Sanitation of olive plants infected by Verticillium dahliae using heat treatments

Verticillium wilt, caused by the fungus Verticillium dahliae, is currently the most important disease affecting olive in the Mediterranean basin. There are no effective treatments for controlling this disease. The use of infected nursery stocks has largely contributed to the spread of the pathogen, and therefore the development of treatments to preventively sanitize the propagation stock is critical in the nursery industry. This study describes novel techniques to achieve this aim. The effects of several temperature–exposure time combinations were evaluated: (i) the survival of pathogen on culture medium (PDA); (ii) the pathogen viability on infected shoots and plants; (iii) the vegetative growth of plants of several cultivars; and (iv) the rooting ability of cuttings. The colonies of the pathogen growing in PDA were killed after 8 h and 60 min of exposure at 40 and 47°C, respectively. Temperatures ≥42°C for at least 2 h were lethal for the pathogen infecting the shoots. Likewise, moist hot air treatments at 42–44°C for 6–12 h eradicated the pathogen, without compromising the viability of the plants. Five olive cultivars were also evaluated and classified according to their thermotolerance as follows: sensitive (Chiquitita), moderately sensitive (Koroneiki, Frantoio and Picual) and heat tolerant (Arbequina). However, the optimized sanitation methods were applicable to all of the cultivars. Finally, heat treatments were applied to unrooted cuttings, which severely affected their rooting ability. Thus, this study developed a hot air treatment to produce V. dahliae‐free olive nursery plants.     

Assessment of somaclonal variability in hop (Humulus lupulus L.) in vitro meristem cultures and clones by molecular methods

In vitro meristem tissue cultures are used for production of virus-free rootstocks of hop (Humulus lupulus L.). Because use of plant tissue cultures is associated with occurrence of somaclonal variability, we assessed somaclonal variability in hop meristem in vitro cultures before and after thermotherapy by different molecular methods (RFLP, RAPD, STS, ISSR and AFLP) and compared it with existing clonal variability of Osvald's clones 31, 72 and 114. No molecular differences were observed between mother plants and in vitro mericlones by RFLP and STS analyses. Amplified molecular differences were found in RAPD and ISSR products of one from five in vitromericlones cvs. Eroica (E5) and Southern Brewer (SB2), respectively. Similarities with mother plants were 0.965 and 0.913 (JSC), respectively. Specific amplified polymorphic products were found for every mericlone and mother plant in AFLP reactions and variability of DNA sequence ranged from 0.824 to 0.993 (JSC). This variability was very similar to determined intra-clonal variability within Osvald's clones 31, 72 and 114 by AFLP analysis. Inter-clonal variability of DNA sequence was exactly higher than intra-clonal variability of DNA sequence in these clones. The molecular differences between Osvald's clone 72 normal and meristem derived were not verifiable. Thermotherapy increased frequency of molecular changes, since amplified differences were found in 14 from 20 in vitro mericlones of cv. Eroica, in 6 from 11 in vitro mericlones of cv. Yeoman and in 15 from 23 in vitro mericlones of cv. Southern Brewer by RAPD and ISSR analyses.     

Cryotherapy of Potato Shoot Tips for Efficient Elimination of Potato Leafroll Virus (PLRV) and Potato Virus Y (PVY)

Viral diseases constitute a major constraint to high yield and high quality production of potato. Potato leafroll virus (PLRV) and Potato virus Y (PVY) are among the most damaging potato viruses and are prevalent in most potato growing areas. In the present study, attempts were made to eliminate PLRV and PVY by three cryogenic protocols, i.e., encapsulation-dehydration, encapsulation-vitrification and droplet. Results showed that both PLRV and PVY could be efficiently eliminated by cryogenic treatments with 83–86% and 91–95% of frequencies of virus-free plantlets obtained for the former and latter, respectively. Frequencies of virus-free plantlets produced by cryogenic treatments were higher than those by meristem culture (56% for PLRV and 62% for PVY) and thermotherapy (50% for PLRV and 65% for PVY), and similar to those by thermotherapy followed by meristem culture (90% for PLRV and 93% for PVY). Survival (75–85%) and regrowth (83–89%) from cryo-treated shoot tips were higher than those from meristem culture (50–55%) and thermotherapy followed by meristem culture (40–50%), but similar to those from thermotherapy (80–87%). The morphology of the plantlets regenerated from cryo-treated shoot tips was similar to that of non-treated plantlets. Thus, cryotherapy would provide an alternative method for efficient elimination of potato viruses, and can be simultaneously used for long-term storage of potato germplasm and for production of virus-free plants.     

