First Report of Leaf Curl on Celery Caused by Colletotrichum acutatum in the United States.

In June 2010 and July 2011, celery (Apium graveolens) samples cv. Tango were submitted to the Penn State Plant Disease Clinic from Franklin and Dauphin Counties, PA, respectively. Plants exhibited curling and twisting of leaves and petioles and dark, brownish-black necrotic lesions at the base of the plant, extending up the petioles. A fungal organism with morphology consistent to Colletotrichum acutatum J.H. Simmonds was isolated from plant lesion tissue excised from the Dauphin Co. sample. Grown on half strength potato dextrose agar (PDA), the colony had gray aerial mycelium and a pink reverse. Conidia were 5.1 to 14.5 × 2.6 to 5.1 µm, aseptate, hyaline, elliptical, with one or both ends slightly pointed, and formed from the mycelium or in dense orange masses of acervuli on the aerial surface of the culture. Setae were not present. To test pathogenicity, five 23-week-old plants of the cv. Sonora and five 11-week-old plants each of the cvs. Tango and Tall Utah were sprayed until runoff with a conidial suspension (1.3 × 106 conidia/ml and 1.4 × 106 conidia/ml, respectively) and 0.025% Tween. One plant of each cv. was sprayed with milliQ water and 0.025% Tween as a control. Plastic bags were sprayed with the conidial suspension (milliQ water for the control), and secured over the individual plants for 24 h to create a humidity chamber. Plants were incubated in a growth chamber with a 16-h photoperiod, 25°C day/18°C night temperatures, and 70% humidity. Post-inoculation, all of the cv. Tango plants exhibited leaf cupping and curling after 7 days and most plants had dark stem lesions after 3 weeks, consistent with celery leaf curl symptoms. Plants of cvs. Tall Utah and Sonora developed malformed leaves and leaf curl symptoms 16 days and 10 days post-inoculation, respectively. None of the control plants developed symptoms. Infected tissue was excised from diseased plants, surface disinfested in 0.5% sodium hypochlorite for 45 s and plated on half strength PDA. Fungal colonies consistent with C. acutatum were recovered from all inoculated celery tissues (except two of the five inoculated cv. Tall Utah plants and the negative controls). To verify morphological identification, the internal transcribed spacer (ITS) rDNA region was amplified and sequenced for our original isolate and those recovered from the inoculated plants using ITS1 and ITS4 primers (2) (GenBank Accession No. JQ794875). Sequence homology revealed 99 to 100% similarity to accessioned isolates of C. acutatum, which included the holotype and a paratype of C. acutatum (Accession Nos. AF411700 and AF411701, respectively). Celery leaf curl has been reported to have caused devastating crop losses on celery in Australia (1, 3) and to our knowledge, C. acutatum causing leaf curl of celery has not been officially reported in the United States. Infected celery plants are unmarketable because of the leaf malformation and eventual plant necrosis caused by C. acutatum. As such, this disease could have serious negative implications for celery growers in the United States. References: (1) J. B. Heaton and S. R. Dullahide. Australas. Plant Pathol. 22:152, 1993. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (3) D. G Wright and J. B. Heaton. Australas. Plant Pathol. 20:155, 1991.

Contamination of fresh and ensiled maize by multiple penicillium mycotoxins.

