Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long-term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana ( Musa spp.), Citrus spp., grapevine ( Vitis vinifera ), Prunus spp., raspberry ( Rubus idaeus ), potato ( Solanum tuberosum ) and sweet potato ( Ipomoea batatas ) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing 'citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.     

Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR

Aim

Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously.

Methods and Results

Test‐systems for real‐time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA^® amplifier.

Conclusions

Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA‐ and DNA‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg).

Significance and Impact of the Study

The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.     

Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X

The STH-2 gene is rapidly activated in potato leaves and tubers following elicitation or infection by Phytophthora infestans. However, its biochemical function remains unknown. In order to ascertain if STH-2 protein is directly involved in the defense of potato against pathogens, the STH-2 coding sequence under the control of the CaMV 35S promoter was introduced into potato plants. Transgenic plants expressing the STH-2 gene were analyzed for an altered pattern of susceptibility to a compatible race of P. infestans and to potato virus X. Results indicate that constitutive expression of the STH-2 gene did not reduce susceptibility of potato to these pathogens.     

A major quantitative trait locus for resistance to Potato leafroll virus is located in a resistance hotspot on potato chromosome XI and is tightly linked to N-gene-like markers.

Potato leafroll virus (PLRV) causes one of the most widespread and important virus diseases in potato. Resistance to PLRV is controlled by genetic factors that limit plant infection by viruliferous aphids or virus multiplication and accumulation. Quantitative trait locus (QTL) analysis of resistance to virus accumulation revealed one major and two minor QTL. The major QTL, PLRV.1, mapped to potato chromosome XI in a resistance hotspot containing several genes for qualitative and quantitative resistance to viruses and other potato pathogens. This QTL explained between 50 and 60% of the phenotypic variance. The two minor QTL mapped to chromosomes V and VI. Genes with sequence similarity to the tobacco N gene for resistance to Tobacco mosaic virus were tightly linked to PLRV.1. The cDNA sequence of an N-like gene was used to develop the sequence characterized amplified region (SCAR) marker N127(1164) that can assist in the selection of potatoes with resistance to PLRV.     

Virus content of seed potato stocks produced in a unique seed potato production scheme

Western Australia has a unique seed potato production scheme which has remained virtually unchanged for more than 60 years, consisting of summer plantings of predominantly one cultivar in wind-exposed coastal swamplands. No rotation is used and the scheme relies on natural winter flooding and 'grazing' by sheep to eliminate unharvested tubers. Stocks are recycled every year with only limited inputs of pathogen-tested seed tubers in recent times. Virus spread in the crop is controlled by selecting large tubers for planting, roguing, aphicide application and growing season inspections. Potato viruses X and S were commonly detected in old seed stocks produced by this scheme attaining 100% infection in some. Both viruses were less frequently found in newly introduced seed stocks. By contrast, potato virus Y was never detected and potato leaf roll virus rarely found.     

甘肃马铃薯种植布局对区域气候变化的响应

基于甘肃省地面气象观测站1961—2008年气象观测资料和马铃薯生长条件,选择最佳小网格推算模型推算出500m×500m的高分辨率的网格序列;确立马铃薯种植适宜性气候区划指标,结合地理信息资料,运用GIS技术,开展马铃薯种植适宜性动态气候区划。结果表明:气候变化使马铃薯最适宜区和适宜区面积分别减小35%和3%,次适宜区和可种植区面积分别扩大18.5%和6.6%,不适宜区面积缩小2.0%。提出了马铃薯应对气候变化建议:各地应根据气候特点,调整作物布局;适当调整播种日期,躲避影响马铃薯产量的春霜冻、块茎形成期的高温危害及伏期干旱等;采取多种农业措施,扩大马铃薯种植面积,提高复种指数。预计随着未来气候进一步变暖,该地区的马铃薯生长发育、产量和结构布局将会继续受到影响,研究成果可为甘肃马铃薯生产以及适应气候变化提供科学参考依据。     

A glasshouse test to assess the sensitivity of potato cultivars to tobacco rattle virus

The sensitivity to tobacco rattle virus of nine potato cultivars and 24 potato clones from the potato breeding programme at SCRI was assessed in a glasshouse pot test and in field trials for two consecutive years. The results and analyses presented indicate that the pot test gives an accurate and reliable estimate of sensitivity to tobacco rattle virus. Methods of assessing severity of expression of disease symptoms were examined. The advantages of such a glasshouse pot test and its application are discussed.     

Survival and Transmission of Potato Virus Y,Pepino Mosaic Virus,and Potato Spindle Tuber Viroid in Water

Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: pepino mosaic virus (PepMV), potato virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.     

