Genetic mapping in (<Emphasis Type="Italic">Populus tomentosa</Emphasis> ×<Emphasis Type="Italic"> Populus bolleana</Emphasis>) and<Emphasis Type="Italic"> P. tomentosa</Emphasis> Carr. using AFLP markers

The AFLP genetic linkage maps for two poplar cultivars were constructed with the pseudo-test-cross mapping strategy. The hybrids were derived from an interspecific backcross between the female hybrid clone TB01 (Populus tomentosa × Populus bolleana) and the male clone LM50 (P. tomentosa). A total of 782 polymorphic fragments were obtained with a PCR-based strategy using 49 enzyme-nested (EcoRI/MseI) primer combinations. Six hundred and thirty two of these fragments segregated in a 1:1 ratio (P<0.01), indicating that these DNA polymorphisms are heterozygous in one parent and null in the other. The linkage analysis was performed using Mapmaker version 3.0 with LOD 5.0 and a maximum recombination fraction () of 0.3. Map distances were estimated using the Kosambi mapping function. In the framework map for LM50 (P. tomentosa), 218 markers were aligned in 19 major linkage groups. The linked loci spanned approximately 2,683 cM of the poplar genome, with an average distance of 12.3 cM between adjacent markers. For TB01 (P. tomentosa × P. bolleana), the analysis revealed 144 loci, which were mapped to 19 major linkage groups and covered about 1,956 cM, with an average distance of 13.6 cM between adjacent markers. These maps covered about 87% and 77% of the estimated genome size of parents LM50 and TB01, respectively. The maps developed in this study lay an important foundation for future genomics research in poplar, providing a means for localizing genes controlling economically important traits in P. tomentosa.Communicated by O. Savolainen     

AFLP and SSR polymorphism in a <Emphasis Type="Italic">Coffea</Emphasis> interspecific backcross progeny [(<Emphasis Type="Italic">C. heterocalyx</Emphasis> × <Emphasis Type="Italic">C. canephora</Emphasis>) × <Emphasis Type="Italic">C. canephora</Emphasis>]

An interspecific cross (BC 1) involving a species with one of the largest genomes in the Coffea genus [Coffea heterocalyx (HET), qDNA = 1.74 pg] and a species with a medium-sized genome [Coffea canephora (CAN), qDNA = 1.43 pg] was studied using two types of molecular markers, AFLP and SSR. One hundred and eighty eight AFLP bands and 34 SSR primer pairs were suitable for mapping. The total map length was 1,360 cM with 190 loci distributed in 15 linkage groups. The results were compared to those obtained previously on an interspecific BC 1 progeny involving a species with a medium-sized genome (Coffea liberica var dewevrei, DEW) and a species with one of the smallest genomes (Coffea pseudozanguebariae, PSE). They are discussed relative to three main points: (1) the relevance of the different marker types, (2) the genomic distribution of AFLP and SSR markers, and (3) the relation between AFLP polymorphism and genome size.Communicated by H.F. Linskens     

Genetic linkage maps of two apple cultivars (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) based on AFLP and microsatellite markers

Genetic linkage maps for two apple cultivars were constructed using AFLP and SSR markers and the pseudo-testcross mapping strategy. The F₁-mapping population was produced by crossing the cultivar Braeburn to the cultivar Telamon and consisted of 257 individuals. Out of the 182 AFLP primer combinations screened, a total of 48 were selected. Using these, 463 AFLP markers segregating 1:1 in the progeny were identified, of which 231 were heterozygous in Telamon and 232 in Braeburn. Eighty-five AFLP markers present in both cultivars (3:1 segregation) were scored in the whole mapping population. Twenty-one SSR primer pairs were tested, which clearly screened 23 loci (some multi-locus markers). This resulted in the identification of 3 loci heterozygous only in Telamon (1:2:1), 5 loci heterozygous only in Braeburn (1:2:1) and 15 loci which were heterozygous in both cultivars (1:1:1:1). Two linkage maps were produced. The Telamon map comprised 259 markers (242 AFLPs and 17 SSRs) divided into 17 linkage groups. The total map length was 1039 cM with a marker density of 4.0 cM. At = 0.05, 8.9% of the mapped loci showed distorted segregation. The Braeburn map consisted of 264 markers (245 AFLPs and 19 SSRs) mapped on 17 linkage groups and spanning 1245 cM. The average distance between two markers was 4.7 cM and segregation distortion was observed for 18.6% of the mapped markers ( = 0.05). Fourty-six markers common to both maps (32 AFLPs and 14 SSRs) allowed the identification of 16 homologous linkage groups. The seventeenth pair of homologous linkage groups from Telamon and Braeburn was identified by 2 SSR markers which were in common to the genetic linkage maps of Fiesta and Discovery, two other apple cultivars.     

