Freezing of edible flowers: Effect on microbial and antioxidant quality during storage.

Edible flowers are a new gourmet product; however, they are not always available all years. Thus, it is essential to find out technologies to guarantee this product for a longer time. Flowers of four species (borage [Borago officinalis], heartsease [Viola tricolor], kalanchoe [Kalanchoe blossfeldiana], and dandelion [Taraxacum officinale]) were subjected to freezing (in their natural form and in ice cubes) and analyzed in terms of visual appearance, the content of flavonoids, hydrolysable tannins, phenolics, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power), and microbial quality after storage for 1 and 3 months. Flowers in ice cubes showed similar appearance to fresh ones during the 3 months of storage, whereas frozen flowers were only equivalent up to 1 month with the exception of kalanchoe. Even though flowers in ice cubes showed good appearance after 3 months of storage, they had the lowest values of bioactive compounds and antioxidant activity. On the contrary, when frozen, the content of bioactive compounds maintained or even increased up to 1 month of storage compared to fresh flowers, except for borage. Furthermore, in both freezing treatments, the microorganisms' counts decreased or maintained when compared to fresh samples, except in dandelion. In general, both treatments may allow keeping the flowers after their flowering times. PRACTICAL APPLICATION: The market of edible flowers is increasing, although they are a very perishable product with short shelf-life. Edible flowers are stored in the cold (frozen or in ice cubes); however, the effect on the bioactive compounds and microbial quality that this treatment may have on borage (Borago officinalis), heartsease (Viola tricolor), kalanchoe (Kalanchoe blossfeldiana), and dandelion (Taraxacum officinale) flowers is unknown. So, the present study was conducted to increase the knowledge about the changes that freezing treatments may have in different edible flowers. The results of the present study underline that each flower has different behavior at frozen and ice cubes storage. However, freezing flowers maintain/increase the contents of bioactive compounds, while ice cubes not. Both treatments are effective in protecting flowers from microorganism growth. So, suggesting that both freezing treatments can be used as a preservative method and may allow keeping the flowers after their flowering times.

Prevalence of Soil-borne Diseases in Kalanchoe blossfeldiana Reveals a Complex of Pathogenic and Opportunistic Fungi.

Greenhouse cultivation of ornamentals is subjected to a high incidence of soil-borne fungal pathogens. In Kalanchoe, these pathogens are responsible for root and stem rot, and for infection of the vascular tissue. Well-known soil-borne pathogens are believed to cause these diseases. Yet, a systematized survey of these pathogens is lacking for Kalanchoe produced commercially. Here, we studied the occurrence of soil-borne fungal pathogens associated with cultivation of Kalanchoe in Denmark and production of cuttings and stock plants in greenhouse facilities located in Turkey and Vietnam. Molecular identification of pathogens complemented mycological identification and pathogenicity testing of the soil-borne fungal pathogens. This study revealed that the fungi Corynespora cassiicola, Thielaviopsis basicola, Fusarium solani, and F. oxysporum are the most prevalent pathogens associated with root and stem rotting and wilt of Kalanchoe under the conditions studied. Furthermore, the study showed that some of the pathogens are part of an infection complex comprising both primary and opportunistic fungal species. The fact that some of the pathogens were present in some regions, while absent in others, suggests how they move between greenhouse facilities on infected plant material. This study generated important information about the soil-borne fungal complex affecting Kalanchoe, which is useful for a better understanding of the biology of the disease and for designing control strategies.

Azotobacter bryophylli sp. nov., isolated from the succulent plant Bryophyllum pinnatum.

