Exogenous application of melatonin improves eradication of apple stem grooving virus from the infected in vitro shoots by shoot tip culture

Production and maintenance of virus-free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of apple stem grooving virus (ASGV) from virus-infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole-3-acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus-infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus-infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus-free, while no virus-free shoots were obtained in shoot tips of the virus-infected shoots cultured without melatonin. Analyses by microtissue direct RT-PCR and RT-qPCR showed that ASGV concentration decreased in shoot tips of the virus-infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the virus-infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus-free plants.     

Shoot tip cryotherapy for plant pathogen eradication

Diseases caused by plant pathogens such as viruses, viroids and phytoplasmas cause huge economic losses of agricultural production and limit the safe movement of plant materials across borders. The use of pathogen-free planting materials provides a strategy for efficient management of these diseases and facilitates the global exchange of genetic resources. Shoot tip cryotherapy is a novel biotechnology method that uses cryogenic procedures to eradicate plant pathogens from the diseased plants. Combining thermotherapy or chemotherapy with shoot tip cryotherapy has further enhanced pathogen eradication efficiency. This review provides updated and comprehensive information on shoot tip cryotherapy and the combination of thermotherapy or chemotherapy with shoot tip cryotherapy for pathogen eradication. Prospects are proposed for future studies.     

吉林、黑龙江毛葱病毒病调查

本研究为明确吉林和黑龙江省毛葱病毒病发生率,从两省5个地区共采集255份毛葱样品。根据毛葱4种主要病毒基因组序列设计特异性引物,对胡葱黄条病毒Shallot yellow stripe virus(SYSV)和青葱X病毒Shallot virus X (SVX)、洋葱黄矮病毒Onion yellow dwarf virus (OYDV)和葱潜隐病毒Shallot latent virus (SLV)进行双重RT-PCR检测。结果表明,229份样品检出病毒,带毒率为89.8%,SLV的检出率最高,达87.06%,OYDV次之,为36.86%,SYSV检出率偏低,为0.78%;同时存在病毒复合侵染,其中双病毒复合侵染为SLV和OYDV,检出率为33.3%;三病毒复合侵染为SYSV、OYDV和SLV,检出率为0.78%,未发现4种病毒复合侵染。本研究为吉林和黑龙江种植区毛葱病毒病防治提供了参考依据。     

Survey and characterization of potyviruses and their strains of Allium species

Nearly 5700 plants of 14 cultivated and 8 wildAllium species and varieties from the Netherlands and other parts of the world, were tested for infection with aphid-borne potyviruses by ELISA, electron microscope decoration tests and/or inoculation onto test plants. This resulted in the detection of two known viruses, viz. leek yellow stripe virus (LYSV) and onion yellow dwarf virus (OYDV), and the discovery and characterization of two new viruses, viz. shallot yellow stripe virus (SYSV) and Welsh onion yellow stripe virus (WoYSV), and of six strains of these viruses. ‘Garlic mosaic’, ‘barlic yellow streak’, ‘onion mosaic’, ‘shallot mosaic’, ‘shallot X’, and ‘shallot yellows’ viruses, incompletely described in the literature, are now reidentified as well-known viruses or as strains or mixtures of such viruses. ‘Garlic yellow stripe virus’ is also a complex containing a potyvirus possibly differing from the viruses found in this survey. The symptoms of the potyviruses studied varied widely and ranged from mild to severe chlorotic to yellow striping of leaves, and they are of little diagnostic importance.LYSV was found in vegetatively propagated pearl onion (A. ampeloprasum var.sectivum) from Europe and Asia. It has decreased in leek crops (A. ampeloprasum var.porrum) in the Netherlands since the 1970, apparently due to resistance in new cultivars. OYDV was common in onion (A. cepa var.cepa) from the former USSR and North Africa, and in European cultivars of shallot (A. cepa var.ascalonicum), with the exception of the highly resistant ‘Santé’, but was not detected during this survey in Asian shallot. European samples of ever-ready onion (A. cepa var.perutile), multiplier onion (A. cepa var.aggregatum) and tree onion (A. cepa var.viviparum) contained OYDV. It was also found in sand leek (A. scorodoprasum) from european gene collections. A strain of OYDV from onion and shallot in Morocco and Spain was virulent on onion and shallot cultivars resistant to common OYDV, as reported early for a similar isolate in the USA.Asian shallot appeared generally infected with the new SYSV, similar to OYDV in host range and symptoms but serologically distinct. It was not detected in onion and shallot from Europe or North Africa. A virulent strain of this virus caused striping in sap-inoculated garlic (A. sativum) and Formosan lily (Lilium formosanum). The new WoYSV, infecting Welsh onion in Indonesia and Japan, was earlier described in Japan as OYDV from rakkyo and Welsh onion. It appeared serologically closely related to SYSV and distantly to OYDV, but differed in its host range.Host-specific strains of LYSV and OYDV were detected in garlic, wild garlic (A. longicuspis), an unidentifiedAllium species (suffix-G), and great-headed garlic (A. ampeloprasum var.holmense) (suffix-GhG)., LYSV-G and OYDV-G infected on average 45% and 73%, respectively, of the garlic samples of worldwide origin. Symptoms of isolates of both strains varied in severity, implying the necessity of serological tests for disease diagnosis and health certification. LYSV-GhG was the cause of yellow striping in 93% of the great-headed garlic plants tested, mainly from the Mediterranean area. One sample was also infected with OYDV-GhG.Many samples from vegetatively propagated crops grown from non-certified planting stock contained a few plants free of potyviruses, implying the possibility to obtain healthy (and possibly resistant) selections of such cultivars avoiding meristem-tip culture. Cross-protection of garlic sets by a mild potyvirus isolate seems to be an alternative to the use of vulnerable virus-free sets.Generally, viruses and virus strains could not be transmitted to anyAllium species other than their natural host, except to the highly susceptible crow garlic (A. vineale). This species, and other predominantly vegetatively propagating wildAllium spp. (field garlic,A. oleraceum; ramsons,A. ursinum; sand leek), were found not to be reservoirs of viruses that might infectAllium crops in the netherlands. Streaking in vegetatively propagated wild leeks (A. ampeloprasum and closely related species) originating from the Mediterranean area and Asia was due to an undescribed miteborne virus. The survey confirmed that spread of potyviruses inAllium crops in the Netherlands is from planting sets, and from a neighbouring crop only if of the same species.     

