Potato leafroll virus purification and antiserum preparation for enzyme-linked immunosorbent assays

Potato leafroll virus (PLRV) was purified from potato foliage and stems with an average yield of 0.14 mg of PLRV/kg of potato. Modifications of an existing purification procedure are reported. Five low dosage (38-118 μg of PLRV) intravenous injections were used to produce a PLRV antiserum for use in enzyme-linked immunosorbent assays (ELISA) from tubers. PLRV was readily detected in ELISA testing of potato tubers and leaves and inPhysalis floridana Rybd. Non-specific reactions were low with all tissues. In parallel tests, a Canadian antiserum produced higher nonspecific reactions with tuber and leaf tissue. The results indicated that the use of low dosage-intravenous injections might be necessary methodology for producing PLRV antiserum for use in ELISA diagnostic tests with tuber tissue where high non-specific reactions have been reported.     

Purification of potato virus A and its detection in potato by enzyme-linked immunosorbent assay (ELISA)

Potato virus A (PVA) was purified fromNicandra physaloides by a simple method that omitted organic solvent clarification and consisted of differential centrifugation followed by equilibrium centrifugation in CsCl. An antiserum was produced that specifically detected PVA in potato leaf sap using either the SDS-agar test or enzyme-linked immunosorbent assay (ELISA). No heterlogous reaction of the antiserum with potato virus Y was detected. Purified PVA was highly infectious; it had an A 258/280 nm absorbance ratio of 1.28. The particles had a normal intact appearance in the electron microscope. Detection of PVA in potato sprouts and foliage by ELISA was compared with the local lesion assay onPhysalis angulata L. plants. ELISA was superior over an indicator plant test when sprout tissue was used. PVA antiserum reacted similarly with mild and crinkle isolates.     

Detection of potato viruses X and S in tissue culture plantlets

The latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) were evaluated in separate studies for their ability to detect potato virus X (PVX) and potato virus S (PVS) in tissue culture plantlets. Healthy and infected clonal lines of several potato cultivars were used. LAT was unsatisfactory because only low levels of agglutination were obtained with infected samples, and because variable and inconsistent results were obtained with both healthy and infected clones. ELISA, however, consistently gave high spectrophotometric readings and intense visual reactions for infected but not for healthy clones. The results indicate that ELISA can be used to detect PVX and PVS in tissue culture plantlets, and in programs where tissue culture is employed, early detection and elimination of infected plantlets is possible.     

Enzyme-linked immunosorbent assay with single or combined antisera for viruses S and X in potato tubers and plants

Enzyme-linked immunosorbent assay (ELISA), with single or combined antisera was effective for diagnosing potato virus S (PVS), potato virus X (PVX) or both viruses in plants grown in the greenhouse or field. In dormant tubers, stolon, middle and apical end composite sampling with or without eyes and sprouted tubers produced reliable positive assays for PVX. Only tuber pieces with sprouts resulted in consistently reliable assays for PVS. Composite sampling of potato foliage was effective for detecting one PVX infected plant in a total of 100 Kennebec, Norland, or Russet Burbank plants. There were some false negative results and greater variability in composite PVS assays, but on average, one PVS infected plant can be detected in composites of 10 Kennebec, Norland, or Russet Burbank plants. Sodium diethyldithiocarbamate (0.01M NaDIECA) in phosphate buffered saline + 0.5% Tween (PBS-T) added to plant extracts enhanced specific reactions for either virus. Onceor twiceused enzyme conjugate was effective in ELISA of either virus from potato foliage.     

Kinetin,thermotherapy, and tissue culture to eliminate potato virus (PVX) in potato

PVX infected plantlets from two potato cultivars grownin vitro with 0.3, 3, 30 or 300 ppm kinetin were exposed to temperatures of 28 or 35 C. After 3 wk, axillary buds were isolated and grown aseptically in organogenic media, followed by PVX testing by ELISA. The serological test was also run on whole plantlets at the end of the kinetin-temperature exposure. No donor plants exposed to 28 C nor the plantlets derived from their buds gave an ELISA (-) reaction, regardless of the kinetin content of the media or that of the cultivar. At 35 C the virus was suppressed to undetectable levels in several whole plantlets. In the cultivar Alpha, 2 out of 6 resulting plantlets after isolation of buds were virus-free in the presence of 3.0 mg/1 kinetin during the treatments. From Atzimba, about 15–40% of the regenerated plants were ELISA (-), without any relationship to the cytokinin content in the media. Heat had a stronger influence on virus elimination than kinetin and the results varied with the cultivar.     

