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Establishment of a new method for improved shoot tip cryopreservation is crucial to facilitate the long-term preservation of plant germplasm as well as the use of cryotherapy for pathogen eradication. The present study reported a vitrification (V) cryo-foil method for shoot tip cryopreservation and virus eradication in apple. Shoot tip regrowth levels after cryopreservation were comparable among V cryo-foil (53 %), V cryo-plate (46 %) and conventional droplet vitrification (Dr-vi, 48 %). The V cryo-foil is more efficient to perform than Dr-vi as more shoot tips can be cryopreserved by one person. In the histological study applying an image-overlaying strategy, shoot tips cryopreserved by V cryo-foil showed a higher survival chance in the youngest leaf primordia than in the apical dome. When V cryo-foil was tested for virus eradication, fifty-five percent (55 %) of cryo-derived shoots were free of the apple stem pitting virus (ASPV), while none and less than 10 % were free of the apple stem grooving virus (ASGV) and the apple chlorotic leaf spot virus (ACLSV), respectively. Thus, these two viruses were efficiently preserved by V cryo-foil cryopreservation. Noticeably, although the shoot regrowth level was reduced to 27 %, a higher frequency (81 %) of ASPV eradication was achieved when a reduced duration of cryoprotectant exposure was applied in V cryo-foil, supporting the use of insufficient cryoprotection for improved virus eradication., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests and personal relationships that may have influenced the work of this study., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

In recent years, kiwifruit viral diseases have become increasingly prevalent in kiwifruit-producing regions of China, significantly impacting both the yield and quality of kiwifruit. This has emerged as a significant constraint on the healthy and sustainable development of the kiwifruit industry. The use of virus-free propagation materials has been proven the most effective strategy for controlling plant viral diseases. In the present study, shoot tip culture, shoot tip cryotherapy, and their combinations with thermotherapy were established to eradicate Actinidia virus A (AcVA), Actinidia virus B (AcVB), and Actinidia chlorotic ringspot-associated virus (AcCRaV) from Actinidia macrosperma . Additionally, the impact of shoot tip size on virus eradication was evaluated. Among the three confirmed viruses, regardless of the procedure, AcVB was the easiest to eradicate, followed by AcVA and AcCRaV. Combining thermotherapy with shoot tip culture or cryotherapy resulted in a higher virus-free frequency (up to 27.3 and 50%, respectively) than shoot tip culture or cryotherapy alone (0 to 20%). Notably, the combination of thermotherapy and 0.5- to 1-mm shoot tip cryotherapy was shown to be the most effective protocol for virus eradication from A . macrosperma , which produced 50% of regenerated shoots free from all the tested viruses. To the best of our knowledge, this is the first report on virus elimination from kiwifruit infected with multiple viruses based on conventional shoot tip culture and shoot tip cryotherapy., Competing Interests: The author(s) declare no conflict of interest.

Realizing the full potential of genome editing for crop improvement has been slow due to inefficient methods for reagent delivery and the reliance on tissue culture for creating gene-edited plants. RNA viral vectors offer an alternative approach for delivering gene engineering reagents and bypassing the tissue culture requirement. Viruses, however, are often excluded from the shoot apical meristem, making virus-mediated gene editing inefficient in some species. Here, we developed effective approaches for generating gene-edited shoots in Cas9-expressing transgenic tomato plants using RNA virus-mediated delivery of single-guide RNAs (sgRNAs). RNA viral vectors expressing sgRNAs were either delivered to leaves or sites near axillary meristems. Trimming of the apical and axillary meristems induced new shoots to form from edited somatic cells. To further encourage the induction of shoots, we used RNA viral vectors to deliver sgRNAs along with the cytokinin biosynthesis gene, isopentenyl transferase. Abundant, phenotypically normal, gene-edited shoots were induced per infected plant with single and multiplexed gene edits fixed in the germline. The use of viruses to deliver both gene editing reagents and developmental regulators overcomes the bottleneck in applying virus-induced gene editing to dicotyledonous crops such as tomato and reduces the dependency on tissue culture., Competing Interests: Competing interests statement:E.E.E. and D.F.V. filed a patent through the University of Minnesota on the use of viruses for plant gene editing. Reviewers C.G. and M.M.M. were co-authors with D.F.V. on a report of a scientific meeting that took place in 2021.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope ® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

A protocol for high-frequency direct organogenesis from root explants of Kachai lemon (Citrus jambhiri Lush.) was developed. Full-length roots (~3 cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L-1) and GA3 (1.0 mg L-1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5-1.0 mg L-1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the scanning electron microscopy (SEM). The expressions of growth regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were elevated in the regenerants obtained from MSN+BAP (1.0 mg L-1)+GA3 (1.0 mg L-1). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA 1.0 mg L-1. Furthermore, indexing of the regenerants confirmed that there was no amplicons detected for Huanglongbing bacterium and Citrus tristeza virus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. This is the first report that describes direct organogenesis from the root explant of Citrus jambhiri Lush. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, its commercial cultivation and genetic conservation., Competing Interests: The authors have declared that no competing interests exist.