Effects of In-furrow and Water-run Oxamyl on Paratrichodorus allius and Corky Ringspot Disease of Potato in the Klamath Basin

Corky ringspot disease (CRS) of potato (Solanum tuberosum) is caused by the tobacco rattle virus (TRV), which is vectored by stubby-root nematodes, Paratrichodorus spp. and Trichodorus spp., and is a significant threat to potato quality and production in many areas of the western United States. Between 2002 and 2005, fields with a history of CRS were planted to potato and treated with various combinations of in-furrow (IF) and chemigated (water run, WR) oxamyl [Methyl N'N'-dimethyl-N-[(methyl carbamoyl)oxy]-1-thiooxamimidate] applications. Soil samples were collected to determine how Paratrichodorus allius populations responded to the various treatment regimes (2002-2004); potato tubers were evaluated for symptoms of CRS in 2004-2005. Applications of oxamyl to potato (1.1 kg a.i./ha) did not cause significant mortality of P. allius but did prevent the populations from increasing. Oxamyl applications that began at 55 days after planting (DAP) or later did not control CRS and were not different from the untreated control. However, application schedules that began early-season, either IF at planting, early WR (33 - 41 DAP), or both, significantly reduced CRS expression in cv. Yukon Gold. Therefore, oxamyl applications must be made early in the growing season to be effective in controlling CRS. Effects of oxamyl on CRS may be due to nematostatic action that suppresses feeding activity during early field season when most virus transmission probably occurs.     

Sweet Potato Virus Disease in Sub-Saharan Africa: Evidence that Neglect of Seedlings in the Traditional Farming System Hinders the Development of Superior Resistant Landraces

Sweet potato virus disease (SPVD) occurs in sweet potato at all localities on the perimeter of Lake Victoria areas surveyed in Uganda and Tanzania, and was particularly common in Kagera District in Tanzania and in Rukungiri District in Uganda. All fields were planted with landraces and the most important control practices, as perceived by farmers, were the planting of cuttings derived from only symptomless parents and destroying diseased plants. Although SPVD-resistant landraces were available, they were perceived by most farmers to have poor and late yields. Most farmers considered that their greatest need was new, more acceptable, SPVD-resistant genotypes. Few farmers had seen either sweet potato seeds (15%) or sweet potato seedlings (11%) and, of those that had, most had ignored them. The lack of seedlings and their neglect by farmers is likely to be hindering the evolution of more acceptable, SPVD-resistant landraces, and is probably responsible for SPVD being a long-term disease problem.     

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.     

Managing soilborne diseases of vegetable crops with a pre-plant soil or substrate amendment of a corn distillation product

The efficacy of condensed distiller's solubles (CDS), a co-product of ethanol production from corn, rich in organic matter, and high in carbon to nitrogen ratio, was tested as a pre-plant amendment against Verticillium wilt of eggplant and potato scab in potato soils from commercial fields and against damping-off diseases of radish and cucumber seedlings in a peat-based mix and muck soil. Eggplants grown in a potato soil amended with CDS (1% mass/mass) showed less Verticillium wilt and increased fresh (37-54%) and dry (31-45%) plant biomass compared to the control in the greenhouse. In a potato soil with medium levels of disease, CDS (1 or 2%) increased the percentage of marketable tubers by 116% under greenhouse, 119% under micro-plot and 75% under field conditions. In the growth room, CDS (1, 2, and 4%) amendment to a peat-based mix infested with Rhizoctonia solani 1 week before planting seeds improved the percentage of healthy radish seedlings (22-72% healthy seedlings compared to 2% in the control). Levels of disease suppression increased with the incubation time prior to planting. Disease control effect of CDS was not consistent between different batches of peat-based mix. In a non-suppressive batch of peat-based mix, disease suppression by CDS was enhanced by a bio-control agent, Trichoderma hamatum 382. In muck soil from a commercial field naturally-infested with Pythium spp., CDS (0.25, 0.5, and 1%) provided protection of cucumber seedlings from damping-off immediately after incorporation, but the maximum protection was seen after 1 week with all three rates. The number of total bacteria was enhanced in the CDS-amended muck soil. In the micro-plots, CDS (0.5 and 1%) as an amendment to muck soil 2 weeks before planting improved the percentage of healthy cucumber seedlings and fresh plant weight compared to the control. CDS is not toxic to the pathogens and disease suppression is believed to be due to biological activity stimulated by CDS in the substrate.     

Development of a genotyping method for potato scab pathogens based on multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S-23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

Introduction a potato cultivar "sprit" as relatively resistant to main fungal pathogens causal agents of early blight and wilting on potato in Iran

Potato (Solanum tubersum L.) is one of the most human food production cultured in Iran especially Zanjan province as a temperate region. Some fungal pathogens caused severely infected on potato tubers or foliage in the majority grown areas and resulted yield losses in potato production. Recent years from 2002 to 2004 infected samples were collected from different potato grown regions in Zanjan province then cultured on PDA after surface sterilization with sodium hypochlorite. Isolated fungal pathogens were identified and study showed the main pathogens with high incidence and frequency were Alternaria solani, Fusarium oxysporum and Verticillium sp. in studied areas. The regions which used convention varieties showed more diseases than other locations which used relatively resistant races. The rate of resistance for 10 international potato varieties was studied by inoculation of them by 10(5) spores suspension of three common fungal pathogens in the field. Study showed Sprit cultivar was more resistant than others to all three common pathogens and Lady-Claire was most susceptible. Yield production of Sprit per unit of land area was also exceeded that of other cultivars by factors of 1.10 to 2.25 respectively. The results of the study helped potato growers to culture Sprit cultivar and have good yield production in Zanjan and Hamedan provinces in this year.     