Efficient elimination of sweetpotato little leaf phytoplasma from sweetpotato by cryotherapy of shoot tips

Shoot tips with 3–4 leaf primordia were excised from in vitro -grown sweetpotato plants ( Ipomoea batatas ) infected with little leaf phytoplasma ( Candidatus Phytoplasma aurantifolia) and subjected to cryotherapy. All plants regenerated from the cryo-treated shoot tips were free of phytoplasma, whereas shoot tip culture or dehydration of shoot tips without subsequent cryotherapy resulted in phytoplasma-free plants at a frequency of only 7–10%. Histological and ultrastructural studies with light and transmission electron microscopy, respectively, indicated that cryotherapy was lethal to all cells except those in the apical dome of the meristem and the two youngest leaf primordia. These surviving parts of the shoot tip contained vascular tissue and sieve elements, but electron microscopy showed no phytoplasma in them. In contrast, an abundance of phytoplasma was found in sieve elements located at the lower, non-surviving parts of the shoot tip 1·0 or 1·5 mm from the apical dome. In the greenhouse, the plants in which phytoplasmas were not detected were healthy-looking, grew vigorously and were readily distinguished from the infected plants that exhibited little leaf and chlorosis symptoms, proliferation of axillary shoots and roots, stunting, and heavily reduced number and size of storage roots. In this study efficient elimination of phytoplasma and production of pathogen-tested plant stocks were achieved with the novel cryotherapy-based approach. The proposed advantage of the technique is that it can be simultaneously used for long-term storage of plant germplasm and for production of pathogen-free plants.     

Essential oil production increased by using virus-free patchouli plants derived from meristem-tip culture

For the production of virus-free patchouli ( Pogostemon cablin ), isolated meristem tips were cultured on a medium supplemented with 0·2 ppm 6-benzylaminopurine. Multiple shoot proliferation was initiated during the culture. Complete plantlets were regenerated by transferring multiple shoots to a medium devoid of phytohormones. Plantlets thus produced were transplanted to soil with a high rate of survival and tested by enzyme-linked immunosorbent assay (ELISA) to check elimination of patchouli mild mosaic virus. Following further propagation by stem cuttings, virus-free plants were grown in the northern and southern parts of Mindanao Island, Philippines. Significant increases in biomass and essential oil yield were observed in virus-free plants grown at both localities. Using gas chromatographic analyses, a full set of major sesquiterpenes was detected in the essential oils recovered from virus-free patchouli plants. Reinfection of healthy clones with the virus took place within 4 months of cultivation in the field.     