Background: Virus-free sugarcane is difficult to achieve due to the multiple vegetative propagation cycles employed commercially. In vitro culture using small (1 mm) meristematic shoot tips has eliminated viruses but survival is low with small explants., Objective: Droplet-Vitrification (D-V) and V-Cryoplate protocols were investigated for the elimination of Sugarcane mosaic virus (SCMV) from large (c. 3 mm) in vitro-derived shoot tips., Materials and Methods: Shoot tips excised from NCo376 and N19 cultivars were exposed to both cryogenic procedures. Virus indexing by RT-qPCR was performed 16 weeks after recovery., Results: Explants exposed to cryo-treatments that recovered and multiplied was 30-92%, while at least 90% of control explants regenerated. No virus was detected in multiplied shoots from either cultivar after D-V and liquid nitrogen immersion. In NCo376, virus was eliminated after D-V without cooling., Conclusion: The preliminary findings suggest that cryotherapy and/or osmotherapy are viable options for SCMV removal from infected plants.

Plant pathogens cause different diseases on crops and industrial plant species that result in economic losses. Pathogen-free plant material has usually been obtained by traditional procedures such as meristem culture, thermotherapy, and chemotherapy. However, there are many limitations of these procedures such as mechanical challenges of meristem excision and low regeneration rate, low resistance to high temperatures, phytotoxicity, and mutagenic effects of the chemicals used in the procedures. Cryotherapy is a newly developed biotechnological tool that has been very effective in virus elimination from economically important plant species. This tool has overcome the abovementioned limitations. This chapter aims to highlight the importance of the cryogenic procedures (vitrification, encapsulation-vitrification, droplet vitrification, two-step freezing, dehydration, encapsulation-dehydration) in order to generate virus-free germplasm.

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85-99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisia tabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected 'healthy' cannabis, Lactuca sativa , and Catharanthus roseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

Arbuscular mycorrhizal (AM) fungi, as root symbionts of most terrestrial plants, improve plant growth and fitness. In addition to the improved plant nutritional status, the physiological changes that trigger metabolic changes in the root via AM fungi can also increase the host ability to overcome biotic and abiotic stresses. Plant viruses are one of the important limiting factors for the commercial cultivation of various crops. The effect of AM fungi on viral infection is variable, and considerable attention is focused on shoot virus infection. This review provides an overview of the potential of AM fungi as bioprotection agents against viral diseases and emphasizes the complex nature of plant-fungus-virus interactions. Several mechanisms, including modulated plant tolerance, manipulation of induced systemic resistance (ISR), and altered vector pressure are involved in such interactions. We propose that using "omics" tools will provide detailed insights into the complex mechanisms underlying mycorrhizal-mediated plant immunity.

Pathogen-free stock plants are required as propagation materials in nurseries and healthy materials are needed in germplasm exchange between countries or regions through quarantine programs. In addition, plant gene banks also prefer to maintain pathogen-free germplasm collections. Shoot tip cryotherapy is a novel biotechnology method whereby cryopreservation methods are used to eradicate obligate pathogens from vegetatively propagated plants. Long-term preservation of pathogens is necessary in all types of virus-related basic research and applications such as antigen preparation for virus detection by immunology-based methods, production of plant-based vaccines, genetic transformation to produce virus-derived resistant transgenic plants, and bionanotechnology to produce nano drugs. Obligate plant pathogens such as viruses and viroids are intracellular parasites that colonize only living cells of the hosts. Therefore, their long-term preservation is difficult. Cryotreatments cannot completely eradicate the obligate pathogens that do not infect meristematic cells and certain proportions of plants recovered from cryotreatments are still pathogen-infected. Furthermore, cryotreatments often fail to eradicate the obligate pathogens that infect meristematic cells. Cryopreservation can be used for the long-term cryopreservation of the obligate plant pathogens. Thus, cryobiotechnology functions as a double-edged sword for plant pathogen eradication and cryopreservation. This review provides updated a synthesis of advances in cryopreservation techniques for eradication and cryopreservation of obligate plant pathogens.

