Elimination of <Emphasis Type="Italic">Plum pox virus</Emphasis> through in vitro thermotherapy and shoot tip culture compared to conventional heat treatment in apricot cultivar Bebecou

The effectiveness of in vitro thermotherapy (explants at 35–37°C for 20 days) in obtaining propagating material of apricot (Prunus armeniaca L.) cultivar Bebecou that is free of Plum pox virus (PPV) was compared to a conventional heat treatment technique (potted plants at 30–35°C for 8 weeks). An improved protocol for in vitro thermotherapy was combined with shoot tip culture. The protocol is 5.8 times more effective than conventional thermotherapy while taking half the time and is likely to have a high commercial impact for nurseries.     

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.     

Double-edged effects of the cryogenic technique for virus eradication and preservation in shallot shoot tips

Plant virus eradication is a prerequisite for the use of virus-free propagules for sustainable crop production. In contrast, virus preservation is required for all types of applied and basic research of viruses. Shoot tip cryopreservation can act as a double-edged strategy, facilitating either virus eradication or virus preservation in cryoderived plants. Here, we tested the efficacies of shoot tip cryopreservation for virus eradication and preservation in shallot (Allium cepa var. aggregatum). In vitro stock shallot shoots infected with onion yellow dwarf virus (OYDV) and shallot latent virus were thermotreated for 0, 2, and 4 weeks at a constant temperature of 36℃ before shoot tip cryopreservation. Results showed that viruses were preserved in recovered shoots when thermotherapy was not applied. Although thermotherapy lowered the regrowth levels of cryotreated shoot tips, the efficiency of virus eradication increased from 5% to 54%. Immunolocalization of OYDV and histological observation of cryotreated shoot tips showed the high frequency of virus preservation was due to the viral invasion of cells close to the apical meristem and the high proportion of cells surviving. Four weeks of thermotherapy drastically decreased the distribution of OYDV, as well as the percentage of surviving cells within the shoot tips, thereby promoting virus eradication. Virus-free plants obtained from combining thermotherapy with cryotherapy showed significantly improved vegetative growth and bulb production. The present study reports how thermotherapy can act as a trigger to facilitate either the safe preservation of Allium viruses or the production of virus-free shallot plants.     

Roles of stolbur phytoplasma and <Emphasis Type="Italic">Reptalus panzeri</Emphasis> (Cixiinae,Auchenorrhyncha) in the epidemiology of Maize redness in Serbia

Maize redness (MR), a disease causing midrib, leaf and stalk reddening and abnormal ear development in maize, has been reported from Serbia, Romania and Bulgaria for 50 years. Recent epiphytotics reduced yields by 40%–90% in southern Banat, Serbia. MR was recently associated with the presence of the stolbur phytoplasma, although the epidemiology of the disease remained unknown. Diseased fields in southern Banat were surveyed for potential vectors of the phytoplasma during 2005 and 2006, and high populations of Reptalus panzeri were found. In affected fields, 20% of the R. panzeri individuals and 85% of symptomatic maize plants carried the stolbur phytoplasma. When stolbur phytoplasma-infected R. panzeri were introduced into insect-free mesh cages containing healthy maize plants, midrib and leaf reddening developed on 48% of plants and stolbur phytoplasma was detected in 90% of the symptomatic plants. No symptoms or phytoplasma-positive plants were found in cages without insects. These data indicate that MR symptoms are associated with the stolbur phytoplasma. Reptalus panzeri is both abundant in affected fields and can transmit the stolbur phytoplasma, indicating the insect is likely to be a major vector of MR.     

植物类病毒脱除技术进展

类病毒是已知最小的植物病原物,可侵染蔬菜、果树以及花卉等多种农作物,并对农业生产造成严重影响。目前,对感染类病毒的植株进行脱毒处理是一种有效的防控措施,本文针对果树、蔬菜和花卉上几种常见的类病毒,综述了茎尖培养、热处理结合茎尖培养、低温处理、超低温处理、化学处理及不含叶原基的顶端分生组织再生等植物类病毒的脱除技术,分析了不同方法的应用效果及所适合脱除的类病毒种类,同时展望了类病毒病害在未来植物病害防控中的重要地位,并提出综合考虑各种因素、制定合理方案、选用适宜方法的类病毒脱除建议。     

Untersuchungen zur Vermehrbarkeit der neuen Zierpflanze Clitoria ternatea L. var. pleniflora Fantz

