The Concept of Microwave Foam Drying Under Vacuum: A Gentle Preservation Method for Sensitive Biological Material.

Microwave vacuum drying as compared to conventional vacuum drying has evinced advantages regarding drying time, while comparable product characteristics were achieved when drying sensitive biological material. Due to the volumetric microwave input, a time reduction of up to 90% is possible. When drying viscous liquids, a foamed structure that remains stable during drying exhibits further advantages as the diffusion-limited third drying step is enhanced by the porous structure. As foams not only have to be thermally resistant during microwave vacuum processing, but also withstand the vacuum, a specific process for foam drying by microwaves under low pressure conditions was developed. Foam formation and stabilization was achieved by using a synergistic mixture of proteins and carbohydrates; Lactobacillus paracasei ssp. paracasei F19 (L. paracasei) served as a model sensitive substance. Investigation of surface activity and foaming properties as a function of L. paracasei concentration revealed a significant positive contribution of the bacterial cells. It was shown that L. paracasei directly adsorbed at the air-water interface. Besides, a structuring of the liquid lamellae was assumed. Moreover, drying time was reduced to at least 50% compared to microwave vacuum drying without foaming. It was further observed that the slight loss in survival was mainly due to the relatively high moisture content and high vacuum levels at the beginning of the process. However, foaming, vacuum application, and final drying, respectively, did not affect viability of the bacterial cells. Thus, by incorporation of lactic acid bacteria into foam structures, drying can be carried out in a fraction of time, and further results in high-product quality. PRACTICAL APPLICATION: The application of continuous foam drying offers an efficient and energy-saving alternative to the currently applied techniques for the processing of sensitive material. The process could be applied for the preservation of starter cultures and probiotics as well as in the pharmaceutical industry, when sensitive material such as therapeutic proteins is dried. This process is especially suitable for freezing-sensitive and thermolabile substances.

Protective effect of sugars on storage stability of microwave freeze-dried and freeze-dried Lactobacillus paracasei F19.

AIMS: Microwave freeze drying (MWFD) in comparison with conventional freeze drying allows for intensification of the preservation process of lactic acid bacteria without imposing additional processing stress. Viability as a function of storage time of microwave freeze-dried Lactobacillus paracasei ssp. paracasei F19 was investigated in comparison to conventionally lyophilized bacteria of the same strain. Furthermore, the impact of the protectants, sorbitol, trehalose and maltodextrin, on shelf life was analysed. METHODS AND RESULTS: The highest inactivation rates of 0·035 and 0·045 day-1 , respectively, were found for cultures without protectants. Thus, all additives were found to exhibit a protective effect during storage with inactivation rates between 0·015 and 0·040 day-1 . Although trehalose and maltodextrin samples were in the glassy state during storage, in contrast to samples containing sorbitol as protectant, the best protective effect could be found for sorbitol with the lowest inactivation rate of 0·015 day-1 . CONCLUSIONS: Due to its low molecular weight, it might protect cells owing to better adsorption to the cytoplasma membrane. Sorbitol additionally shows antioxidative properties. Storage behaviour of microwave freeze-dried cultures follows the typical behaviour of a product dried by conventional lyophilization. No significant influence of the drying technique on storage behaviour was detected. SIGNIFICANCE AND IMPACT OF THE STUDY: General findings concerning storage behaviour in freeze drying are likely to be applicable in MWFD with only slight adjustments.

Discrimination between mild and severe Citrus tristeza virus isolates with a rapid and highly specific real-time reverse transcription-polymerase chain reaction method using TaqMan LNA probes.

Severe isolates of Citrus tristeza virus (CTV) inducing seedling yellows (SY) and/or stem pitting (SP) in grapefruit or sweet orange are a major threat for the citrus industry worldwide. Identification of these CTV variants was achieved by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) using a general primer set and three TaqMan locked nucleic acids (LNA) probes targeting sequences characteristic of severe, mild (non-SY, non-SP), and T36-like isolates. Successful amplification was achieved from fresh or silica-desiccated CTV-infected samples and all isolates but one reacted with one or more probes. Standard curves using RNA transcripts homologous to the three probes allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) copies. RT-PCR assays with homologous and heterologous transcript RNA mixes demonstrated that each probe reacted only with its cognate sequence which was detected even at ratios below 2.5%. Analysis of 56 pathogenically distinct CTV isolates from 20 countries showed that mild isolates reacted only with the mild probe, whereas severe SP and SY isolates reacted with the severe-SP or the T36-like probes, respectively, and often with a second probe. This procedure can be useful to identify and control potentially dangerous CTV isolates in areas affected only by mild isolates.

Preferential accumulation of severe variants of Citrus tristeza virus in plants co-inoculated with mild and severe variants.

The viral population in sweet orange plants, either healthy or pre-inoculated with the asymptomatic isolate of Citrus tristeza virus (CTV) T32, and then graft- or aphid-inoculated with the stem-pitting isolate T318, was characterized with respect to symptom expression, reaction with monoclonal antibody MCA13, single-strand conformation polymorphism (SSCP) of genes p18 and p20, bi-directional RT-PCR, and dot-blot hybridisation. All plants inoculated with T318, with or without pre-inoculation, showed stem pitting, reacted with MCA13, had the SSCP profile characteristic of this isolate, and in bi-directional RT-PCR yielded a 450-bp DNA product associated with severe isolates, indicating that T32 afforded no protection against T318. The latter isolate had two main sequence variants, the minor one of which was indistinguishable from the main T32 sequence, and both were detected in most plants that were graft-inoculated with T318. However, the T32 variant was not detected in plants that were aphid-inoculated only with T318 and also showed stem pitting. This suggested an association of symptoms with the major T318 sequence and preferential transmission of this variant by aphids. The T318-specific variant accumulated more than the T32 variant in plants in which both were replicating, suggesting a higher fitness of the former. Our results clearly emphasize the potential threat of severe CTV variants in areas where mild isolates are presently predominant.