Maintenance,symptoms and distribution of potato viruses X,S, Y,A, M and leafroll in potato tissue culture plantlets

Maintaining potato viruses X, S, Y, A, M and leafroll in tissue culture plantlets is a convenient, cost and space effective alternative to the use of greenhouse plants. Of these six viruses, only certain strains of PVX induced symptoms in tissue culture plantlets. Nevertheless, all infected tissue culture plants were found to be more reliable than greenhouse grown plants as virus-infected controls in enzyme-linked immunosorbent assay (ELISA) testing. Another advantage of maintaining viruses in tissue culture plantlets was the elimination of contamination by other viruses or other pathogens. Leaves, stems, and roots of virus infected plantlets were tested separately for antigen levels by ELISA. In these tests, the stems and leaves of all but PVA infected tissue culture plants consistently gave positive ELISA values. In contrast, root tissue from PVY infected tissue culture plantlets was not reliable for PVY detection. In all cases, the viruses detected in the original source material were detected in the resulting tissue culture plantlets.     

Mechanical and rheological properties of calcium carbonate-filled ethylene propylene diene elastomer reinforced by metallic acrylate salt

High performance ethylene propylene diene methylene elastomer (EPDM)/calcium carbonate (CaCO₃)/aluminum triacrylate (ALTA) hybrids have been prepared by a melt compounding process. The mechanical properties of the peroxide cured EPDM/CaCO₃/ALTA vulcanizates were investigated by tensile, hardness, resilience, abrasion and fatigue life tests. The results showed that the ALTA can greatly improve the modulus at 100 %, tensile strength, abrasion resistance and fatigue life of the EPDM/CaCO₃/ALTA vulcanizates, while retaining their high elongation at break. ALTA as a reactive filler had accelerating effect on the vulcanization reaction of and increased the crosslink density of the EPDM/CaCO₃ composites. This phenomenon is due to increasing the ionic bonds arise from both homo-polymerization of ALTA and graft co-polymerization of it onto the EPDM. Scanning electron microscopy (SEM) revealed the presence of ALTA causes to the roughness of the fracture surface of CaCO₃-filled EPDM compounds demonstrating high interaction between the fillers and EPDM improved by introduction of ALTA. Dynamic mechanical analysis revealed that the glass transition temperature (T _g ) and dissipation peak (tan δ) of EPDM composites decreases with increasing ALTA content. Decrease in tan δ value and inward shifting of T _g were related to improved interaction of filler and EPDM.     

A reevaluation of bromoethane in comparison to rindite for the post-harvest detection of potato virus Y in tubers by ELISA

A reevaluation of breaking tuber dormancy with bromoethane to increase the concentration of potato virus Y in the tuber revealed a positive response, by ELISA testing, to the treatment. The degree of response increased with the maturity of the tuber. Response to treatment with rindite was generally stronger, although differences were slight.     

Comparison of latex agglutination,enzyme-linked immunosorbent assay,and indicator plants for detection of potato viruses S and X in potatoes

Potato virus S (PVS) was consistently detected by the enzyme-linked immunosorbent assay (ELISA) and the latex agglutination test (LAT) at dilutions (PVS sap: healthy sap) of 1:499 and 1:19, respectively. Mechanical inoculation ofNicotiana debneyi gave variable results with detection possible 38% of the time at dilutions of 1:499. Potato virus X (PVX) was consistently detected by ELISA and LAT at dilutions (PVX sap: healthy sap) of 1:199 and 1:49, respectively. Mechanical inoculation ofGomphrena globosa with PVX resulted in consistent detection of the virus at a dilution of 1:499.     

Bison,a new red-skinned potato variety

Bison, a new red potato, was introduced by North Dakota State University. This new red variety has smooth tuber type and bright red skin color. Bison yields somewhat less than Norland and Red Pontiac but the advantage of Bison over these two varieties is its uniformity and bright red color. Bison is about medium in total solids and makes chips comparable in color to Norchip but lighter in color than Kennebec. Bison is resistant to race 0 of the late blight organismPhytophthora infestans, but susceptible to race 1–4.     