Nowadays, only little information regarding the climber Clitoria ternatea L. var. pleniflora Fantz as “new ornamental plant” does exist in the German-speaking area. This plant is from interest because of its characteristic attractive, bright blue flowers. The vegetative propagation of Clitoria ternatea was investigated at the Institute for Horticultural Sciences, Humboldt-University of Berlin. The objective was to study how different types of cuttings could influence the rooting, proliferation and growth behaviour of greenhouse plants cultivated in two different seasons (summer and autumn). It was found that either stem or tip cuttings from the lower and middle zones of the mother plants with a fully developed leaf are suitable for summer or autumn propagation. Also, it was shown that the cutting variants affected the branching behaviour and further plant growth. Nearly 90% of the trial plants remained with a single shoot. However, in the vegetation period 11% of the plants originating from tip cutting and 14% of the plants originating from stem cuttings could be found with two branches. In autumn, the formation of lateral shoots was strongly declining. Here, the shoot length was particularly influenced by using different types of cutting treatments during propagation. The stem cuttings of the first batch planted clearly developed plants with longer shoots compared to those growing from tip cuttings. In the second batch planted, a lower shoot length growth was generally recorded (means of 5 to 6?cm for the internodes after three-month cultivation). In conclusion, the results regarding the propagation show that C.?ternatea can be recommended as a new ornamental container plant. However, further studies should follow in order to investigate possible photoperiod effects as well as suitable cultivation measures.     

Role of <Emphasis Type="Italic">Cacopsylla pyri</Emphasis> in the epidemiology of pear decline in Spain

The frequency of pear decline-positive insects and transmission of pear decline (PD) phytoplasma by Cacopsylla pyri in Spain has been studied. Psyllids were used for experiments on phytoplasma transmission both to healthy Pyrus communis trees and to an artificial feeding medium. Over a period of 1 year, about 100 psyllids were collected monthly from pear trees, cv. Williams, using the beating tray method, and tested for the presence of PD phytoplasma. Results indicate that the frequency of PD positive psyllids changes through the year and that C. pyri transmits the pear decline associated disease agent. Phytoplasma transmission was also effective under laboratory conditions using a feeding medium. The relationship between PD positive Cacopsylla pyri, Pear decline phytoplasma transmission and the sex of the vector was also evaluated. Although the percentage of PD positive psyllids was similar in both genders, PD phytoplasma transmission by females was significantly higher than by males. Since the sex ratio (male/female) was less than 1:1 for most of the year, these results should be taken into consideration for controlling Pear decline in Mediterranean climates.     

Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture

Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT–PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.     

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.     

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。     

离体黑果枸杞再生途径的研究

以黑果枸杞的叶片和茎段为外植体,采用MS为基本培养基,研究激素对经历愈伤组织诱导、丛生芽诱导以及叶片直接诱导小植株再生途径的影响。实验结果表明,诱导茎段、叶片形成愈伤组织的适宜培养基分别为MS+2,4-D0.3 mg·L~(-1)、MS+2,4-D0.4 mg·L~(-1),其诱导率均为100%;诱导茎段、愈伤组织分化形成丛生芽的适宜培养基分别为MS+6-BA0.2 mg·L~(-1)+KT0.1 mg·L~(-1)、MS+6-BA0.5 mg·L~(-1),而愈伤组织分化出的丛生芽均发生玻璃化现象,其增殖系数分别为32.3倍、47.1倍;诱导叶片分化形成植株的适宜培养基为MS+NAA0.01 mg·L~(-1),其再生植株诱导率为33.3%。结论:黑果枸杞再生能力强,以上途径均能形成再生植株,其最佳的离体繁殖途径为茎段诱导丛生芽形成再生植株。     

Molecular characterization and phylogeny of phytoplasma associated with bunchy top disease in its new host Okra (<Emphasis Type="Italic">Abelmoschus esculentus</Emphasis>) in India reveal a novel lineage within the 16SrI group

Okra plants with bunchy top disease were found to be prevalent during the period of August–October 2009 in New Delhi, India. The common symptoms observed were shortening of internodes, aggregation of leaves at the apical region, reduced leaf lamina, stem reddening, fruit bending, phyllody and stunting of plants. The disease incidence ranged from 2–60% accompanied by significant reductions in production of both flowers and seeds. Nested polymerase chain reaction targeting phytoplasma specific 16S rDNA and rp genes revealed all symptomatic plants to be positive for phytoplasma. Homology searches depicted its closest identity to phytoplasmas of 16SrI ‘Candidatus Phytoplasma asteris’, like the Sugarcane yellows and Periwinkle phyllody phytoplasmas. Profiles for 16S rDNA obtained with 10 restriction endonucleases, differed in TaqI sites for two phytoplasma isolates (BHND5 & 10) from the standard pattern of 16SrI-B subgroup, the latter was seen in the case of isolate BHND1. Restriction fragment analysis of rp genes with AluI, Tsp509I matched with patterns of the rpI-B phytoplasmas. Phylogenetic reconstruction of rp genes revealed okra bunchy top phytoplasma (BHND1) as a divergent isolate, the subsequent sequence analysis of which showed the presence of a novel BslI site. These significant differences suggest that multiple phytoplasma strains are affecting okra, one of which is a diverging lineage within the 16SrI-B group while others represent a new 16SrI subgroup not reported so far. Additionally, this is the first report of a phytoplasma associated disease in okra plants worldwide.     