Toxins produced by Penicillium species are reported in maize silage and have been associated with health problems in cattle. Our objectives were to evaluate the prevalence and dynamics of patulin (PAT), mycophenolic acid (MPA), cyclopiazonic acid (CPA), and roquefortine C (ROC) in fresh and ensiled maize. To achieve these objectives we developed a high-performance liquid chromatography method coupled with mass spectrometry to detect all four toxins simultaneously in silage. In addition we collected weather data, information on agronomic practices, and silage fermentation characteristics for each study site. Silage was collected at harvest and after ensiling in 2001 and 2002 from 30 Pennsylvania dairies. The average concentration of toxins (range in parentheses) was: PAT 0.08 microg/g (0.01 to 1.21), MPA 0.16 microg/g (0.02 to 1.30), CPA 0.12 microg/g (0.02 to 1.43), and ROC 0.38 microg/g (0.01 to 5.71). ROC was the most frequently detected toxin (60%), followed by MPA (42%), CPA (37%), and PAT (23%). Of 120 samples tested, 15% contained no detectible levels of toxin, 25% were contaminated with one toxin, 32% with two, 18% with three, and 10% with all four toxins. All four mycotoxins were found in freshly harvested material, contradicting the belief that Penicillium toxin formation occurs exclusively during storage. We observed that weather conditions during specific growth stages of the crop affected the final concentration of toxins in freshly harvested maize. In ensiled material, PAT levels were affected by concentrations of propionic and isobutyric acids. Based on our data, Penicillium mycotoxins can form while the crop is in the field and after ensiling, suggesting that preventative measures should begin prior to ensiling.

Microbiological and molecular determination of mycobiota in fresh and ensiled maize silage.

The mycobiota of fresh and ensiled maize was studied with culturing techniques and a DNA sequence-based approach. Freshly chopped and ensiled maize were collected for 2 y from 12 farms in Pennsylvania. Samples were plated on selective media and isolates identified by morphology and sequences of the internal transcribed spacer regions of rDNA, 800-900 bp of the 5' end of the translation elongation factor 1-alpha gene and a portion of the rodA gene (Aspergillus fumigatus only). ITS regions were amplified from total silage DNA, cloned, sequenced and compared to fungal ITS sequences in GenBank with the BLAST-N algorithm. For samples analyzed by both methods, the molecular technique detected a greater number of species than selective plating. Plating recovered several Penicillium and Fusarium species and Aspergillus fumigatus, while molecular analysis detected Alternaria, Penicillium and Fusarium species. Data from both methods found that Fusarium and Penicillium were the dominant mycotoxigenic fungi in silage, while yeast made up the majority all fungi recovered or detected. Known mycotoxigenic species often accounted for 50% or more of the total number of species isolated or detected at each site. Viable Fusaria were not isolated from or detected in ensiled maize, suggesting that Fusarium species do not survive the ensiling process. Results from this study suggest that given the numerous species of fungi present in silage with mycotoxin producing ability, there is a strong possibility that silage may be contaminated with multiple toxins simultaneously.

Relationships Between Weather Conditions, Agronomic Practices, and Fermentation Characteristics with Deoxynivalenol Content in Fresh and Ensiled Maize.

The deoxynivalenol (DON) content of maize silage was determined in samples collected at harvest and after ensiling in 2001 and 2002 from 30 to 40 Pennsylvania dairies. Information on cultural practices, hybrid maturity, planting, and harvest date was collected from each site. Site-specific weather data and a corn development model were used to estimate hybrid development at each site. Correlation analysis was used to assess the relationship between weather data, hybrid development, cultural practices and preharvest DON. Fermentation characteristics (moisture, pH, and so on) of ensiled samples were measured to study their relationship to postharvest DON contamination. No significant difference (P ≤ 0.05) was noted between the numbers of samples containing DON in 2001 and 2002, although concentration was higher in 2002 samples. A positive correlation was observed between DON concentration of harvest samples and daily average temperature, minimum temperature, and growing degree day during tasselling, silking, and milk stages. A negative correlation was observed between daily average precipitation at blister stage and DON concentration in harvest samples. Samples from no-till or minimum-till locations had higher DON concentrations than moldboard or mixed-till locations. Harvest samples had higher DON concentration than ensiled samples, suggesting that some physical, chemical, or microbiological changes, resulting from ensiling, may reduce DON in storage.

Immobilon-Ny+ nucleic acid blotting membrane: an advanced nylon membrane optimized for superior fixation and reprobing.