Quantitative trait loci identified for sugar related traits in a sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp.) cultivar × <Emphasis Type="Italic">Saccharum officinarum</Emphasis> population

The identification of markers linked to quantitative trait loci (QTLs) for increased sugar accumulation could improve the effectiveness of current breeding strategies in sugarcane. Progeny from a cross between a high sucrose producing cultivar, (denotes Australian plant breeding rights), and a Saccharum officinarum clone, IJ76-514 were grown in two field experiments in different years, and evaluated in the early and mid-season phases of crop maturity, to identify robust QTLs in affecting sucrose content in cane. Using an extensive genetic map constructed for with over 1,000 AFLP and SSR markers, a total of 37 QTLs were identified for brix and pol of which, 16 were detected in both experiments. Of these 37 QTL, 30 were clustered into 12 genomic regions in six of the eight homo(eo)logous groups. Each QTL explained from 3 to 9% of the phenotypic variation observed. Both positive and negative effects were identified and the location of the QTLs on linkage groups belonging to the same homo(eo)logy group suggested that a number of the QTLs were allelic forms of the same genes. Of the 37 QTLs identified, the majority were significant in both early and mature cane, but 8 were identified as early specific QTLs and 9 as mature cane QTLs. In total, 97 interactions were significant (P<10⁻⁵) and these were localised to 32 genomic regions of which 6 were detected with both years’ data. Models including all the QTLs explained from 37 to 66% of the total phenotypic variation, depending on the trait. The results will be subsequently applied in marker assisted breeding. denotes variety covered by Australian plant breeding rights.     

Screening interspecific hybrids of<Emphasis Type="Italic"> Populus</Emphasis> (<Emphasis Type="Italic">P. ciliata</Emphasis> ×<Emphasis Type="Italic"> maximowiczii</Emphasis>) using AFLP markers

Hybrids of Populus ciliata × maximowiczii are very vigorous and outperform both the parents in growth performance and yield. Genetic evaluation of 24 of these interspecific hybrids along with the two mother trees (Populus ciliata), and five male-parent (Populus maximowiczii) genotypes was carried out using the AFLP marker assay. Eight AFLP primer combinations detected 428 markers, of which 280 (66%) were polymorphic. Genetic relationships within the samples were evaluated by generating the similarity matrix based on Jaccards coefficient. The phenetic dendrograms, as well as the PCO plots, separated the hybrids and the two parent species into three distinct clusters. The hybrids grouped closer to the P. ciliata (female parent) cluster as compared to the P. maximowiczii (male parent) cluster. The hybrid cluster contained internal groupings, which correlated to some extent with growth performance. The four best performing hybrids (42m1, 65m1, 23m2, Cm2-5-20/91) formed a distinct sub-cluster. Data from a single primer combination was sufficient for distinguishing the hybrids from the parents and assigning paternity. The hybrids showed 22 markers that were absent in P. ciliata but were monomorphically present in all the hybrids, suggesting outcrossing and common paternity. Further, these 22 markers were found in all the P. maximowiczii genotypes confirming it as the male parent. These male-specific markers can be converted to SCAR markers and used for rapid screening of the P.ciliata × maximowiczii hybrids. The primer combination E-AAC × M-CAA was identified as most suitable for ascertaining true hybridity. AFLP proves to be a useful tool for screening of P. ciliata × maximowiczii hybrids at the early stages of development.Communicated by H.F. Liskens     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Characterization of mixed disomic and polysomic inheritance in the octoploid strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis>) using AFLP mapping