Increased plant tolerance against chrysanthemum yellows phytoplasma (‘Candidatus Phytoplasma asteris’) following double inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif

The aim of this work was to assess the effects of a combined inoculum of a rhizobacterium and an arbuscular mycorrhizal (AM) fungus on plant responses to phytoplasma infection, and on phytoplasma multiplication and viability in Chrysanthemum carinatum plants infected by chrysanthemum yellows phytoplasma (CY). Combined inoculation with Glomus mosseae BEG12 and Pseudomonas putida S1Pf1Rif resulted in some resistance to phytoplasma infection (about 30%), delayed symptom expression in nonresistant plants, improved growth of the aerial part of the infected plants (+68·1%), and altered root morphology (root tip number: +49·9%; branching degree: +82·8%). Combined inoculation with the two beneficial microorganisms did not alter CY multiplication and viability. In inoculated and infected plants, phytoplasma morphology was typical of senescent cells. A more active and efficient root system in double‐inoculated plants probably mediated the effects of the two rhizospheric microorganisms in the infected plants. The practical application of rhizospheric microorganisms for mitigating phytoplasma damage, following evaluation under field conditions, represents an additional tool for the integrated management of phytoplasmosis.     

Transmission and detection of toria [<Emphasis Type="Italic">Brassica rapa</Emphasis> L. subsp. <Emphasis Type="Italic">dichotoma</Emphasis> (Roxb.)] phyllody phytoplasma and identification of a potential vector

Phyllody disease associated with 16SrIX phytoplasma was observed in the range of 4.1–11% in 10 different lines of toria [Brassica rapa L. subsp. dichotoma (Roxb.)] in experimental fields of the Indian Agricultural Research Institute, New Delhi, India during 2008 and 2009. The toria phyllody (TP) phytoplasma was detected in all the symptomatic and 13.3% of asymptomatic toria plants by nested PCR. The phytoplasma was detected in midrib, flower part, siliquae, stem, and root of infected plants as well as seeds. TP was transmitted by grafting and by dodder to toria and nine other rapeseed/mustard species as confirmed by nested PCR. However, symptoms of phytoplasma infection were induced only in toria, yellow sarson [Brassica rapa L. subsp. trilocularis (Roxb.)], brown sarson [Brassica rapa L. subsp. sarson (Prain)], rapeseed (B. napus subsp. oleifera), and rocket or taramira (Eruca sativa) but not in mustard (B. juncea), black mustard (B. nigra), Ethiopian mustard (B. carinata), B. tournefortii and white mustard (Sinapis alba). Transmission of TP phytoplasma to periwinkle (Catharanthus roseus) was successful only through dodder, but no transmission to tomato (Lycopersicon esculentum) or brinjal (Solanum melongena) was found. TP phytoplasma was detected in Laodelpax striatellus, an abundant planthopper in toria fields, which indicates that this planthopper may be a potential vector for TP phytoplasma.     

Sensitive Detection of a Phytoplasma Associated with Little Leaf Symptoms in Withania somnifera

Withania somnifera is an important medicinal plant native to the Indian-sub continent. Owing to the presence of a number of precious alkaloids, flavonoids and withanolides, it is widely used in the Indian and African systems of medicines. It is severely affected by phytoplasma present in the sieve tubes of phloem. With a view to micropropagate phytoplasma-free W. somnifera plants, an efficient and effective nested PCR-based system was developed for detection of associated phytoplasmas. Universal primers, designed from the 16S rDNA sequences of phytoplasmas, were applied in direct/nested-PCR. Total DNA extracts from leaf tissues of 33 suspected symptomatic and 11 non-symptomatic plants were subjected to direct PCR. The direct PCR products were subsequently employed as templates in nested PCR. The nested PCR could reamplify direct PCR products yielding a DNA fragment of 1.4 kb. A phytoplasma was detected in all the diseased plants and not from the healthy looking plants. Further, it was sensitive enough to amplify phytoplasma DNA obtained from crude DNA diluted up to 2500 times from naturally infected plants and also from various stages of in vitro-propagated diseased plants. Identical restriction fragment polymorphism enzyme profiles were obtained following restriction enzyme digestion of nested PCR products, obtained from five different plants, by EcoRI, AluI and RsaI restriction endonucleases. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro-generated plants and probably identification of, as yet unknown, hosts and vectors of phytoplasma associated with phytoplasma disease of W. somnifera.     