The use of charged nylon membranes in nucleic acid blotting applications has become an important factor in the success of hybridization-based assays. Retention of nucleic acids on these membranes is promoted by baking at 80 degrees C under vacuum or by exposure to short wavelength UV light, with the latter method preferred. Immobilon-Ny+ is an advanced, positively charged nylon membrane that has been optimized to show superior retention of target DNA and RNA under hybridization conditions. Higher signal levels are obtained in these assays compared to competitive membranes, even after 13 cycles of probing. This report illustrates the superior performance of Immobilon-Ny+ in 32P and chemiluminescent hybridization assays on blotted DNA and RNA.

Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking.

The data presented here (Fig. 2, Table 2) clearly demonstrate that wetting of Immobilon-P is not required for immunodetection of transferrin. It can be inferred that the immobilized transferrin undergoes sufficient rehydration at the molecular level to permit epitope recognition by the antibodies, even when the surrounding areas of PVDF remain hydrophobic. The hydrophobic blot procedure was compatible with blots prepared by both tank and semidry transfer and with the substrates BCIP/NBT and 4CN. Additionally, the binding specificity of antitransferrin antiserum was not altered in the hydrophobic blot protocol. Given the diversity of blocking agents, antibodies, and visualization systems available, optimization for specific reagent combinations may be necessary. The most important parameters will be the concentration of blocking agents and detergents used since either, depending on chemical properties and concentration, may cause the membrane to wet out during incubation. This procedure is not applicable to nitrocellulose membranes since surfactants are used in their manufacture specifically to cause wetting in aqueous buffers.

Abscisic acid enhances the transcription of wheat-germ agglutinin mRNA without altering its tissue-specific expression.

We used RNA gel-blot analysis, in-situ hybridization, and nuclear run-on transcription to examine the effects of exogenous abscisic acid (ABA) on the spatial distribution of mRNA for the lectin wheat-germ agglutinin (WGA) in developing wheat (Triticum aestivum L. cv. Marshall) embryos and seedlings. When analyzed by RNA gel blots, both developing embryos and seedlings exhibited higher steady-state levels of WGA mRNA after ABA treatment. As determined by in-situ hybridization, incubation of developing embryos in 0.1 mM ABA resulted in accumulation of WGA mRNA in the epidermal and subepidermal cell layers of the radicle and seminal roots and throughout the rootcap and coleorhiza. This spatial distribution was identical to that in control embryos. Nuclear run-on transcription assays indicated that at least part of this increase is attributable to transcriptional induction. Thus, exogenous ABA is capable of inducing increased WGA mRNA accumulation only in cells where it is expressed during normal embryogenesis. When seeds were germinated in the absence of ABA, WGA mRNA was detected only in the rootcap. In contrast, seeds imbibed and germinated in the presence of ABA for 3 d exhibited a spatial distribution of WGA mRNA similar to that observed in developing embryos treated with ABA. In contrast, when ABA was added to 3-d-old seedlings, WGA mRNA was not detected in regions of the root beyond the rootcap. We conclude that exogenous ABA, when applied continuously from imbibition, causes retention of the embryo-specific pattern of WGA mRNA distribution and that the spatial pattern of WGA mRNA expression in roots does not change when ABA is added after germination.

Synthesis of the low molecular weight heat shock proteins in plants.

Heat shock of living tissue induces the synthesis of a unique group of proteins, the heat shock proteins. In plants, the major group of heat shock proteins has a molecular mass of 15 to 25 kilodaltons. Accumulation of these proteins to stainable levels has been reported in only a few species. To examine accumulation of the low molecular weight heat shock proteins in a broader range of species, two-dimensional electrophoresis was used to resolve total protein from the following species: soybean (Glycine max L. Merr., var Wayne), pea (Pisum sativum L., var Early Alaska), sunflower (Helianthus annuus L.), wheat (Triticum aestivum L.), rice (Oryza sativa L., cv IR-36), maize (Zea mays L.), pearl millet (Pennisetum americanum L. Leeke, line 23DB), and Panicum miliaceum L. When identified by both silver staining and incorporation of radiolabel, a diverse array of low molecular weight heat shock proteins was synthesized in each of these species. These proteins accumulated to significant levels after three hours of heat shock but exhibited considerable heterogeneity in isoelectric point, molecular weight, stainability, and radiolabel incorporation. Although most appeared to be synthesized only during heat shock, some were detectable at low levels in control tissue. Compared to the monocots, a higher proportion of low molecular weight heat shock proteins was detectable in control tissues from dicots.