Within family Baculoviridae, members of the Betabaculovirus genus are employed as biocontrol agents against lepidopteran pests, either alone or in combination with selected members of the Alphabaculovirus genus. Epinotia aporema granulovirus (EpapGV) is a fast killing betabaculovirus that infects the bean shoot borer (E. aporema) and is a promising biopesticide. Because occlusion bodies (OBs) play a key role in baculovirus horizontal transmission, we investigated the composition of EpapGV OBs. Using mass spectrometry-based proteomics we could identify 56 proteins that are included in the OBs during the final stages of larval infection. Our data provides experimental validation of several annotated hypothetical coding sequences. Proteogenomic mapping against genomic sequence detected a previously unannotated ac110-like core gene and a putative translation fusion product of ORFs epap48 and epap49. Comparative studies of the proteomes available for the family Baculoviridae highlight the conservation of core gene products as parts of the occluded virion. Two proteins specific for betabaculoviruses (Epap48 and Epap95) are incorporated into OBs. Moreover, quantification based on emPAI values showed that Epap95 is one of the most abundant components of EpapGV OBs., Competing Interests: The authors have declared that no competing interests exist.

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58-60%) than in 0.5-mm-ones (30-38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRV-preserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Plant cryopreservation has provide secure back-ups of germplasm collections of vegetatively propagated crops. Often, recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes. The present study investigated the effects of Apple stem grooving virus (ASGV) on shoot proliferation of in vitro stock cultures and recovery of cryopreserved shoot tips of 'Gala' apple. Results showed that virus infection reduced shoot proliferation of in vitro stock cultures and cell ability to regenerate normal shoots in cryopreserved shoot tips. Virus infection increased total soluble protein, total soluble sugar and free proline levels and altered endogenous levels of indoleacetic acid (IAA) and zeatin riboside (ZR), but induced severe cell membrane damage and caused alternation in mitochondria shape of the in vitro stock shoots. The altered levels of IAA and ZR were most likely to be responsible for the reduced shoot proliferation of in vitro stock culture. Cell damage and alternations in mitochondria shape in ASGV-infected shoot tips were most likely responsible for the reduced cell ability to regenerate normal shoots following cryopreservation. To the best of our knowledge, this is the first study on effects of virus infection on recovery of cryopreserved shoot tips. Results reported here emphasize that healthy in vitro stock cultures should be used for cryopreservation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Apple stem grooving virus (ASGV), a difficult-to-eradicate virus from apple propagative materials, causes serious damage to apple production. The use of virus-free plants has been and is an effective strategy for control of plant viral diseases. This study aimed to eradicate ASGV from virus-infected in-vitro-cultured shoots of four apple cultivars and one rootstock by combining thermotherapy with cryotherapy. In vitro stock shoots infected with ASGV were thermo-treated using an alternating temperature of 36°C (day) and 32°C (night). Shoot tips were excised from the treated stock shoots and subjected to cryotherapy. Results showed that, although thermotherapy did not influence shoot survival rates, it reduced shoot growth and proliferation of in vitro shoots. Shoot regrowth rates decreased while virus eradication frequencies increased in cryo-treated shoot tips as time durations of thermotherapy increased from 0 to 6 weeks. Shoot regrowth and frequency of virus eradication were positively and negatively correlated, respectively, with the size of shoot tips. The protocol established here yielded shoot regrowth rates and virus eradication frequencies of 33 to 76% and 30 to 100%, respectively, in the four apple cultivars and one rootstock. Thermotherapy altered virus distribution patterns, subsequently resulting in production of a larger virus-free area in the thermo-treated shoot tips. Many cells in the top layers of apical dome and some cells in the youngest leaf primordia survived in cryo-treated shoot tips; these cells were most likely free of virus infection. Thus, plants regenerated from the procedure of combining thermotherapy with cryotherapy were free of ASGV, as judged by reverse-transcription polymerase chain reaction. To the best of our knowledge, this is the widest-spectrum technique reported thus far for the production of ASGV-free plants and provides a novel biotechnology for the production of virus-free plants in Malus spp.

Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) are several major viral pathogens of apple trees, responsible for substantial damage to the world's apple industry. This study aimed to evaluate the effectiveness of the encapsulation-dehydration cryopreservation technique to eradicate these viral pathogens from in vitro shoot tips excised from 'Marubakaido' apple rootstock cultures. Axillary shoot tips were excised from in vitro cultures, encapsulated in alginate beads, precultured in MS salts, dehydrated in a laminar flow hood, immersed in liquid nitrogen, then warmed and recovered on medium. After LN exposure, in vitro rooting and acclimatization, recovered 'Marubakaido' plants exhibited 52% survival and 35% regrowth without callus formation. After 8 months of regrowth, PCR analyses revealed that all the plants were free of ACLSV and ASPV, but 2 out of 20 recovered plants were still infected with ASGV. This is the first report in Brazil of the application of cryotherapy to eradicate viral complexes in Malus. Cryotherapy can facilitate the production of virus-free plants by producing high quality plant material., (Copyright © 2018 Elsevier B.V. All rights reserved.)