Efficiency of eradication of four viruses from garlic (Allium sativum) by meristem-tip culture

Mechanical inoculation tests and ELISA with sap from garlic plants used for sanitation by meristem-tip culture revealed four viruses, viz. garlic common latent virus (GCLV) (carlavirus), the garlic strains of leek yellow stripe virus (LYSV-G), onion yellow dwarf virus (OYDV-G) (aphid-borne potyviruses), and onion mite-borne latent virus (OMbLV-G) (taxonomically unassigned virus). The same tests performed on explants grownin vitro showed elimination efficiencies of 100% for LYSV-G, 92% for OYDV-G, 62% for GCLV, and less then 54% for OMbLV-G.Meristem tips excised from garlic cloves and bulbils, 0.15–1.0 mm in size, were tested for regeneration and efficiency of virus elimination after transfer to Murashige and Skoog medium. Successful regeneration into plantlets was obtained with 71% of the meristems from cloves and 72% of those from bulbils, but virus elimination was easiest from cloves: 38% of all explants from cloves and 25% of those from bulbils were virus-free. The efficiency of elimination increased with increasing weight of the cloves, irrespective of the virus. Small tip size seemed to favour virus elimination, but sizes smaller than 0.4 mm led to increasing failure of regeneration.Micropropagation was most successful when cytokinins were omitted from the medium and the garlic shoot was split. Multiplication factors of 3–6 were obtained.     

京郊大蒜病毒病的研究及其鳞茎中病毒的脱除

 京郊大蒜病毒病发生普遍,主要症状为条纹花叶、矮化和叶片扭曲畸形。病体细胞中含大量线状病毒粒体和风轮状内含物。线状病毒粒体长度范围为250-1875nm,以长度550-800nm粒体居多。其中长700-800nm的粒体被鉴定为大蒜花叶病毒(GMV),回接脱毒大蒜叶片产生条纹花叶症状。血清学鉴定表明GMV与洋葱黄矮病毒(OYDV)有近缘关系。长度500-600nm的粒体可能为大蒜潜隐病毒(GLV),回接脱毒大蒜不产生花叶症状。下述两种病毒为京郊大蒜的主要病毒。此外,个别标样含烟草花叶病毒,但不是大蒜的主要病毒。对800nm以下线状病毒粒体归属尚待研究。
应用营养茎尖、生殖茎尖和根尖分生组织培养技术,可以脱去上述主要病毒,获得脱毒大蒜。     

Mite-borne virus isolates from cultivated Allium species,and their classification into two new rymoviruses in the family Potyviridae

While testing several samples of onion and of vegetatively propagated garlic, sand leek and shallot from a number of countries, virus isolates with unusually flexuous particles were obtained by mite (Aceria tulipae) or sap transmissions. No aphid-borne poty-or carlavirus was transmitted by mites, and mite-borne virus isolates could not be transmitted by aphids. The mite-borne isolates did not react with antisera to aphid-borne potyviruses ofAllium spp. or with the Agdia potyvirus group monoclonal. In contrast to the mite-borne onion and garlic mosaic viruses reported in the literature, our mite-borne isolates induced no visible or only very mild symptoms inAllium spp., except isolates from shallot ‘Santé’ which caused diffuse striping. Heavily mite-infested test plants or plant samples showed streaking and malformation due to mite feeding (tangle-top). The mite-borne virus isolates could be classified with test plants and a discriminating antiserum into three groups, representing two viruses and a strain of one of them. They are tentatively named onion mite-borne latent virus (OMbLV), garlic strain of this virus (OMbLV-G), and shallot mite-borne latent virus (SMbLV). Mite transmission, length of virus particles (ca. 700 to 800 nm), and the presence of granular inclusion bodies in infected tissue indicate that the viruses belong to the mite-borne genusRymovirus of the familyPotyviridae. OMbLV from shallot and onion, and OMbLV-G from garlic and sand leek, can be assayed onChenopodium murale but differ in their natural hosts. They are very common. SMbLV, to whichC. murale does not react, was isolated from shallot originating from Asia and Russia.     

