Potato Leaf Tissue Research Articles

A plant cell wall-associated kinase encoding gene is dramatically downregulated during nematode infection of potato

ABSTRACT Plant cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs) have been increasingly recognized as important regulators of plant immunity against various plant pathogens. However, the role of the WAK/WAKL family in plant-nematode interactions remains to be determined. Here, we analyzed a WAK-encoding gene (Soltu.DM.02G029720.1) from potato (Solanum tuberosum). The Soltu.DM.02G029720.1 encoded protein contains domains characteristic of WAK/WAKL proteins and shows the highest similarity to SlWAKL2 from tomato (S. lycopersicum). We thus named the gene as StWAKL2. Phylogenetic analysis of a wide range of plant WAKs/WAKLs further revealed close similarity of StWAKL2 to three WAK/WAKL proteins demonstrated to play a role in disease resistance. To gain insights into the potential regulation and function of StWAKL2, transgenic potato lines containing the StWAKL2 promoter fused to the β-glucuronidase (GUS) reporter gene were generated and used to investigate StWAKL2 expression during plant development and upon nematode infection. Histochemical analyses revealed that StWAKL2 has specific expression patterns in potato leaf and root tissues. During nematode infection, GUS activity was mostly undetected at nematode infection sites over the course of nematode parasitism, although strong GUS activity was observed in root tissues adjacent to the infection region. Furthermore, mining of the transcriptomic data derived from cyst nematode infection of Arabidopsis roots identified a few WAK/WAKL genes, including a StWAKL2 homologue, found to be significantly down-regulated in nematode-induced feeding sites. These results indicated that specific suppression of WAK/WAKL genes in nematode-induced feeding sites might be crucial for cyst nematodes to achieve successful infection of host plants. Further studies are needed to uncover the role of WAK/WAKL genes in plant defenses against nematode infection.

Biocontrol of potato early blight and suppression of Alternaria grandis sporulation by Clonostachys spp.

AbstractEarly blight, caused by Alternaria solani and A. grandis, severely affects potato crops around the world, and few management options apart from intensive fungicide use are available. In this work, we tested whether biocontrol treatments with different species of the mycoparasitic fungus Clonostachys could reduce early blight severity on the foliage of pot‐grown potatoes of cultivar Ágata, and affect the survival and sporulation of the causal pathogen A. grandis. Twenty isolates from five Clonostachys species were screened in a preliminary biocontrol trial, and nine of them were selected and tested further in two confirmation trials. Treatments with three isolates from three species (C. chloroleuca, C. pseudochroleuca, C. rhizophaga) resulted in a significant reduction in disease severity in both confirmation trials, with control efficacy ranging from 88.7% to 92.9% in the first trial, and from 83.1% to 84.7% in the second trial. All Clonostachys isolates used in the confirmation trials survived on potato leaf tissues for at least 15 days, and resumed growth when subjected to high humidity, resulting in mycoparasitic overgrowth and significant reduction of the pathogen sporulation. Our results provide evidence that different species of Clonostachys, not only the ubiquitous C. rosea, are sources of useful candidates for the biocontrol of potato early blight and possibly other plant diseases caused by fungal pathogens.

Cultivar-specific nutritional status of potato (Solanum tuberosum L.) crops.

Gradients in the elemental composition of a potato leaf tissue (i.e. its ionome) can be linked to crop potential. Because the ionome is a function of genetics and environmental conditions, practitioners aim at fine-tuning fertilization to obtain an optimal ionome based on the needs of potato cultivars. Our objective was to assess the validity of cultivar grouping and predict potato tuber yields using foliar ionomes. The dataset comprised 3382 observations in Québec (Canada) from 1970 to 2017. The first mature leaves from top were sampled at the beginning of flowering for total N, P, K, Ca, and Mg analysis. We preprocessed nutrient concentrations (ionomes) by centering each nutrient to the geometric mean of all nutrients and to a filling value, a transformation known as row-centered log ratios (clr). A density-based clustering algorithm (dbscan) on these preprocessed ionomes failed to delineate groups of high-yield cultivars. We also used the preprocessed ionomes to assess their effects on tuber yield classes (high- and low-yields) on a cultivar basis using k-nearest neighbors, random forest and support vector machines classification algorithms. Our machine learning models returned an average accuracy of 70%, a fair diagnostic potential to detect in-season nutrient imbalance of potato cultivars using clr variables considering potential confounding factors. Optimal ionomic regions of new cultivars could be assigned to the one of the closest documented cultivar.

Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato

In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6–98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

Metabolic diversity in sweet potato (Ipomoea batatas, Lam.) leaves and storage roots

Sweet potato (Ipomoea batatas, Lam.) is an important root vegetable in developing countries. After its domestication in Neotropical America, human migration led to the distribution of the sweet potato plant throughout the world. Both leaf and storage root are high in compounds of nutritional value. Yet, the storage roots are of particular value due to their significant content of provitamin A (β-carotene). The breeding effort for elite sweet potato lines led to the reduction of genetic diversity and the potential to improve other traits. The focus of the present study was to assess the metabolic diversity of 27 sweet potato cultivars including landraces and improved varieties. A metabolite profiling approach was optimised for sweet potato leaf and storage root tissue and 130 metabolites identified with three different analysis platforms. The data highlighted a lack of correlation between storage root phenotype and leaf metabolism. Furthermore, the metabolic diversity of storage roots was based on the secondary metabolism, including phenylpropanoids and carotenoids. Three cultivars of three different flesh colouration (yellow, orange and purple) showed a significant difference of the primary metabolism. This data demonstrates the value of metabolite profiling to breeding programs as a means of identifying differences in phenotypes/chemotypes and characterising parental material for future pre-breeding resources.

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.)

BackgroundStarch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism.ResultsWe identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis.ConclusionsThis study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR.

Time-course investigation of Phytophthora infestans infection of potato leaf from three cultivars by quantitative proteomics.

Potato late blight is one the most important crop diseases worldwide. Even though potato has been studied for many years, the potato disease late blight still has a vast negative effect on the potato production [1], [2], [3]. Late blight is caused by the pathogen Phytophthora infestans (P. infestans), which initiates infection through leaves. However, the biological activities during different stages of infection are poorly described, and could enable novel or improved ways of defeating late blight infection [4]. Therefore, we investigated the interactions between P. infestans (mixed strain culture) and potato (Solanum tuberosum). Three commercially available field potato cultivars of different resistance to late blight infection; Kuras (moderate), Sarpo Mira (highly resistant) and Bintje (very susceptable) were grown under controlled green house conditions and inoculated with a diversity of P. infestans populations.We used label-free quantitative proteomics to investigate the infection with P. infestans in a time-course study over 258 h. Several key issues limits proteome analysis of potato leaf tissue [5], [6], [7]. Firstly, the immense complexity of the plant proteome, which is further complicated by the presence of highly abundant proteins, such as ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Secondly, plant leaf and potato, in particular, contain abundant levels amounts of phenols and polyphenols, which hinder or completely prevent a successful protein extraction. Hitherto, protein profiling of potato leaf tissues have been limited to few proteome studies and only 1484 proteins have been extracted and comprehensively described [5], [8], [9]. We here present the detailed methods and raw data by optimized gel-enhanced label free quantitative approach. The methodology enabled us to detect and quantify between 3248 and 3529 unique proteins from each cultivar, and up to 758 P. infestans derived proteins. The complete dataset is available via ProteomeXchange, with the identifier PXD002767.

Photosynthetic responses of potato to Colorado potato beetle injury and differences in injury between adult males and females

AbstractEstablishing rates of injury to plants and the physiological impact of this injury provides essential data in the development of economic injury levels, but variation of sex effects is not often considered. Here, we examined injury by the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), larvae and adult males and females on potato, Solanum tuberosum L. (Solanaceae). Specifically, we looked for adult sex differences between males and females in injury rates (= leaf consumption rates), and examined the impact of all types of injury (larval, adult male, and adult female) on gas exchange parameters of remaining potato leaf tissue. Experiments were conducted in the field and in growth chambers on Frito‐Lay proprietary and Pike chipping‐potato varieties at pre‐blooming and blooming stages. We found no change in photosynthetic rates on remaining (uninjured) leaf tissue infested with male, female, or fourth‐stage larva of Colorado potato beetle. However, when the midrib was cut in trials with male beetles, the remaining tissue above the injury exhibited photosynthetic rate reductions as a result of stomatal limitations. These findings are consistent with the pattern that we and other researchers have observed with gross tissue removal by various insects on other plant species. Adult females consumed more tissue than males, and temperature was positively correlated with feeding rates for both sexes. Sex‐related differences in feeding rate are most important to studies quantifying consumption rates for economically important species because of its potential impact on resulting economic injury level calculations.