A study of surface morphology and structure of cotton fibres with soluble immobilized-cellulase treatment

Our previous studies have demonstrated that cotton fabrics treated with soluble immobilized-cellulase showed considerably lower degradation and higher retention of tensile strength than those treated with free cellulase. It is important to investigate the surface morphology and structure of cotton fibres for understanding the enzymatic degradation. In this study, the effects of the soluble immobilized-cellulase on the surface morphology and structure of cotton fibres were investigated. The ultrastructural changes in the fibre surfaces were inspected using Tapping-Mode Atomic Force Microscopy (TM-AFM) and scanning electron microscopy (SEM), and the results showed that the smooth surfaces could be obtained after the immobilized-cellulase treatment, and no obvious damage was observed on the fibre surfaces. The hydrogen bonds in the certain depth area beneath the fibre surface were investigated using Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy (ATR-FTIR) after the cellulase treatments. Furthermore, the result of fibre accessibility indicated that the hydrolysis occurring in the interior of the cotton fibres was limited during the immobilized-cellulase treatment. Crystalline index (CI) of the cotton fibres treated with free cellulase was slightly higher than that of fibres treated with immobilized cellulase.     

A rapid and simple method for determination of potato chloroplast DNA type

A procedure for restriction enzyme analysis of the potato chloroplast DNA (ctDNA) is described. The advantages of this method are: 1) rapid determination of ctDNA type, 2) no ultracentrifugation, and 3) low cost of analyses. This method makes it easy to distinguish the ctDNA types of wild and cultivated potato accessions.     

Pre and early post-emergence weed control with Paraquat in potatoes

Studies using Paraquat herbicide for early post-emergence control of broadleaved and grass weeds in Katahdin and Russet Burbank potatoes were conducted in Maine during four growing seasons. All rates and times of application of Paraquat gave good commercial control of grass and broadleaf weeds when compared to Premerge and Dowpon treatment as checks. Paraquat applied to Katahdins 2 weeks after ground crack reduced the yield of tubers but did not significantly affect specific gravity. Yield and specific gravity of Russet Burbank was reduced by Paraquat applied one and 2 weeks after ground crack. Paraquat can be used effectively for weed control in Katahdin up to one week after ground crack without crop damage. In Russet Burbank it appeared that application at ground crack was about as late as Paraquat could be applied without affecting yield or specific gravity of tubers.     

A two-step ELISA for rapid,reliable detection of potato viruses

The reliability of the standard double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) was compared with a shorter, two-step DAS procedure in which sample and conjugate were mixed and incubated together in one step. The two assays were compared using beet western yellows virus and potato leafroll, M, S, X, and Y viruses. The two-step procedure was more sensitive,i.e., it detected small quantities of virus with greater statistical reliability than the standard procedure. At high virus concentrations, the standard produced stronger ELISA reactions than the two-step assay, but both assays were reliable. Since all of the viruses tested withstood high incubation temperatures, the incubation period for the two-step procedure could be reduced to 1 hr at 30 or 37 C. Therefore, assays could be completed within 2 hr using the two-step procedure compared with 2 days for the standard procedure. Reliable results were achieved with samples prepared by grinding tissues in buffer or, more simply, by adding pure, pressure extracted juice directly to conjugate in assay wells. Coating plates with gamma globulins or with F(ab′)₂ fragments of gamma globulins gave equally reliable results with all viruses except potato leafroll, where coating with gamma globulins was superior.     

A rapid method for determining blackspot susceptibility of potato clones

Two methods of determining susceptibility of potato clones to blackspot were compared: (1) bruising by weight dropping and (2) bruising by abrasive peeling. A highly significant positive correlation was obtained between the intensity of enzymatic discoloration following abrasive peeling and the amount of blackspot that developed by weight dropping (r=0.93). Abrasive peeling was more rapid than the weight-dropping method. Tuber samples were abraded 30 sec and the amount of enzymatic discoloration evaluated after 24 hr. The need for individually bruising and hand peeling of tubers was eliminated with this method. Because of the rapidity of the abrasive peeling method, it can be used effectively in potato breeding programs to screen large numbers of clones for blackspot susceptibility. Results indicate that tuber maturity affects enzymatic discoloration and blackspot susceptibility. Immature tubers, dug while the vines are still green, are more resistant to blackspot than mature tubers. Tuber maturity therefore must be considered when screening clones for susceptibility to blackspot.     