Induction of heat resistance in Fusarium oxysporum and Verticillium dahliae caused by exposure to sublethal heat treatments

The effects of sublethal heat treatments on heat resistance were studied forFusarium oxysporum f.sp.dianthi (Fod) andVerticillium dahliae (Vd), one isolate of each pathogen. Treatments of propagule suspensions of Fod at 55°C and of Vd at 45°C for 30 min were survived by less than 0.001% and 0.01% of the propagules, respectively. Pretreatment of suspensions of Fod at 45°C increased survival of the 55°C treatment up to 0.73% of the propagules and pretreatment of suspensions of Vd at 40°C increased survival of the 45°C treatment up to 0.40%. Induction of heat resistance was dependent on duration of the exposure to the sublethal temperature. With Fod, this duration was shorter for propagules from old cultures than for those from young cultures. Pretreatment at 45°C of a suspension of an 1-week-old culture of Fod induced resistance when lasting 30 min or longer, but not when 20 min or shorter. With Vd, the duration of the pretreatment inducing heat resistance depended on type of culture — white or black — due to differences in microsclerotia formation. Implications of induced heat resistance for control of plant diseases by thermotherapy are discussed.     

Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir

Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.     

In vitro screening for resistance to apple proliferation in Malus species

A screening system for apple proliferation resistance was developed, based on in vitro graft‐inoculation with the causal agent ‘Candidatus Phytoplasma mali’. For this, in vitro cultures of the field‐resistant apomictic genotypes Malus sieboldii, H0909, D2212 and the susceptible Malus × domestica genotypes Golden Delicious and rootstock M9 were established, as well as in vitro cultures of Rubinette and Golden Delicious infected with ‘Ca. P. mali’ strains PM4 and PM6, respectively. Healthy in vitro shoots were inoculated by micrografting with infected shoots used as graft tip. After 6 weeks graft contact no significant differences for graft quality were observed between healthy and infected grafts. Mortality of grafts and transmission rates were not significantly different among the different genotypes. The phytoplasma concentration in inoculated shoots was determined at different times post‐inoculation (p.i.) by quantitative real‐time PCR. Infected M. sieboldii and D2212 had lower phytoplasma concentration than the susceptible controls and showed no symptoms. H0909 showed an intermediate behaviour exhibiting lower phytoplasma concentrations with strain PM4 but growth was affected. The dynamics of phytoplasma concentration reached a maximum at 6–8 months p.i. for all genotypes but the values for 3–5 and 10–12 months p.i. were similar. The resistance of M. sieboldii and D2212 was confirmed in vitro. A significant difference in phytoplasma concentration was observed between strains PM4 and PM6.     

A New Natural Planthopper Vector of Stolbur Phytoplasma in the Genus Pentastiridius (Hemiptera: Cixiidae)

A new disease of sugar beet called Syndrome des Basses Richesses, which appeared in Burgundy and Franche-Comté, France, in 1991, is of uncertain aetiology. However, evidence for aerial transmission of the disease, symptom similarity with yellow wilt and preliminary results of phytoplasma detection, support the hypothesis of a phytoplasma being associated to the disease. A search for a natural phytoplasma vector, was conducted in Franche-Comté in 1997 and 1998, in an area where sugar beet crops had been affected since 1996. A cixiid, tentatively identified as Pentastiridius beieri, not described in the preceding years and not formerly reported as a phytoplasma vector, was present in sugar beet plots in high populations from June to August in 1997 and 1998. Individuals were captured and used for transmission experiments to periwinkle and sugar beet seedlings. They were further tested for the presence of a phytoplasma in their body, using PCR amplification of 16S rDNA of phytoplasmas. In 1997 and 1998, from 2% to 13.3% of the individuals carried a stolbur phytoplasma and insects which tested positive, appeared to have transmitted, through feeding, a stolbur phytoplasma to periwinkles and to sugar beets. This cixiid, whose vectoring capacity of stolbur phytoplasma to plants, is now clearly demonstrated, is available for experimental inoculations, in order to examine the role of phytoplasmas in the Syndrome des Basses Richesses, through the observation of symptom expression in phytoplasma-inoculated plants.     

<Emphasis Type="Italic">In vitro</Emphasis> screening for sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>L.) resistant calli to <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> fungal culture filtrate

Diaporthe helianthi the causal agent of sunflower (Helianthus annuus) stem canker, causes significant reductions in yield and oil content in most sunflower-growing areas. With the aim of enhancing host resistance, we selected in vitro sunflower calli against culture filtrates of two pathogen isolates (7/96 and 101/96). This technique may be an effective and rapid tool to discriminate the most virulent D. helianthi isolate and to screen for host resistance in the early stage of a breeding programme. Further investigation on the mechanisms involved in defence pathways showed no induction of salicylic acid and pathogenesis-related proteins in calli, indicating that the host resistance is not associated with Systemic Acquired Resistance but probably other biochemical mechanisms.