Rapid Laboratory Evaluation of Fungicides against Phytophthora infestans Causing Late Blight of Potato

Aim: To develop method for rapid and simple laboratory evaluation of fungicides against Phytophthora infestans, the causal pathogen of late blight of potato. Study Design: Descriptive statistics and analysis of variance. Each treatment was replicated thrice. Place and Duration of Study: Department of Plant Protection, Palli-Siksha Bhavana (Institute of Agriculture), Visva-Bharati, India in February, 2014. Methodology: Small portion of potato leaf tissue from advancing margin of late bight lesion is placed on water since Phytophthora infestans (Mont.) de Bary grow well in water producing hypha and sporangium. Using this property, performance of fungicides on mycelial growth and sporangia production/formation was assayed by placing a small bit of infected leaf tissue in different Short Research Article Mondal et al.; JSRR, 4(2): 168-173, 2015; Article no. JSRR.2015.019 169 concentration of fungicides. Results: Among the seventeen fungicides tested, chlorothalonil, fenamidone + mancozeb and tricyclazole inhibited mycelial growth and sporangia production but dimethomorph, tebuconazole + trifloxystrobin inhibited sporangia production only. Metalaxyl + mancozeb totally inhibited mycelial growth at higher concentration. Conclusion: This method can be useful for evaluating and comparing performance of different fungicides and even same formulations of a fungicide, produced by different companies within a very short time period. The experimental work can be completed within 48 –72h if late blight infected leaves are available.

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure

Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15–30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG–Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.

Effects of drying on caffeoylquinic acid derivative content and antioxidant capacity of sweet potato leaves

Caffeoylquinic acid (CQA) derivatives are known to possess antioxidative potential and have many beneficial effects on human health. The present study compared the CQA contents and antioxidant activities of aerial parts of sweet potato plants. The effects of drying methods (freeze drying, and drying at 30°C, 70°C, and 100°C) on these two parameters of the first fully expanded leaves were also assessed. The results indicated that the CQA derivatives were detectable in leaves, stem, and flowers of sweet potato plants (varied from 39.34 mg/g dry weight to 154.05 mg/g dry weight), with the leaves (particularly expanding and first fully expanded leaves) containing more CQA derivatives than other aerial plant parts. The expanding and first fully expanded leaves also exhibited greater antioxidant activities than other aerial plant parts, possibly due to their higher contents of CQA derivatives. Drying method significantly affected the content of CQA derivatives in dried sweet potato leaf tissues. Drying treatments at both 70°C and 100°C significantly reduced the CQA derivative content and antioxidant activity in the first fully expanded leaves. Among the tested drying methods, the freeze-drying method demonstrated the preservation of the highest amount of CQA derivatives (147.84 mg/g) and antioxidant property. However, 30°C cool air drying was also a desirable choice (total CQA derivative content was reduced to only 129.52 mg/g), compared to 70°C and 100°C hot air drying, for commercial-scale processing of sweet potato leaves, if the higher operation cost of freeze drying was a major concern.

Invasion of Solanum tuberosum L. by Aspergillus terreus: a microscopic and proteomics insight on pathogenicity.

BackgroundAspergillus terreus is one of the most harmful filamentous fungal pathogen of humans, animals and plants. Recently, researchers have discovered that A. terreus can cause foliar blight disease in potato (Solanum tuberosum L.). We used light and scanning electron microscopy, and performed proteomics analysis in an attempt to dissect the invasion process of A. terreus in this important crop.ResultsMicroscopic study revealed that invasion of leaf tissue is marked by rapid germination of A. terreus phialidic conidia (PC) by 4 h after inoculation. By 8 h after inoculation, primary germ tubes from PC differentiated into irregular protuberance, often displayed stomata atropism, and failed to penetrate via the epidermal cells. Colonization of leaf tissues was associated with high rate of production of accessory conidia (AC). These analyses showed the occurrence of a unique opposing pattern of AC, tissue-specific and produced on melanized colonizing hyphae during the infection of leaf tissue. A significant proteome change hallmarked by differential expression of class I patatin, lipoxygenase, catalase-peroxidase complex, and cysteine proteinase inhibitor were observed during tuber colonization. These proteins are often involved in signal transduction pathways and crosstalk in pathogenic responses.ConclusionA. terreus abundantly produced AC and multipolar germinating PC to invade potato leaf tissue. Additionally, A. terreus differentially induced enzymes in potato tuber during colonization which facilitates rapid disease development.

An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y.

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

Protein Profiling in Potato (Solanum tuberosum L.) Leaf Tissues by Differential Centrifugation

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Highly sensitive field test lateral flow immunodiagnostics of PVX infection

A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.