RNA interference (RNAi) is a conserved mechanism against viruses in plants and animals. It is thought to inactivate the viral genome by producing virus-derived small interfering RNAs (vsiRNAs). Rice black-streaked dwarf virus (RBSDV) is transmitted to plants by the small brown planthopper (Laodelphax striatellus), and seriously threatens production of rice in East Asia, particularly Oryza sativa japonica subspecies. Through deep sequencing, genome-wide comparisons of RBSDV-derived vsiRNAs were made between the japonica variety Nipponbare, and the indica variety 9311. Four small RNA libraries were constructed from the leaves and shoots of each variety. We found 659,756 unique vsiRNAs in the four samples, and only 43,485 reads were commonly shared. The size distributions of vsiRNAs were mostly 21- and 22-nt long, and A/U bias (66-68%) existed at the first nucleotide of vsiRNAs. Additionally, vsiRNAs were continuously but heterogeneously distributed along S1-S10 segments of the RBSDV genome. Distribution profiles of vsiRNA hotspots were similar in different hosts and tissues, and the 5'- and 3'-terminal regions of S4, S5, and S8 had more hotspots. Distribution and abundance of RBSDV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. Degradome analysis found 25 and 11 host genes appeared to be targeted by vsiRNAs in 9311 and Nipponbare. We report for the first time vsiRNAs derived from RBSDV-infected rice., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.

Plants have evolved a limited repertoire of NB-LRR disease resistance ( R ) genes to protect themselves against myriad pathogens. This limitation is thought to be counterbalanced by the rapid evolution of NB-LRR proteins, as only a few sequence changes have been shown to be sufficient to alter resistance specificities toward novel strains of a pathogen. However, little is known about the flexibility of NB-LRR R genes to switch resistance specificities between phylogenetically unrelated pathogens. To investigate this, we created domain swaps between the close homologs Gpa2 and Rx1 , which confer resistance in potato ( Solanum tuberosum ) to the cyst nematode Globodera pallida and Potato virus X , respectively. The genetic fusion of the CC-NB-ARC of Gpa2 with the LRR of Rx1 (Gpa2 CN /Rx1 L ) results in autoactivity, but lowering the protein levels restored its specific activation response, including extreme resistance to Potato virus X in potato shoots. The reciprocal chimera (Rx1 CN /Gpa2 L ) shows a loss-of-function phenotype, but exchange of the first three LRRs of Gpa2 by the corresponding region of Rx1 was sufficient to regain a wild-type resistance response to G. pallida in the roots. These data demonstrate that exchanging the recognition moiety in the LRR is sufficient to convert extreme virus resistance in the leaves into mild nematode resistance in the roots, and vice versa. In addition, we show that the CC-NB-ARC can operate independently of the recognition specificities defined by the LRR domain, either aboveground or belowground. These data show the versatility of NB-LRR genes to generate resistance to unrelated pathogens with completely different lifestyles and routes of invasion., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

This study aimed to develop a methodology for eliminating cassava frogskin disease (CFSD) from in vitro shoot tip culture by associating thermotherapy and tetracycline. Cuttings from different accessions (BGM0232, BGM0315, BGM0464, BGM584, BGM0841, and BGM1342), infected with CFSD according to visual inspection of the disease symptoms, were used for cleaning. To verify the absence of other diseases, the plants were indexed for Cassava common mosaic virus - CsCMV (by ELISA) and Cassava vein mosaic virus - CsVMV (by polymerase chain reaction, PCR), proving that the accessions were free of these viruses, except for BGM0315 and BGM0464, which were infected with CsVMV. Subsequently, the cuttings were submitted to different tetracycline concentrations for 3 min, and then subjected to thermotherapy under different temperatures (35°, 38°, 40°, 45°, and 55°C). Shoots of 2 cm were harvested, and their surfaces were sterilized in a laminar flow chamber. Subsequently, the shoot tips of different sizes were removed (0.2, 0.4, 0.5, and 1.0 mm) for inoculation in a culture medium with tetracycline at the same concentrations in which the cuttings were dipped. After 60 days of cultivation, the explants were transferred to a multiplication medium without antibiotics. Thirty days after the transfer, the viability of the regenerated plants was evaluated, which were then acclimatized for 70 days in a greenhouse and transferred to the field. After 7 months, a visual analysis of the symptomatic roots and a PCR analysis were held to prove the elimination of CFSD and CsVMV from the accessions infected with these viruses (BGM0315 and BGM0464), respectively. Most of the treatments resulted in 100% cleaning of CFSD-infected plants. From accessions that were also infected with CsVMV, only 2% of the plants remained infected, also demonstrating the cleaning efficiency of this protocol for this disease.