The complete nucleotide sequence of a severe stem pitting isolate of Citrus tristeza virus from Spain: comparison with isolates from different origins.

The genomic RNA of the severe stem pitting Citrus tristeza virus (CTV) isolate T318A from Spain (19252 nt) was completely sequenced. It showed strong sequence similarities with the severe isolates SY568 from California and NUagA from Japan, and distant relationships with mild non-stem pitting isolates T385 from Spain and T30 from Florida. Contrasting with other severe CTV isolates, T318A had a predominant sequence variant even in the highly variable 5'-terminal untranslated region, in which a unique sequence variant (type II) previously associated with severe stem pitting isolates was detected. The high homogeneity of the T318A population suggests that the sequence obtained is probably responsible for the symptoms induced and makes it a useful tool to delimit pathogenicity determinants.

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.

Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host.

Viroids, small single-stranded circular RNAs endowed with autonomous replication, are unique systems to conduct evolutionary studies of complete RNA genomes. The primary structure of 36 progeny variants of peach latent mosaic viroid (PLMVd), evolved from inoculations of the peach indicator GF-305 with four individual PLMVd cDNAs differing in their pathogenicity, has been determined. Most progeny variants had unique sequences, revealing that the extremely heterogeneous character of PLMVd natural isolates most probably results from the intrinsic ability of this RNA to accumulate changes, rather than from repeated inoculations of the same individual trees under field conditions. The structure of the populations derived from single PLMVd sequences differed according to the observed phenotype. Variant gds6 induced a reproducible symptomatic infection and gave rise to a more uniform progeny that preserves some parental features, whereas variant gds15, which induced a variable phenotype, showed a more complex behaviour, generating two distinct progenies in symptomatic and asymptomatic individual plants. Progenies derived from variants esc10 and Is11, which incited latent infections, followed a similar evolutionary pattern, leading to a population structure consisting of two main groups of variants, one of which was formed by variants closely related to the parental sequence. The evolution rate exhibited by PLMVd, considerably higher than that reported for potato spindle tuber viroid, may contribute to the fluctuating symptomatology of the severe PLMVd natural isolates. However, the polymorphism observed in PLMVd progenies does preserve some structural and functional elements previously proposed for this viroid, supporting the fact that they act as constraints limiting the genetic divergence of PLMVd quasispecies generated de novo.

Pear Blister Canker Viroid: Host Range and Improved Bioassay with Two New Pear Indicators, Fieud 37 and Fieud 110.

An investigation was conducted to improve the biological detection of pear blister canker viroid (PBCVd), which over a period of 2 to 3 years induces pear blister canker disease on the perry pear (Pyrus communis) cv. A20. PBCVd was not detected by dot blot hybridization or bioassay in any of the tested species of Amelanchier, Aronia, Cotoneaster, Crataegus, and Pyracantha. However, some species of Chaenomeles, Cydonia, and Sorbus, five out of 13 species of Malus, 15 Pyrus species, and 16 commercial pear cultivars were susceptible to PBCVd, although none developed symptoms. Only in perry pear seedlings did approximately 5% of the population react to infection with pure PBCVd strains by developing petiole, leaf, or bark necrosis 2 to 3 years (cv. A20) or 3 to 5 months (cv. Fieudière) after inoculation. The selected Fieud 37 and Fieud 110 clones are proposed as PBCVd indicators to replace A20. Results from bioassays on the indicators and from detection by a PBCVd-cRNA probe were essentially in agreement except for some Malus species.

Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implications of the existence of constraints limiting the heterogeneity of viroid quasispecies.

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.

Peach latent mosaic viroid (PLMVd) can adopt hammerhead structures in both polarity strands. In the course of a study on the variability of this viroid a natural sequence variant has been characterized in which the hammerhead structure of the plus polarity strand has the sequence CCGA instead of the conserved uridine turn motif CUGA present in the catalytic pocket of all natural hammerhead structures. The viroid RNA containing this mutant hammerhead structure, but not those with the two other possible substitutions, U-->A and U-->G, in the same position of the catalytic pocket, showed significant self-cleavage activity during in vitro transcription. Moreover, the corresponding full-length PLMVd cDNA was infectious and the mutation was retained in a fraction of the viroid progeny. These results indicate that the sequence flexibility of the hammerhead structure, acting in vitro and in vivo , is higher than anticipated and provide relevant data for a deeper insight into the catalytic mechanism of this class of ribozymes and into the structure of the uridine turn motif.

Pear blister canker viroid: sequence variability and causal role in pear blister canker disease.

The sequences of several cDNA clones of pear blister canker viroid (PBCVd) P1914T and P47A isolates have been determined. Seven out of eight P1914T clones analysed have a constant sequence which differs at six positions from that of the P2098T isolate reported previously. The remaining P1914T clone (8) has a single nucleotide substitution. The same six changes have been also observed in most of the ten P47A clones sequenced. However, some P47A clones show additional variability in positions on both strands of the central conserved region (CCR) and in another conserved sequence at the left-terminal region. This is the first report of a change affecting the upper strand of a viroid CCR. Reasons why such a change is tolerated are discussed. Infectivity bioassays have demonstrated that PBCVd is the causal agent of PBC disease.