A new filamentous virus in shallot

Serological analysis (ELISA, immunoblotting, and immunoelectron microscopy) of the partially purified virus preparations obtained from shallot plants (selection sample no. 83) inoculated with the Mongolian isolate of onion yellow dwarf potyvirus (OYDV) revealed neither an OYDV coat protein antigen nor virions decorated by the corresponding antiserum. At the same time, these preparations contained many flexuous filamentous virus particles (FFVP) with a modal length of 780 nm. It is suggested that shallot no. 83 is resistant at least to the Mongolian isolate of OYDV, while the discovered FFVPs are the virions of some other viral agent. The nature and origin of a new virus are now under study in this laboratory     

Further Evidence that Shallot Yellow Stripe Virus (SYSV) Is a Distinct Potyvirus and Reidentification of Welsh Onion Yellow Stripe Virus as a SYSV Strain

ABSTRACT An antiserum to shallot yellow stripe virus (SYSV) was raised and used in combination with a range of other antisera to potyviruses of Allium spp. in electron microscopic decoration experiments. The serological results corroborated an earlier finding that the type isolates of SYSV and Welsh onion yellow stripe virus (WoYSV) are closely related to each other and only distantly related to onion yellow dwarf (OYDV) and leek yellow stripe (LYSV) viruses, the two other major potyviruses infecting Allium spp. Moreover, the decoration results indicated that Japanese potyviruses named OYDV and Wakegi yellow dwarf virus are isolates of SYSV. Sequence analysis of the 3'-terminal regions of the SYSV and WoYSV ge-nomes revealed coat protein (CP) amino acid and 3'-nontranslated region (3'-NTR) nucleotide sequence identities of 95 and 89%, respectively. The CP amino acid and 3'-NTR nucleotide sequences of these viruses differed from those of OYDV and LYSV by >25 and >67%, respectively. The serological and molecular studies showed that SYSV and WoYSV are different strains of a potyvirus distinct from OYDV and LYSV. For priority reasons, we propose that these strains together with the Wakegi-type isolates of OYDV described in Japan be referred to as SYSV and that SYSV isolates from Allium spp. other than shallot be designated as the Welsh onion strain of SYSV (SYSV-Wo).     

苹果脱毒技术研究进展

苹果（Malus pumila Mill.）普遍感染病毒。目前, 培育无病毒原种母本树, 建立用于繁殖接穗和营养系砧木的母本园, 栽植无病毒苗木, 是防治病毒病的根本措施。本文针对常见的4 种苹果病毒及1 种类病毒, 综述了茎尖培养、热处理、化学处理、微茎尖嫁接以及低温处理脱除苹果病毒方法的研究进展, 分析了不同方法的应用效果, 及所适合脱除的病毒种类, 以期为我国苹果病毒脱除技术研究提供参考信息。     

Testing garlic cloves and bulblets for onion yellow dwarf virus by ACP-ELISA

Onion yellow dwarf virus (OYDV) was detected in cloves and aerial bulblets of garlic (Allium sativum) at levels as high as or higher than in leaves of plants grown from tested cloves. It is recommended to test bulblets or a few cloves per bulb before planting to determine if all cloves of a bulb are virus-free. This aids in early detection and allows a more thorough testing of stock than field testing.     

Leek yellow stripe virus and its relationships to onion yellow dwarf virus; characterization,ecology and possible control

Since 1970 yellow stripe disease of leek (Allium porrum) has developed epidemically in the south-eastern part of the Netherlands coincident with increasing year-around cultivation of the crop. Many autumn and winter crops now become totally infected. Apparently similar attacks, first reported in Germany in 1937, are increasingly attracting attention in various European countries. This paper describes the leek yellow stripe virus (LYSV) as a new potyvirus related to onion yellow dwarf virus (OYDV), which was so far incompletely described. LYSV is hardly infectious to onion (A. cepa) and shallot (A. ascalonicum) and OYDV behaves similarly on leek. The leek virus further differs from OYDV in not being infectious toA. fistulosum and in causing distinct local lesions onChenopodium amaranticolor andC. quinoa. The two viruses closely resemble each other in external symptoms in their respective hosts, in persistence of infectivity in expressed sap, and in particle morphology and length (LYSV 820 nm; OYDV 833 nm). Intracytoplasmic inclusion bodies slightly differ. Further biophysical characters of the two viruses, such as sedimentation coefficient (OYDV 143 S), buoyant density in CsCl (LYSV 1.326; OYDV 1.306, or 1.258 in Cs₂SO₄), and molecular mass of coat protein subunit (LYSV 34000; OYDV 30000 dalton), are characteristic of the potyvirus group, but do not assist in judging their relationships. Serologically they are only distantly related if at all. The leek virus is not seed-borne. It is aphid-transmitted in the non-persistent manner and its main epidemic build-up is during late summer and autumn. The sole sources of infection are nearby leek crops. Awaiting the development of resistant leek cultivars, it is advised to avoid sowing leek seed beds and planting spring crops near overwintering leek, and to remove infected plants showing up during summer.     