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Plant transformation with the wild type Ri plasmid T-DNA of Agrobacterium rhizogenes is a promising method for breeding of compact plants and has been the subject of numerous studies. However, knowledge concerning the isolated functions of single genes and ORFs from the plasmid is limited. The rolB and ORF13 oncogenes of A. rhizogenes show considerable promise in plant breeding, but have not been comprehensively studied. Detailed information regarding the morphological impact of specific genes of the Ri plasmid will allow for optimized targeted breeding of plants transformed with the wild type Ri plasmid T-DNA. rolB and ORF13 were recombined into the genome of Arabidopsis thaliana using Gateway ® cloning and the effect on plant growth was assessed biometrically throughout the plants' life cycle. rolB-lines exhibited dwarfing, early necrosis of rosette leaves, altered leaf and flower morphology, and developed an increased number of inflorescences per rosette area compared to the wild type. ORF13-lines were extremely dwarfed, attaining only ca. 1% of the rosette area of the wild type, leaf and flower size was reduced, and the shape modified. The study documents that the traits inferred by the rolB oncogene yield plants with increased formation of generative shoots, but also result in some degree of premature senescence of vegetative organs. The extreme dwarfism seen in ORF13-lines indicate that this oncogene may be more important in the dwarfing response of plants transformed with the wild type Ri plasmid T-DNA than previously assumed and that transformation with this oncogene induces a very compact phenotype., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Heat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)-Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy., Results: At first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. 'Jinshui no. 2') shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5'-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C., Conclusions: Collectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.

Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in 'Borami' collected from Chilgok and Gyeongsan and in 'Secret Pink' from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the 'Yellow Cap' collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

In this study of three grapevine leafroll-associated virus 3 (GLRaV-3) genetic variants in two grapevine rootstock hosts, GLRaV-3 detection was shown to be affected by the virus distribution, titre, and the genetic variant. Group VI and NZ2 GLRaV-3 variants had reduced detectability compared with the group I variant. Differences in the genomic and subgenomic RNA (sgRNA) expression levels, and differences in the level of expression between the genetic variants were also observed. The observed differences in virus titre and sgRNA expression levels suggest differences in plant-virus interactions by the various GLRaV-3 genetic variants.

A new bisegmented dsRNA virus has been detected in shoots of blue spruce with bud blight disease symptoms and infection by Cucurbitaria piceae (Bortw.) fungus. The virus genome consists of two segments that are 2071 and 2257 nt long, encoding the putative RNA polymerase and capsid protein, respectively. Rosellinia necatrix partitivirus 1 is a closely related virus with 45 % amino acid sequence identity in the polymerase, and crimson clover cryptic virus 2 has 36 % amino acid sequence identity in the capsid protein. Based on taxonomic criteria, the virus should be classified as a member of the genus Betapartitivirus (family Partitiviridae). The name Cucurbitaria piceae partitivirus 1 and acronym CpPV1 are proposed.

Background: There is an urgent need in Kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. An in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries., Methods: Malus sieversii Ledeb. M. Roem. and Malus domestica Borkh. accessions were initiated in vitro for virus identification and elimination. Reverse transcription and multiplex PCR were used to test for five viruses. PVS2 vitrification was used as a tool for cryotherapy., Results: Four viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) were detected in 17 accessions. Tomato ringspot virus (ToRSV) was not detected. ACLSV affected 53.8% of the accessions, ASPV 30.8%, ASGV 5.1%, and ApMV was found only in 'Aport Alexander'. Cryotherapy produced virus-free shoot tips for seven of nine cultivars tested. Six cultivars had 60-100% elimination of ACLSV., Conclusions: An in vitro collection of 59 accessions was established. Virus elimination using cryotherapy produced virus-free shoots for seven of nine cultivars and is a promising technique for developing a virus-free apple collection.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Virus tropism is a result of interactions between virus, host and vector species, and determines the fate of an infection. In this study, we examined the infection process of Citrus tristeza virus (CTV) in susceptible and resistant species, and found that the tropism of CTV is not simply phloem limited, but tissue specific. In resistant species, virus infection was not prevented, but mostly restricted to the roots. This phenomenon was also observed after partial replacement of genes of one CTV strain from another, despite both parental strains being capable of systemic infection. Finally, the roots remained susceptible in the absence of viral gene products needed for systemic infection of shoots. Our results suggest that all phloem cells within a plant are not equally susceptible and that changes in host or virus may produce a novel tropism: restriction by the host to a location where further virus spread is prevented., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Grapevine vein clearing virus (GVCV) is a new badnavirus in the family Caulimoviridae that is closely associated with an emerging vein-clearing and vine decline disease in the Midwest region of the United States. It has a circular, double-stranded DNA genome of 7,753 bp that is predicted to encode three open reading frames (ORFs) on the plus-strand DNA. The largest ORF encodes a polyprotein that contains domains for a reverse transcriptase (RT), an RNase H, and a DNA-binding zinc-finger protein (ZF). In this study, two genomic regions, a 570-bp region of the RT domain and a 540-bp region of the ZF domain were used for an analysis of the genetic diversity of GVCV populations. In total, 39 recombinant plasmids were sequenced. These plasmids consisted of three individual clones from each of 13 isolates sampled from five grape varieties in three states. The sequence variants of GVCV could not be phylogenetically grouped into clades according to geographical location and grape variety. Codons of RT or ZF regions are subject to purifying selection pressure. Quantitative polymerase chain reaction assays indicated that GVCV accumulates abundantly in the petioles and least in the root tip tissue. Upon grafting of GVCV-infected buds onto four major grape cultivars, GVCV was not detected in the grafted 'Chambourcin' vine but was present in the grafted 'Vidal Blanc', 'Cayuga White', and 'Traminette' vines, suggesting that Chambourcin is resistant to GVCV. Furthermore, seven nucleotides were changed in the sequenced RT and ZF regions of GVCV from a grafted Traminette vine and one in the sequenced regions of GVCV from grafted Cayuga White but no changes were found in the sequenced regions of GVCV in the grafted Vidal Blanc. The results provide a genetic snapshot of GVCV populations, which will yield knowledge important for monitoring GVCV epidemics and for preventing the loss of grape production that is associated with GVCV.