A Gram-stain-negative, aerobic, nitrogen-fixing bacterium, designated strain L461T, was isolated from leaves of Bryophyllum pinnatum growing at the South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated it as a member of the genus Azotobacter closely related to Azotobacter beijerinckii JCM 20725T (97.82 % similarity) and Azotobacter chroococcum ATCC 9043T (97.34 %). Its major fatty acid components were C16 : 1 ω9c and C16 : 0. Its predominant isoprenoid quinone was Q-9. Its major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid, phospholipid and one unknown lipid. Its DNA G+C content was 64.9 mol% (Tm). DNA-DNA relatedness values between strain L461T and the reference strains of A. beijerinckii and A. chroococcum were 46.43 and 28.23 %, respectively. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting, and comparison of cellular fatty acids distinguished strain L461T from the closely related Azotobacter species. Based on these data, the novel species Azotobacter bryophylli sp. nov. is proposed, with the type strain L461T (=KCTC 62195T=GDMCC 1.1250T).

Paenibacillus bryophyllum sp. nov., a nitrogen-fixing species isolated from Bryophyllum pinnatum.

A nitrogen-fixing, endospore-forming bacterium, designated strain L201T was isolated from the leaves of Bryophyllum pinnatum growing in South China Agricultural University. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain L201T is affiliated with the genus Paenibacillus, and closely related to Paenibacillus albidus Q4-3T (97.4%), Paenibacillus odorifer DSM 15391T (97.3%) and Paenibacillus borealis DSM 13188T (97.2%). The main fatty acids components was anteiso-C15:0 (48.1%). The predominant isoprenoid quinone was MK-7. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The G+C content of strain L201T was 43.9%. DNA-DNA relatedness between L201T and the reference strain was 29.8%. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting and comparison of cellular fatty acids distinguished strain L201T from the closely related Paenibacillus species. Based on these data, the novel species Paenibacillus bryophyllum sp. nov. is proposed, with the type strain L201T(= KCTC 33951 T = GDMCC 1.1251 T).

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced.

Brucella abortus efp gene is required for an efficient internalization in HeLa cells.

Numerous chromosomal virulence genes (chv) have been shown to play an important role in the ability of Agrobacterium tumefaciens to transform plants. The A. tumefaciens chvH gene encodes a protein similar in sequence to the Escherichia coli elongation factor P (EF-P). In A. tumefaciens this factor is required for tumor formation and for full expression of the vir genes, exerting its activity at a post-transcriptional level. Cross-complementation assays suggest that the chvH gene and the efp gene of E. coli are functionally homologous. We have cloned and characterized the efp homolog gene in Brucella abortus which has 45% identity to A. tumefaciens chvH and 35% identity to E. coli efp. The gene complemented detergent sensitivity and virulence in the chvH A. tumefaciens mutant, suggesting that both genes are functionally homologous; the growth rate in complex medium also increased to wild type levels. An efp mutant in B. abortus 2308 grew slower in complex media and showed more sensitivity to detergents. Infection assays in J774 macrophage like cells revealed no significant differences between the wild type and the efp mutant strains. The recovery of this mutant from spleens of inoculated mice was equivalent compared to that of the parental strain suggesting that B. abortus efp is not required for virulence in an animal model. However the efp mutant revealed significant differences at 1 h-4 h post-infection in HeLa infection assays compared to the wild type strain, indicating that cellular internalization was affected in non-professional phagocytes. Double immunofluorescence assays for detecting extracellular and intracellular bacteria, demonstrated that the mutant attaches to HeLa cells as the wild type but is deficient in the internalization process, thus indicating that efp is involved in the penetration of Brucella in non-professional phagocytes.

Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua / Methods for the detection of Agrobacterium from plant, soil and water samples

El género Agrobacterium incluye especies ftopatógenas que inducen la formación de agallas en el cuello o la proliferación de raíces en cabellera en más de 600 especies de dicotiledóneas, y especies no patógenas cuyo hábitat natural es el suelo. Como no es posible erradicar a las especies patógenas y habida cuenta de que más del 80 % de las infecciones puede provenir de viveros, es importante evitar la diseminación de la enfermedad. Por ello, el objetivo de este trabajo ha sido desarrollar técnicas sensibles y precisas que, aisladamente o combinadas, permitan detectar la presencia de especies y biovares de Agrobacterium a partir de muestras de material vegetal, suelo y agua. Se comprobó que con la estrategia combinada de realizar aislamientos en los medios semiselectivos D1, D1-M y YEM-RCT; PCR multiplex sobre el gen 23S ADNr; PCR específca sobre los genes virC1 y virC2 y bioensayos en plántulas de pimiento cv. California Wonder y en hojas cortadas de kalanchoe, se reduce la posibilidad de obtener falsos negativos y/o falsos positivos. Por lo expuesto, esta combinación de técnicas constituye una herramienta adecuada para el diagnóstico de cepas patógenas de Agrobacterium a partir de distintos tipos de muestras.