Improved PCR detection of potyviruses in <Emphasis Type="Italic">Allium</Emphasis> species

Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RT-nested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5–27.3 ng ml⁻¹. The RT-nested-PCR done after DAS-ELISA was 10² times more sensitive than the DAS-ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 10⁴ times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS-ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR.     

New mite-borne virus isolates from rakkyo,shallot and wild leek species

Flexuous viruses were transmitted from rakkyo (Allium chinense) and wild leek species (especiallyA. commutatum) to plants of crow garlic (A. vineale), by transfer of dry bulb mites. By electron microscope decoration tests using three antisera and by inoculations onto test plants, it was concluded that from each of the two natural host species at least two viruses were isolated. The viruses from wild leeks are both pathogenic onAllium spp. and may be of economic importance. Decoration tests on a virus mixture from shallot obtained earlier, revealed another new mite-borne virus in this species. The mite-borne viruses ofAllium spp. appear to be very common; they are largely diverse and their identification remains difficult.     

Detection and elimination of viruses infecting sweet potatoes in Hungary

Sweet potato has been grown in Hungary for the last three decades, and its popularity is increasing among farmers and consumers. Its production is hampered by pests and diseases due to poor agricultural practices, such as the use of virus-infected propagation materials. We tested the presence of 15 viruses by PCR and quantitative PCR in 110 sweet potato plants collected from seven regions in Hungary. Seven viruses in single or multiple infections associated with a wide range of foliar symptoms were detected: sweet potato chlorotic stunt virus (SPCSV), sweet potato virus G (SPVG), sweet potato virus C (SPVC), sweet potato feathery mottle virus (SPFMV), sweet potato virus 2 (SPV2), sweet potato leaf curl virus (SPLCV), and sweet potato pakakuy virus (SPPV). This is the first report on the occurrence of the begomovirus SPLCV in sweet potatoes in Hungary. The infectivity and identity of these viruses were confirmed through bioassays (grafting to Ipomoea setosa) and sequencing of the PCR-amplified sections of their genomes, respectively. Due to the necessity for virus-free sweet potato propagation material in Hungary, virus elimination was carried out successfully in five out of six genotypes important for Hungarian farmers using heat treatment and meristem tip culture. All five viruses detected in the plants before heat treatment were removed except SPPV, which persists after heat treatment. Production and strict regulation of virus-free sweet potato propagation materials are recommended to avoid exacerbating the virus situation and protect Hungarian farmers from further losses.     

Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue blot immunoassay

Sugarcane yellow leaf virus (SCYLV), a member of the Luteoviridae , is implicated in the sugarcane disease known as yellow leaf syndrome (YLS), which is characterized by yellowing of the leaf midrib followed by leaf necrosis and possible growth suppression. YLS is distributed worldwide and susceptible cultivars are commonly infected with SCYLV. However, not all cultivars infected with SCYLV show symptoms of YLS and some cultivars that show symptoms do so sporadically. Since it is difficult to obtain virus-free plants of susceptible cultivars, it has not been possible to study the factors involved in SCYLV infection nor the effects of infection on plant growth and yield. A tissue blot immunoassay was used to visualize in vivo presence of the virus so that virus-infected and virus-free plants could be distinguished. Meristem tip cultures were used to produce virus-free plantings of six SCYLV-susceptible sugarcane cultivars. Nearly all of the regenerated sugarcane lines remained virus-free over a period of up to 4 years, whether grown in isolated fields or in the glasshouse. Experimental re-infection of the virus-free plants by viruliferous aphids demonstrated that meristem tip culture did not affect susceptibility of sugarcane to SCYLV. Improved diagnosis and production of virus-free plants of SCYLV-susceptible cultivars will facilitate research to quantify the effect of the virus on yield and to analyse the processes involved in disease development.     

甘薯脱毒技术及增产效果研究

作者于１９８８～１９９５年研究了甘薯病毒及甘薯脱毒技术,明确了侵染山东甘薯的主要病毒种类是甘薯羽状斑驳病毒和甘薯潜隐病毒,在国内首次分离侵染甘薯的烟草花叶病毒,探明了其生物学特性。筛选出适合山东甘薯茎尖培养基的最佳激素配比,浓度,ｐＨ值。探索了脱毒薯的增产机理和增产效果,提出了组织培养,茎尖苗检测,脱毒薯速率与推广应用的配套技术规程,培养出徐薯１８等１２个品种的脱毒苗,平均增产４２．９％出干率提高