Preparation and characterization of glass fiber-coir hybrid composites by a novel and facile Prepreg/Press process

The present study explored the preparation of glass fiber-coir reinforced unsaturated polyester resin hybrid (GCU) composites with a novel Prepreg/Press fabrication process. Flexural, impact and thermal-mechanical properties of GCU composites were investigated. Coir reinforced unsaturated polyester resin (CU) composites was also prepared with the same process to explore the enhancement effect of glass fabric on the mechanical properties of coir-based composites. The effect of fabrication pressure on the mechanical properties of CU and GCU composites was examined. Micromorphology and interfacial reaction of the composites were analyzed. It is shown that GCU composites fabricated with the Prepreg/Press process have excellent flexural strength (185.0 MPa), MOE (18.3 GPa), and impact strength (67.2 kJ/m²). The mechanical properties of GCU composites increased with the increase of applied pressure up to 0.8 MPa in the Prepreg/Press process. However, further increase of applied pressure led to the decrease in mechanical properties. The addition of glass fabrics to GCU composites showed 419 % improvement in flexural strength, 708 % improvement in MOE and 562 % improvement in impact strength over coir-based composites. The micromorphology study proved that the poor interfacial bonding between coir and matrix led to the low mechanical properties of coir-based composites.     

Coastal Russet: A mid-season Russet-skinned potato cultivar

Coastal Russet (tested as B9596-2) is a clone with mid-season maturity that produces russet skin tubers that are long and slightly flattened. Tuber yields in the eastern portions of the United States have been considerably greater than for BelRus. Tubers are well-shaped, have shallow eyes, and are attractive in appearance. Tuber appearance and size make this cultivar very well suited for the tablestock market. It is not suitable for processing. Coastal Russet is highly resistant to tuber net necrosis caused by late current season infection with leafroll and is tolerant to common scab.     

Manufacture and performance of O-carboxymethyl chitosan sodium salt/cellulose fibers in N-methylmorpholine-N-oxide system

A series of O-carboxymethyl chitosan sodium salt (NaCMCh) with different degree of substitution (DS) of -CH₂COONa agent were successfully prepared by altering the reaction temperature and time. Both fourier transform infrared spectroscopy (FTIR) and ¹³C-Nuclear Magnetic Resonance (¹³C-NMR) were used to study the structure of NaCMCh. And the impact of DS on the antibacterial activity of NaCMCh was investigated. Then, the NaCMCh with optimal antibacterial activity was selected to prepare NaCMCh/cellulose fibers in N-methylmorpholine-N-oxide (NMMO) system. The structure, crystallization behavior, thermal property and morphology of obtained fibers were carefully studied with FTIR, Wide-angle X-ray diffraction (WAXD), differential scanning calorimetry (DSC), thermal gravimetric (TG) and scanning electron microscope (SEM), respectively. Meanwhile, the antibacterial activity as well as mechanical properties of resultant fibers was also investigated. The results demonstrated that the fibers had strong antibacterial activity against Escherichia coli (E. coli), acceptable mechanical properties and good water retention.     

Hybridization between Gp. Tuberosum Haploids and 1EBN wild potato species

Diploid wild potato species, classified as 1EBN (Endosperm Balance Number), do not cross with tetraploid or diploid forms ofS. tuberosum Gp. Tuberosum. The crossing of 2n pollen producing 1EBN clones as well as chromosome-doubled 1EBN clones with Gp. Tuberosum haploids was initiated to overcome this hybridization barrier. The screening of eleven 1EBN species,S. brachistotrichum, S. bulbocastanum, S. cardiophyllum, S. chancayense, S. commersonii, S. etuberosum, S. fernandezianum, S. jamesii, S. mochicense, S. pinnatisectum andS. trifidum, resulted in the identification of 2n pollen producers in eight of the species examined. Direct hybridization with Gp. Tuberosum haploids utilizing the above-mentioned crossing scheme was successful withS. chancayense andS. commersonii. The hybrids obtained, however, were male and female sterile. Abnormal microsporogenesis in the hybrids was postulated to be the result of an interaction between Gp. Tuberosum cytoplasm and nuclear genes contributed by the 1EBN male parent. Analyses of the growth of 1EBN species’ pollen tubes in the stigma/style of Gp. Tuberosum haploids were also conducted. Interspecific incompatibilities were observed with the most severe forms found with the use of species in the Series Etuberosa. The use of the Endosperm Balance Number theory can aid in designing crosses which overcome barriers to successful endosperm development. Sterilities in the hybrids obtained as well as interspecific incompatibilities will also need to be addressed, however, before 1EBN species germplasm can be successfully utilized for the improvement of the cultivated potato.