Study on the Relationship Between the Toxin of Phytophthora infestans (Mont.) de Bary and Resistance of Potato

Plant pathogenic fungi produce mycotoxins which may lead host plants to produce disease symptoms and may have a significant positive role in the course of disease development. The putative Phytophthora infestans toxins were prepared by culturing in liquid medium (60 g rye and 10% tomato juice L- 1) for 1 mon at 17°C, filtering through four layers of cheesecloth and being precipitated by ammonium sulfate. The resulted putative toxin solutions were used to study the effects on 3 potato varieties (both leaf and tuber tissues). The results show that potato leaves and tubers exhibit symptoms similar to the late blight resulted from P. infestans infection. Potato varieties reacting differentially to both the toxin dilutions and toxins produced by different P. infestans isolates suggested the presence of toxin-mediated specificities between potato and P. infestans. Potato leaf and tuber tissues have different and contrary reactions to the toxin. Toxin-mediated specificities are likely present between potato and P. infestans.

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low-cost assay for the quantification and in planta monitoring of Phytophthora infestans growth. In this study, we describe a SYBR Green-based quantitative real-time PCR (qPCR) method for the quantification of P. infestans. The method is based on a simultaneous plant-pathogen DNA purification followed by a qPCR in which the relative quantification of pathogen and plant DNA is performed. Besides assuring an accurate quantification, the use of a plant gene provides a reliable indicator of sample quality, allowing the exclusion of inappropriate samples. By applying this methodology, we were able to detect P. infestans in potato leaf and tuber tissue before the first symptoms of the disease were observed and to monitor the in planta growth of the pathogen for 6 days. This is a reliable low-cost assay that provides rapid, accurate and sensitive quantification of the late blight pathogen, allowing the in planta monitoring of P. infestans growth. The quantitative nature of the assay described in this study may be useful in plant breeding programmes and basic research. The method is appropriate for the comparison of cultivars with different, and even subtle, degrees of pathogen resistance and in the screening of new anti-oomycete compounds. The method can be easily adapted to tomato and the model plant Nicotiana benthamiana.

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.

Detection of potato spindle tuber viroid (PSTVd) in minute amounts of potato (Solanum tuberosum L.) leaf tissue by hybridization techniques and, together with potato viruses, by multiplex RT-PCR

Potato spindle tuber viroid (PSTVd) is one of the major constraints for the improvement of potato production worldwide. Sensitive and sophisticated diagnostic procedures such as multiplex RT-PCR will help to control the spread of this and other pathogens, but are technically demanding and expensive. We therefore analyzed the usefulness of hybridization techniques for detection of PSTVd in minute amounts of tissue from potato plants raised through in vitro culture, which is the routine way of potato production. Tissue print, dot blot and Northern blot hybridization with a digoxigenin-labeled PSTVd specific riboprobe allowed detection of PSTVd on a nylon membrane at a minimum quantity of 10 pg per spot or, correspondingly, in RNA extracted from microgram quantities of potato leaf tissue. These findings underlined the usefulness of hybridization techniques in PSTVd diagnosis. The application of multiplex RT-PCR for PSTVd detection in the presence of five potato viruses clearly demonstrated the advantages of this technique, but its potential drawbacks, when used under field conditions in developing countries, must not be underestimated.

Cloning, Expression and Characterization of a Non-Specific Lipid Transfer Protein Gene from Potato

Plant non-specific lipid transfer proteins(nsLTPs) are abundantlipid binding proteins with small molecules that play several biological roles including antimicrobial defense,signaling,cell wall loosening,proteinase inhibiting and so on. A new cDNA clone,named StLTPa1,corresponding to potato nsLTP gene was isolated from a cultivated potato(Solanum tuberosum) infected with Ralstonia solanacearum. The sequence analysis showed that the cloned cDNA contained 636 bp nucleotide which encoded a type I nsLTP of 91 amino acids. The StLTPa1 gene is well conserved in the coding region with 60–92% identity at the amino acid level,and 75–85% identity at the nucleotide sequence level compared with other Solanaceae nsLTPs. The transcripts of the gene accumulated in potato leaf and stem tissues induced by R. solanacearum and showed dominant differential expression between resistant and susceptible genotypes. In situ hybridization results showed that the StLTPa1 mRNA was localized in phloem cells of vascular tissues in potato leaf and stem tissues after pathogen infection. Salicylic acid,methyl jasmonate and abscisic acid induced StLTPa1 gene expression in different manners. These three signal molecules could produce a long-term effect on the StLTPa1 gene expression during the early stages of potato—R. solanacearum interaction. These results thus showed that the StLTPa1 may be pathogen-responsive gene in plant defenses and involve in a complex gene regulation network.