv., 2D + 16E in Atalantia roxburghiana Hook. f., 18E in Swinglea glutinosa (Blanco) Merr., 2D + 16E in Aegle marmelos (L.) Corr., 4D + 14E in Afraegle paniculata (Schum.) Engl., 2B + 2D + 13E + 2Dst in Feronia limonia (L.) Swing. and 3B + 9D + 6E in Feroniella oblata Swing. The advanced genera in each tribe and subtribe such as Merrillia and Feroniella showed heterochromatin (CMA-positive bands) rich chromosome configuration in general. Swinglea glutinosa without heterochromatin rich chromosome, which is considered to be the most primitive chromosome configuration, seems to be the key genus for clarifying the evolution of the tribe Citreae.

First Report of 'Candidatus Liberibacter asiaticus' from Atalantia buxifolia in Guangdong, China.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host 'Candidatus Liberibacter asiaticus', the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of 'Ca. L. asiaticus'. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of 'Ca. L. asiaticus' from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of 'Ca. L. asiaticus' from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of 'Ca. L. asiaticus'.

Aromatic Plants from Western Cuba. VI. Composition of the Leaf Oils of Murraya exotica L., Amyris balsamifera L., Severinia buxifolia (Poir.) Ten. and Triphasia trifolia (Burm. f.) P. Wilson

The chemical composition of the leaf oils of Murraya exotica L., Amyris balsamifera L., Severinia buxifolia (Poir.) Ten. and Triphasia trifolia (Burm. f.) P. Wilson from Cuba were studied by GC and GC/MS. Sixty-one compounds of the oil of M. exotica were identified of which β-caryophyllene (24.1%) was the major constituent. In the oil obtained from A. balsamifera, 56 compounds were identified of which valerianol (43.8%) was the major constituent. Sixty-seven components were identified in the oil of S. buxifolia and the major constituents were (E)-β-santalol (26.9%) and α-santalene (22.3%), while 81 compounds were identified in the oil of T. trifolia and the major constituent was germacrene B (16.3%).

Citrus tristeza virus replicates and forms infectious virions in protoplasts of resistant citrus relatives

Citrus tristeza virus (CTV) is the most economically important viral disease of citrus worldwide. Cultivars with improved CTV tolerance or resistance are needed to manage CTV-induced diseases. The citrus relatives Poncirus trifoliata (L.) Raf., Swinglea glutinosa (Blanco) Merr., and Severinia buxifolia (Poir) Ten. are potential sources of CTV resistance, but their resistance mechanisms are poorly characterized. As a first step to examine the mechanisms of resistance to CTV in these citrus relatives and selected Citrus × Poncirus hybrids, it was necessary to develop methods for protoplast isolation and viral inoculation to allow examination of CTV multiplication in this range of citrus varieties and relatives. Leaf and/or cultured cell protoplasts were isolated and inoculated with four biologically distinct CTV isolates. Northern-blot hybridization analyses for progeny RNAs and immuno-electron microscopy assays for newly produced virions showed that CTV replicated and produced infectious particles in protoplasts from all of the resistant plants tested. These results suggest that resistance to CTV observed at the plant level results from a lack of virus movement and/or some induced resistance response, rather than lack of viral multiplication at the cellular level.

Sources of Resistance to Diaprepes Weevil in Subfamily Aurantiodeae, Rutaceae

Commercially used citrus rootstocks can all be seriously damaged by larvae of the sugar cane root weevil, Diaprepes abbreviatus (L.). Six greenhouse challenge experiments were used to compare the resistance of 24 selections of citrus rootstocks and related species to this weevil by measuring root damage, and larval growth and survival. The commercial rootstocks tested were found to be susceptible, while at least seven other species within the subfamily Aurantiodeae were observed to be significantly more resistant. The species Balsamocitrus dawei Stapf. was most resistant to weevil larvae, exhibited less root damage than commonly used rootstock cultivars, and significantly depressed larval growth and survival. The species Glycosmis pentaphylla (Retz.) Correa, Microcitrus australis (Planch.) Swing., Eremocitrus glauca (Lindl.) Swing., Severinia buxifolia (Poir.) Tenore, Triphasia trifolia (Burm. f.) P. Wils., and Citrus hystrix DC. suffered as much damage from the weevil as common rootstock cultivars but significantly depressed growth of larvae feeding on them. One new hybrid rootstock, HRS-801, also significantly depressed D. abbreviatus larval growth, but this effect has not yet been verified as having significance in a long-term or field situation. Several strategies are discussed for developing citrus rootstocks resistant to D. abbreviatus.

Identification of the Chinese box orange (Severinia buxifolia) as an alternative host of the bacterium causing citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2‐3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3‐4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBBfree psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.