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly diffcult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identifcation techniques by using a set of semi-selective and differential culture media in combination with a specifc PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specifc PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

[Methods for the detection of Agrobacterium from plant, soil and water samples]. / Métodos para la detección de Agrobacterium a partir de muestras de material vegetal, suelo y agua.

The genus Agrobacterium includes phytopathogenic bacteria that induce the development of root crown galls and/or aerial galls at the base of the stem or hairy roots on more than 600 species of plants belonging to 90 dicotyledonous families and non-pathogenic species. These bacteria being natural soil inhabitants are particularly difficult to eradicate, which is a problem in nurseries where more than 80% of infections occur. Since early detection is crucial to avoid the inadvertent spread of the disease, the aim of this work was to develop sensitive and precise identification techniques by using a set of semi-selective and differential culture media in combination with a specific PCR to amplify a partial sequence derived from the virC operon, as well as a multiplex PCR on the basis of 23SrDNA sequences, and biological assays to identify and differentiate species and biovars of Agrobacterium obtained either from soil, water or plant samples. The combination of the different assays allowed us to reduce the number of false positive and negative results from bacteria isolated from any of the three types of samples. Therefore, the combination of multiplex PCR, specific PCR, isolations in semi-selective D1, D1-M and YEM-RCT media combined with bioassays on cut leaves of Kalanchoe and seedlings of California Wonder pepper cultivar constitute an accurate tool to detect species and biovars of Agrobacterium for diagnostic purposes.

Development of SCAR markers and PCR assays for single or simultaneous species-specific detection of Phytophthora nicotianae and Pythium helicoides in ebb-and-flow irrigated kalanchoe.

Phytophthora nicotianae and Pythium helicoides are important water-borne oomycete pathogens of irrigated ornamentals particularly ebb-and-flow irrigated kalanchoe in Japan. We developed novel PCR-based sequence characterized amplified region markers and assays for rapid identification and species-specific detection of both pathogens in separate PCR reactions or simultaneously in a duplex PCR.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Agrobacterium tumefaciens type IV secretion protein VirB3 is an inner membrane protein and requires VirB4, VirB7, and VirB8 for stabilization.

Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.

Development of microsatellite markers for Pythium helicoides.

A strategy combining dual-suppression PCR and thermal asymmetric interlaced PCR was used to determine sequences flanking microsatellite regions in Pythium helicoides. The primer pairs were designed to amplify loci containing (AC)n, (GA)n, (AGC)n, (CAC)n(CAA)n, (TCA)n and (CTTT)n repeats from the P. helicoides nuclear genome. The PCR products of each primer pair, amplified from three representative isolates collected from different hosts and locations, were cloned and sequenced. Different degrees of polymorphism were detected among these microsatellite markers. The numbers of alleles were 6, 2, 4, 11, 4 and 4 in YL-AC, YL-AGC, YL-CAA, YL-CTTT, YL-GA and YL-TCA, respectively. Allele analysis of 30 P. helicoides isolates showed length polymorphisms in all loci, except for YL-AC, using capillary electrophoresis. Thus, we have developed a simple method for designing PCR primers to amplify microsatellite markers from P. helicoides.

Transformation of the plant Kalanchoë daigremontiana using Agrobacterium tumefaciens.

Kalanchoë daigremontiana can be stably transformed using the Agrobacterium tumefaciens-mediated T-DNA transfer method, as described here. Sterilized plant tissue is cocultivated with an A. tumefaciens suspension, transformants are selected and the shoots are grown in rooting medium and then in soil. Plant phenotypes can be examined approximately 3 mo after transfer of plants to soil.