The sequence alignments of five Tunisian isolates of Prunus necrotic ringspot virus (PNRSV) were searched for evidence of recombination and diversifying selection. Since failing to account for recombination can elevate the false positive error rate in positive selection inference, a genetic algorithm (GARD) was used first and led to the detection of potential recombination events in the coat protein-encoding gene of that virus. The Recco algorithm confirmed these results by identifying, additionally, the potential recombinants. For neutrality testing and evaluation of nucleotide polymorphism in PNRSV CP gene, Tajima's D, and Fu and Li's D and F statistical tests were used. About selection inference, eight algorithms (SLAC, FEL, IFEL, REL, FUBAR, MEME, PARRIS, and GA branch) incorporated in HyPhy package were utilized to assess the selection pressure exerted on the expression of PNRSV capsid. Inferred phylogenies pointed out, in addition to the three classical groups (PE-5, PV-32, and PV-96), the delineation of a fourth cluster having the new proposed designation SW6, and a fifth clade comprising four Tunisian PNRSV isolates which underwent recombination and selective pressure and to which the name Tunisian outgroup was allocated.

In the present study, Yam mosaic virus (YMV) could be efficiently eliminated by cryotherapy in Dioscorea opposita. Shoot apices were precultured for 16 h with 0.3 M sucrose, encapsulated in sodium alginate and dehydrated for 4 h prior to direct immersion in liquid nitrogen. Up to 90 percent of the plants regenerated from cryopreserved shoot tips were YMV-free, whereas only 40% of those regenerated using meristem culture were YMV-free. YMV-free yam plantlets could be propagated in vitro through nodal stem culture, with sequential subculturing at 6-week intervals on medium containing 0.5 mg per liter kinetin. The microtubers formed at the bottom and axil of the explants, incubated at 30 degreeC after being chilled (4 degree C) for 3 months, could be sprouted successfully under in vivo conditions. Healthy plants were established without any damaging symptoms of the virus. Thus, cryotherapy provides an alternative method for efficient elimination of yam viruses, and could be simultaneously used for long-term storage of yam germplasm and for the production of virus-free plants.

Horticultural crops are economically valuable for sustainable agricultural production. Plant diseases caused by Pathogens including virus, phytoplasma and bacterium have been a great threat to production of horticultural crops. The efficient use of pathogen-free plant materials has overcome the menace of plant diseases and has sustained crop production. Cryotherapy of shoot tips, a novel application of cryopreservation technique, has become a new plant biotechnology tool for plant pathogen eradication. When compared with the traditional methods, cryotherapy of shoot tips produces high frequency of pathogen-free plants, which is independent of shoot tip size and cryogenic methods. Cryotherapy of shoot tips has six major steps to produce pathogen-free plants: (1) introduction of infected plant materials into in vitro cultures; (2) excision of shoot tips; (3) cryotherapy; (4) post-culture for plant regeneration; (5) indexing of pathogens in regenerated plants after cryotherapy; and (6) establishment of pathogen-free nuclear stock plants. The key steps 2, 3, and 4 are similar to cryopreservation, and play a major role in obtaining high pathogen eradication frequency.

Fresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 10(6) RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 10(6) to 2 × 10(6) PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (10(5) to 10(6) RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (10(1) to 10(3) PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (10(5) to 10(6) PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.