Development of a Genotyping Method for Potato Scab Pathogens Based on Multiplex PCR

Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S–23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.     

Ectopic expression of a UDP-glucose:phenylpropanoid glucosyltransferase leads to increased resistance of transgenic tobacco plants against infection with Potato Virus Y

Transgenic tobacco plants over-expressing a salicylate- and pathogen-inducible glucosyltransferase (TOGT) acting on various phenylpropanoids show enhanced resistance against infection with potato virus Y (PVY). The transgenic plants are characterized by a several-fold increased glucosyltransferase activity in leaves as well as in roots. Under non-infectious conditions profiles of phenylpropanoids in leaves of transgenic lines were similar to that of controls. Feeding experiments with leaf-discs demonstrated a higher capacity for glucosylation of the coumarin scopoletin. After inoculation with PVY the transgenic lines showed similar formation of necrotic leaf lesions but significantly decreased levels of virus coat-protein when compared with control plants. Thus, our results imply that the activity of TOGT and the subsequent accumulation of glucosylated coumarins represent an important step in the cascade of events resulting in confinement of viral pathogens.     

Flight take-off performance of Colorado potato beetle in relation to potato phenology

The flight take-off frequency of adult Colorado potato beetles, Leptinotarsa decemlineata (Say), from potato plants, Solanum tuberosum L. 'Red Pontiac' at the bloom stage of development was 2.2-2.5-fold that of Colorado potato beetle from plants at the vegetative stage. Tests were conducted in a flight chamber over a period of 3 h. Prefeeding Colorado potato beetles for 48 h on potato plants at the bloom or at the vegetative stage before placing them into the flight chamber resulted in the same significantly higher flight take-off frequency from potato plants at the bloom stage than from plants at the vegetative stage. These results demonstrate that the factor in potato plants in bloom that stimulates the flight take-off of the Colorado potato beetle is independent of the feeding history of the beetles and begins acting only when the beetles are in the presence of the plant. According to these results, the dispersal of adult Colorado potato beetles from potato fields in bloom to younger potato fields with plants at the vegetative stage, previously reported in the literature, is at least partly explained by the effect of plant phenology on the frequency of flight take-off. Results confirm the value of planting potato fields of similar phenology over as wide an area as possible to reduce Colorado potato beetle dispersal between fields. Results also imply that staggering the planting dates of conventional potato refuge areas near Colorado potato beetle transgenic or conventionally resistant potato fields is a sound management practice, because it promotes the movement of wild beetles over to the adjacent younger resistant crops.     

A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens

Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.     

Persistence of Phoma exigua xar. foveata and Polyscytalum pustulans in dry soils from potato stores in relation to reinfection of stocks derived from stem cuttings

The incidence of Phoma exigua var. foveata and Polyscytalum pustulans in dry soil and dust from potato stores was assessed at 10 farms in Scotland producing virus-tested stocks of seed potatoes derived from stem cuttings (VTSC). Samples were collected on three occasions during 1976: during the storage period (February-March), soon after the potatoes were planted (May) and just before the new crop was lifted and stored (August-October). Both pathogens were detected at all three sampling times but P. exigua var. foveata was more frequently detected at the last one than P. pustulans. Soil and dust from various sites within the stores, such as floors, ledges, graders and boxes, were contaminated by these pathogens. Propagules of both pathogens remained airborne for at least 12–17 min after the floors were swept. P. exigua var. foveata remained viable for at least 16 months in dry soil from tuber surfaces stored at 4–6°C. When VTSC tubers were dusted before planting with a dry store soil contaminated by P. exigua var. foveata the gangrene potential of the progeny tubers was much greater than that from untreated tubers or from tubers treated with an uncontaminated soil. The role of inoculum surviving in dry soil is discussed in relation to reinfection of VTSC stocks.     

Use of resistant and susceptible potato cultivars in the trap cropping of potato cyst nematodes, Globodera pallida and G. rostochiensis

The effects of planting date and growing period of potato cultivars on their efficiency as trap crops for potato cyst nematodes (PCN) were studied. Plots were planted with susceptible or resistant cultivars in April, June and August and these were grown for 5, 6 or 7 wk before removal of the plants by hand lifting. Crops planted in June provided the best overall reductions in PCN population density of up to 95%, with cv. Santé significantly more effective than the other cultivars. Population reductions from the August planting were only slightly less than from planting in June but the tuber yields obtained were much greater: Maris Piper and Maris Bard produced 16.4 and 21.4 t ha^-1 respectively, with 37% and 43% respectively, of a size useful for canning (i.e. between 20 and 40 mm diameter).