Production of an antimicrobial substance against Cryptococcus neoformans by Paenibacillus brasilensis Sa3 isolated from the rhizosphere of Kalanchoe brasiliensis.

An antifungal substance produced by Paenibacillus brasilensis strain Sa3 was preliminary characterized and showed to be stable after treatment with different enzymes and organic solvents and at a wide range of pH, and presented a molecular weight between 3 and 10 kDa. In vitro antagonism of this strain towards Cryptococcus neoformans was investigated by optical and electronic microscopic analyses and a fungicidal effect on C. neoformans was observed. Ultrastructural analysis showed intense changes on the fungus when it was paired cultured with strain Sa3, mainly the detachment of the capsule from the cell wall and the presence of altered organelles in the cytoplasm. This novel antifungal substance produced by P. brasilensis Sa3 may represent a new insight in antifungal therapy mainly against emergent fungi. Also, prospective studies on rhizobacteria of plants as Kalanchoe brasiliensis may offer a potential source for the discovery of bioactive compounds with medical value.

The VirB5 protein localizes to the T-pilus tips in Agrobacterium tumefaciens.

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) mediates the transfer of single-stranded DNA and protein virulence factors into plant cells, and also determines the assembly of the T-pilus, which is believed to play a role in host recognition. The T-pilus is composed of the major component VirB2 and the minor component VirB5. Using immuno-electron microscopy we detected the major component VirB2 along the entire length of detached T-pili, but not on cell-bound T-pili or on the cell surface. In contrast, the minor T-pilus component VirB5 was detected on the tips of cell-bound T-pili as well as on the ends of detached T-pili and on the cell surface. To gain further insights into the role of VirB5 we introduced changes at its C terminus. C-terminal deletions of up to four amino acids and alanine replacements did not abolish T-pilus formation and incorporation of the VirB5 variants at the tip, although they did impact the length of T-pili. Also, these changes differentially affected the ability of the T4SS to transfer DNA into plant and bacterial recipients, suggesting differential effects on host-cell specificity. The data presented here suggest that VirB5 localizes at the T-pilus tip, and provide novel insights into its role during the type IV secretion process.

VirB1* promotes T-pilus formation in the vir-Type IV secretion system of Agrobacterium tumefaciens.

The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.

Lon protease of the alpha-proteobacterium Agrobacterium tumefaciens is required for normal growth, cellular morphology and full virulence.

The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper a lon mutant of the alpha-proteobacterium Agrobacterium tumefaciens C58 has been characterized. Unlike lon mutants of Escherichia coli, the lon mutant of A. tumefaciens grows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease of A. tumefaciens shares high homology with its counterparts in E. coli and in Sinorhizobium meliloti, and functionally complements an E. coli lon mutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of Lon(At) corresponding to the ATP-binding site and the active site serine of the E. coli Lon protease abolish complementation of phenotypes of the A. tumefaciens and E. coli lon mutants. The nucleotide sequence upstream of A. tumefaciens lon contains an element similar to the consensus sigma(32) heat-shock promoter of E. coli. Northern and Western blot analyses indicated that expression of lon is induced by elevated temperature, albeit to a much lower level than that of groEL. The lon mutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system.

Molecular characterization of the Agrobacterium tumefaciens DNA transfer protein VirB6.

The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.

Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of IncQ plasmids between Agrobacterium cells and the establishment of IncQ plasmids in plant cells.

The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.

Aecidium kalanchoe sp. nov., a new rust on Kalanchoe blossfeldiana (Crassulaceae).

A rust fungus found on cultivars of Kalanchoe blossfeldiana (Crassulaceae) is described as a new species, Aecidium kalanchoe sp. nov., and compared to the other described rusts on members of the Crassulaceae. Only one other rust is known to parasitize Kalanchoe spp. A DNA sequence of A. kalanchoe suggests that the teleomorph is related to Puccinia.