Cytoplasmic distribution of heat shock proteins in soybean.

Previous analyses of the distribution of heat shock (hs) proteins in soybean (Glycine max L. Merr., var Wayne) have demonstrated that a fraction of the low molecular weight hs protein associates with ribosomes during hs. To more specifically characterize the nature of this association, isokinetic centrifugation of ribosomes through sucrose gradients was used to separate monosomes from polysomes. The present analysis demonstrated that hs proteins were bound to polysomes but not monosomes. Treatment of polysomes with puromycin, K(+), and Mg(2+), which caused dissociation of ribosomes into 40S and 60S subunits, also caused dissociation of the hs proteins. Using the procedure of Nover et al. (1983, Mol. Cell Biol, 3: 1628-1655), a hs granule fraction was also isolated. As in tomato cells, hs granules from soybean seedlings contained the low molecular weight hs proteins as a primary component and a number of other non-hs proteins of relative molecular mass 30 to 40 kilodaltons and 70 to 90 kilodaltons. On metrizamide gradients they exhibited a buoyant density of 1.20 to 1.21 grams per cubic centimeter, typical of ribonucleoprotein particles. Heat shock granules were characterized as unique cytoplasmic particles based on protein composition and buoyant density. Isopycnic centrifugation of ribosome preparations demonstrated that they contained hs granules, but the hs proteins bound to polysomes were not released by KCI/EDTA treatment. Thus, the polysome-bound hs proteins and the granule-bound hs proteins appear to represent two distinct populations of hs proteins in the cytoplasm. Heat shock granules were not distinguishable from ribosomes at the level of resolution used in transmission electron microscopy.

Wheat-germ agglutinin is synthesized as a glycosylated precursor.

The biosynthesis and processing of wheat-germ agglutinin (WGA) were studied in developing wheat (Triticum aestivum L. cv. Marshall) embryos using pulse-chase labeling, subcellular fractionation and immunocytochemistry. A substantial amount of newly synthesized WGA was organelle-associated. Isolation of WGA on affinity columns of immobilized N-acetylglucosamine indicated that it was present in a dimeric form. When extracts from embryos pulse-labeled with [(35)S]cysteine were fractionated on an isopycnic sucrose gradient, radioactivity incorporated into WGA was detected at a position coincident with the endoplasmic reticulum (ER) marker enzyme NADH-cytochromec reductase. The WGA in the ER could be slowly chased into the soluble, vacuolar fraction, with a half-life of approx. 8 h. Immunolocalization studies demonstrated the accumulation and distribution of WGA throughout the vacuoles.Four forms of the WGA monomer were characterized using immunoaffinity purification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In-vitro translation of polyadenylated RNA isolated from developing wheat embryos produced a polypeptide with Mr 21 000. In-vivo labeling of embryos with radioactive amino acids resulted in the formation of a polypeptide of Mr 23 000 and the mature monomer of Mr 18000. When [(3)H]mannose was used in labeling studies, only the polypeptide of Mr 23 000 was detected. In-vivo labeling in the presence of tunicamycin yielded an additional polypeptide of Mr 20 000. These results indicate that WGA is cotranslationally processed by the removal of a signal peptide and the addition of a glycan, presumably at the carboxy-terminus (N.V. Raikhel and T.A. Wilkins, 1987, Proc. Natl. Acad. Sci. USA 84, 6745-6749). The glycosylated precursor of WGA is post-translationally processed to the mature form by the removal of a carboxyl-terminal glycopeptide.