Many plant viruses encode proteins that suppress RNA silencing, but little is known about the activity of silencing suppressors in roots. This study examined differences in the silencing suppression activity of different viruses in leaves and roots of Nicotiana benthamiana plants. Infection by tobacco mosaic virus, potato virus Y and cucumber mosaic virus but not potato virus X (PVX) resulted in strong silencing suppression activity of a transgene in both leaves and roots, whereas infection by beet necrotic yellow vein virus (BNYVV) and tobacco rattle virus (TRV) showed transgene silencing suppression in roots but not in leaves. For most viruses tested, viral negative-strand RNA accumulated at a very low level in roots, compared with considerable levels of positive-strand genomic RNA. Co-inoculation of leaves with PVX and either BNYVV or TRV produced an increase in PVX negative-strand RNA and subgenomic RNA (sgRNA) accumulation in roots. The cysteine-rich proteins (CRPs) BNYVV p14 and TRV 16K showed weak silencing suppression activity in leaves. However, when either of these CRPs was expressed from a PVX vector, there was an enhancement of PVX negative-strand RNA and sgRNA accumulation in roots compared with PVX alone. Such enhancement of PVX sgRNAs was also observed by expression of CRPs of other viruses and the well-known suppressors HC-Pro and p19 but not of the potato mop-top virus p8 CRP. These results indicate that BNYVV- and TRV-encoded CRPs suppress RNA silencing more efficiently in roots than in leaves.

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

The possibility of eradicating the pelargonium flower break virus (PFBV) and pelargonium line pattern virus (PLPV) by cryotherapy of axillary shoot apices was investigated using five Pelargonium cultivars. Viruses were detected by DAS-ELISA and their location was determined by immunolocalization. Apex culture did not permit elimination of PFBV and only 15 percent regenerated plants of 'Stellar Artic' cultivar were ELISA PLPV-negative. Plants regenerated from cryotherapy-treated apices were tested by DAS-ELISA after a 3-month in vitro culture period. Viruses were not detected in 25 percent and 50 percent of the plants tested for PFBV and PLPV, respectively. However, immunolocalization carried out on apices originating from cryopreserved shoot tips sampled from DAS-ELISA negative plants showed that they were still virus-infected. Using immunolocalization, PFBV and PLPV could be detected in Pelargonium apices, even in the meristematic dome. However, viral particles were more numerous in basal zone cells than in meristematic cells. Our results demonstrate that PFBV and PLPV are present within meristematic cells and that cryopreservation can partly reduce the quantity of these viruses in Pelargonium plants but not eliminate them totally. Additional knowledge on localization and behaviour of viruses during cryopreservation is essential to optimize cryotherapy and plant genetic resource management.

RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3' overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.

Background: Tomato yellow leaf curl virus (TYLCV) is a member of the family Geminiviridae, genus Begomovirus. To test the infectivity of TYLCV in tomato plants, an improved protocol for inoculation of in vitro-cultured tomato plants was developed., Results: A TYLCV isolate was cloned, sequenced and used to construct a 1.8-mer infectious clone. Three weeks old microshoots of TYLCV-susceptible tomato plants were inoculated with Agrobacterium tumefaciens harboring the infectious clone for the TYLCV isolate. After two weeks, the TYLCV symptoms started to appear on the in vitro-inoculated plants and the symptoms became more severe and pronounced eight weeks post-inoculation. The method was used efficiently to uncover the resistance mechanism against TYLCV in Solanum habrochaites accession LA 1777, a wild tomato known for its high resistance to whitefly and TYLCV., Conclusions: The reported in vitro-inoculation method can be used to screen tomato genotypes for their responses to TYLCV under controlled conditions and it will be a useful tool for better understanding of the TYLCV biology in tomato plants.

Piriformospora indica is a root endophytic fungus with plant-promoting properties in numerous plant species and induces resistance against root and shoot pathogens in barley, wheat, and Arabidopsis. A study over several years showed that the endophyte P. indica colonised the roots of the most consumed vegetable crop tomato. P. indica improved the growth of tomato resulting in increased biomass of leaves by up to 20%. Limitation of disease severity caused by Verticillium dahliae by more than 30% was observed on tomato plants colonised by the endophyte. Further experiments were carried out in hydroponic cultures which are commonly used for the indoor production of tomatoes in central Europe. After adaptation of inoculation techniques (inoculum density, plant stage), it was shown that P. indica influences the concentration of Pepino mosaic virus in tomato shoots. The outcome of the interaction seems to be affected by light intensity. Most importantly, the endophyte increases tomato fruit biomass in hydroponic culture concerning fresh weight (up to 100%) and dry matter content (up to 20%). Hence, P. indica represents a suitable growth promoting endophyte for tomato which can be applied in production systems of this important vegetable plant not only in soil, but also in hydroponic cultures.