Potato virus X eradication from potato meristem tips held at 30°C

Summary Thermotherapy of PVX infected potato plants at 30°C followed by in vitro tip culture at 24°C was more or less successful depending on the duration of the heart treatment. Virus inactivation in meristematic tissues was much more efficient when the heat treatment was applied to mature plants rather than to meristem tips cultured in viro. By combining 30°C treatment of potato plants with 30°C tip culture. PVX eradication was not improved but the final proportion of PVX-free plantlets was increased because the tip population developed faster, giving a higher number of rooted plantlets at 30°C than at 24°C.     

广西葡萄良种脱毒研究进展

建立葡萄脱毒技术并繁育和推广葡萄良种脱毒苗木,对于广西葡萄产业的健康可持续发展极为重要。本研究团队在广西最先开展了葡萄脱毒技术研究。目前已建立了10种葡萄主要病毒的RT-PCR检测方法,并建立了热处理结合茎尖培养的葡萄脱毒技术,且已成功对‘阳光玫瑰’葡萄、‘夏黑’葡萄和‘巨峰’葡萄三个优良品种的带毒单株进行了脱毒。研究成果有助于进一步推动广西葡萄产业的健康可持续发展。     

Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy

Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL~(–1); R15 and R25, respectively), thermotherapy(37°C; T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were 68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.     

湿度对田间自然热罩防治柑橘黄龙病的影响

比较加湿处理后热罩和自然条件下热罩对黄龙病的防治效果,结果表明自然条件处理后30 d叶片平均病菌浓度下降了85％以上,而加湿条件下叶片平均病菌浓度仅下降3.7％,与自然条件处理间差异达到显著水平;自然条件热罩处理后90 d叶片平均病菌浓度下降了97％以上,而加湿处理病树叶片平均病菌浓度下降了38％,与自然条件处理间的差异达到极显著水平。对照组叶片平均病菌浓度处理后30 d和90 d均有略微增加;加湿未热罩处理叶片平均病菌浓度在处理后30 d和90 d分别增加了4.5倍和8.7倍,但与对照组无显著差异。本研究结果表明加湿热罩处理病树显著减弱了其杀灭黄龙病菌效果,同时表明增加土壤湿度对黄龙病菌繁殖可能具有一定的促进作用。     

热处理结合茎尖组织培养脱除唐菖蒲病毒的研究

应用热处理与茎尖组织培养相结合的方法，对唐菖蒲携带的ＣＭＶ，ＢＹＭＶ，ＴＭＶ进行脱病毒研究，脱毒试管苗进行了生物测定和电镜观察，对流免疫电泳测定，间接酶联免疫吸附法测定，结果表明，热处理结合茎尖组织培养的方法脱病毒效果优于单独进行茎尖组织培养的效果。４２℃处理５ｄ的茎尖组织培养试管苗脱病毒的综合效果最佳，同时改进了唐菖蒲组织培养中的脱分化及生根培养基，缩短了培养周期，使一个茎尖殖体一年内可扩繁３０     

Attempts to eliminate <Emphasis Type="Italic">Candidatus</Emphasis> phytoplasma phoenicium from infected Lebanese almond varieties by tissue culture techniques combined or not with thermotherapy

Elimination of Candidatus phytoplasma phoenicium from two infected Lebanese varieties of almond by using different tissue culture techniques is reported. Except for the oxytetracycline therapy which totally inhibited the development of explants, stem cutting cultures associated with thermotherapy, shoot tip cultures associated or not with thermotherapy, and shoot tip micrografting were all suitable, either for shoot regeneration or for elimination of phytoplasma from the two varieties. However, stem cutting culture coupled with thermotherapy seemed to be the most effective for regeneration of phytoplasma-free plantlets.     

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.