A two-way pseudo-testcross strategy, combined with Single Dose Restriction Fragment (SDRF) marker analysis, was used for genetic mapping in the octoploid cultivated strawberry Fragaria x ananassa (2n = 8 x = 56). Based on a 113 full-sib progeny from a cross between the variety Capitola and the clone CF1116, we generated two parental maps using Amplified Fragment Length Polymorphism (AFLP) markers. Ninety two percent of the markers (727 out of 789) showed ratios corresponding to simplex markers (the majority being SDRF markers), and 8% (62 out of 789) fitted a multiplex ratio. Linkage maps were first established using SDRF markers in coupling phase. The female map comprised 235 markers distributed among 43 co-segregation groups, giving a map size of 1,604 cM. On the male map, 280 markers were assigned to 43 co-segregation groups, yielding a map size of 1,496 cM. Once the co-segregation groups were established, their association was tested using repulsion-phase markers. In total, taking into account associations representing the same linkage groups, 30 linkage groups were detected on the female side and 28 on the male side. On the female map, 68.3% of the pairwise marker linkages were in coupling versus 31.7% in repulsion phase, and the corresponding figures on the male map were 72.2% and 27.8%, respectively. In addition, both groups linked only in the coupling phase and groups linked in the repulsion phase were characterized. The observations suggest that the meiotic behavior of the F. x ananassa genome is neither fully disomic nor fully polysomic, but rather mixed. The genome may not be as completely diploidized as previously assumed.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Enzymatic synthesis of alkyl glucosides using <Emphasis Type="Italic">Leuconostoc</Emphasis> <Emphasis Type="Italic">mesenteroides</Emphasis> dextransucrase

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl α-d-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl α-d-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl α-d-glucopyranoside and ethyl α-d-glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl α-d-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants. Young-Min Kim and Byung-Hoon Kim contributed equally to this work.     

Bioinformatics Characterization of Potential New Beta-Glucuronidase from <Emphasis Type="Italic">Streptococcus</Emphasis> <Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

<Emphasis Type="Italic">Sphingomonas rosea</Emphasis> sp. nov. and <Emphasis Type="Italic">Sphingomonas swuensis</Emphasis> sp. nov., rosy colored <Emphasis Type="Italic">β</Emphasis>-glucosidase-producing bacteria isolated from soil

Two strains PB196^T and PB62^T of Gram-negative, non-motile, and non-spore-forming bacteria, were isolated from soil in South Korea and characterized to determine their taxonomic positions. 16S rRNA gene sequence analysis showed that the two strains belonged to the genus Sphingomonas. The highest degree of sequence similarity of strain PB196^T was found with PB62^T (98.9%), Sphingomonas humi PB323^T (98.9%), Sphingomonas kaistensis PB56^T (98.2%), and Sphingomonas astaxanthinifaciens TDMA-17^T (98.0%). The highest degree of sequence similarity of strain PB62^T was found with Sphingomonas humi PB323^T (98.8%), Sphingomonas astaxanthinifaciens TDMA-17^T (98.2%), and Sphingomonas kaistensis PB56^T (98.1%). Chemotaxonomic data revealed that they possessed ubiquinone-10 (Q-10) as common in the genus Sphingomonas, that the predominant fatty acids were summed feature 7 (C_18:1 ω7c/ω9t/ω12t), summed feature 4 (C_16:1 ω7c/C_15:0 iso 2OH), C_16:0, and C_17:1 ω6c, and that they contained sphingoglycolipid, phosphatidylglycerol (PG), and phosphatidyle-thanolamine (PE) in common but they showed difference for diphosphatidylglycerol (DPG). Based on these data, PB196^T (=KCTC 12339^T =JCM 16604^T) and PB62^T (=KCTC 12336^T =JCM 16605^T =KEMB 9004-005^T) should be classified as type strains of two novel species, for which the names Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov. are proposed, respectively.