Infection caused by Cucumber mosaic virus (CMV) is one of the most important viral diseases of pepper worldwide. Young pepper seedlings were inoculated mechanically with CMV-Fny (Fast New York) isolate and were kept in growth chambers at 20-25 degrees C for symptom and fruit development. All inoculated plants developed severe mosaic symptoms and produced fruit except one. Mature seeds were isolated from fruits harvested from CMV-infected plants. Total RNA was extracted from pepper seeds and analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using CMV sub-group IA specific primers. Analysis of individual whole seeds showed that seed-borne infection of CMV in pepper ranged from 95 to 100%. Further seed-growth tests were performed in Petri dishes and CMV was detected in both seed coat and embryo. Seed coat infection of CMV ranged from 53 to 83% while that of the embryo ranged from 10 to 46%. Seed-growth tests in pots were also performed and the rate of seed transmission was approximately 10 to 14%. This is the first report of CMV seed transmission in pepper., (Published by Elsevier B.V.)

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Delta 2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Delta 2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Delta 2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Delta 2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Delta 2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Delta 2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Delta 2b, whereas accumulation of Delta 2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

Cryotherapy is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. It eliminates plant pathogens such as viruses, phytoplasmas and bacteria by briefly treating shoot tips in liquid nitrogen using cryopreservation protocols. Healthy plants are regenerated from the surviving pathogen-free meristematic tissue. The method facilitates treatment of large numbers of samples and is independent of shoot tip size. It has the potential to replace more traditional methods like meristem culture.

Virus-induced gene silencing (VIGS) is increasingly being used as a reverse genetics tool to study functions of specific plant genes. It is especially useful for plants, such as soybean, that are recalcitrant to transformation. Previously, Bean pod mottle virus (BPMV) was shown to be an effective VIGS vector for soybean. However, the reported BPMV vector requires in vitro RNA transcription and inoculation, which is not reliable or amenable to high-throughput applications. To increase the efficiency of the BPMV vector for soybean functional genomics, a DNA-based version was developed. Reported here is the construction of a Cauliflower mosaic virus 35S promoter-driven BPMV vector that is efficient for the study of soybean gene function. The selection of a mild rather than a severe BPMV strain greatly reduced the symptom interference caused by virus infection. The DNA-based BPMV vector was used to silence soybean homologues of genes involved in plant defense, translation, and the cytoskeleton in shoots and in roots. VIGS of the Actin gene resulted in reduced numbers of Soybean mosaic virus infection foci. The results demonstrate the utility of this new vector as an efficient tool for a wide range of applications for soybean functional genomics.

In this study, sugar beet tissue culture clones were used to screen rhizomania resistant genotypes. At first, explants derived from shoot tips of sugar beet seedlings were transferred to shoot tip elongation media after surface sterilization. Then, the grown shoots were transferred to media containing various hormonal combinations NAA, BA, IBA and GA3 for multiplication, growth and rooting. Later, the clones were transferred to soil-peatmoss mixture were adapted to greenhouse conditions. For screening clones against rhizomania, the genotypes of adapted clones were selected and inoculated to rhizomania-infested soil. This experiment was in a randomized complete block design with three replicates (three inoculation times) in greenhouse. Adapted plants were transferred to the soil containing rhizomania virus. All infested soils were diluted 3 to 7 with sand. After two months, infested plants were examined by DAS-ELISA test also optical densities of the samples were analyzed by SAS program. Significant differences among genotypes and blocks were observed. Genotypes were classified to few groups (ranked from completely susceptible to completely resistant). The difference between blocks was because of difference of inoculation time temperature. Use of clones of each genotype caused an increase in selection accuracy of resistant genotypes. By use of this method, chance of escaping from inoculation factor decrease and researchers can determine to be resistance of plants with high level of confidence and apply in breeding programs.

A study was undertaken to determine whether cocoa swollen shoot virus is transmitted by seeds, to improve the robustness of quarantine procedures for international exchange and long term conservation of cocoa germplasm. PCR/capillary electrophoresis, using cocoa swollen shoot virus primers designed from the most conserved regions of the six published cocoa genome sequences, allowed the detection of cocoa swollen shoot virus in all the component parts of cocoa seeds from cocoa swollen shoot virus-infected trees. PCR/capillary electrophoresis revealed the presence of cocoa swollen shoot virus in seedlings raised from seeds obtained from cocoa swollen shoot virus-infected trees. The high frequency with which the virus was transmitted through the seedlings suggested that cocoa swollen shoot virus is transmitted by seeds. This has serious implications for cocoa germplasm conservation and distribution.

Investigations were undertaken on the use of somatic embryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis.

Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.