Biofortification of tomato (Solanum lycopersicum) fruit with the anticancer compound methylselenocysteine using a selenocysteine methyltransferase from a selenium hyperaccumulator

Methylselenocysteine (MeSeCys) is an amino acid derivative that possesses potent anticancer activity in animals. Plants that can tolerate growth on soils with high Se content, known as Se hyperaccumulators, do so by converting inorganic Se to MeSeCys by the enzyme selenocysteine methyltransferase (SMT). A cDNA encoding the SMT from a Se hyperaccumulator was overexpressed in tomato (Solanum lycopersicum). Transgenic plants were provided with selenite or selenate to the roots during fruit development, and liquid chromatography-mass spectrometry was used to show that MeSeCys accumulated in the fruit but not in the leaves. Depending on the transgenic line and Se treatment, up to 16% of the total Se in the fruit was present as MeSeCys. MeSeCys was produced more effectively from selenite on a percentage conversion basis, but greater accumulation of MeSeCys could be achieved from selenate due to its better translocation from the roots. MeSeCys was heat stable and survived processing of the fruit to tomato juice.     

Specific serum antibody responses in channel catfish (Ictalurus punctatus) provide limited protection against Streptococcus ictaluri challenge

Passive immunization studies were conducted to determine the role of specific antibodies in immunity to Streptococcus ictaluri. Adult channel catfish (Ictalurus punctatus) were injected i.p. with tryptic soy broth as control or with 1.5 × 10(7)colony-forming units (cfu) S. ictaluri/fish at 0, 30, and 60 d, and serum was collected 90 d after the original challenge. Fish were passively immunized by i.p. injection with serum from the tryptic soy broth (TSB) control group, anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (SSI), or heat-inactivated anti-S. ictaluri serum from fish immunized three times and sampled at 90 d (HISSI). These passively immunized fish were then challenged 72 h later with 1.5 × 10(8)cfu S. ictaluri/fish. Over 21 d, the mean cumulative percent survival was 43.3 (TSB), 63.3 (SSI), and 50.0 (HISSI). A significant difference in cumulative percent survival was noted between the TSB and the HISSI groups, and significant differences were noted between these groups and the SSI group. Serum obtained from immunized fish 72 h after passive immunization exhibited increased anti-S. ictaluri antibody levels. Twenty-one days after the challenge, the HISSI and SSI group antibody levels significantly increased above their corresponding pre-challenge levels. No significant (r(2)=0.0806; P<0.5985) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results indicate that both specific anti-S. ictaluri antibodies and non-specific immune responses are important for protection against S. ictaluri.     

Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils

Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA.     

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.     

Nitrogen and phosphorus availability from liquid dairy manure to potatoes

The effective use of manure nutrients requires an accurate assessment of their availability to the specific crops being grown. A fertilizer equivalence approach was used to evaluate the availability of nitrogen (N) and phosphorus (P) from liquid dairy manure to potatoes in field experiments conducted in northeast Wisconsin in 2000, 2001, and 2002. Crop responses to moderate and high rates of liquid dairy manure (93,500 and 187,000 L ha^?1) were compared to results obtained from N or P fertilizer applied at five rates (0 to 269 kg ha^?1). Availability estimates were made using the fertilizer equivalence method based on tuber yield, harvested tuber N or P concentration and uptake, petiole nitrate or total P concentration, and soil nitrate or extractable P phosphate levels. Results showed an apparent availability of manurial N from 10% to 40%, with an overall average across all 3 years and all parameters of 28.6% for the lower rate and 24.6% for the high rate. These values are slightly less than average availability measured where corn has been used as the test crop. Although all parameters did not show responses to P fertilizer additions in these trials, where estimates were possible, P availability ranged from 20% to 90% with an overall average across all 3 years of 55.4% for the low and 50.5% for the high manure rates. Despite potato being more shallow-rooted and somewhat less efficient in N and P use compared to corn, it appears that only minor adjustments to manurial nutrient availability estimates are warranted when potatoes are grown.     

Temporal disease dynamics and relative importance of sexual and asexual reproduction of grape downy mildew (Plasmopara viticola) in an isolated vineyard in the North Georgia Mountains,USA

The North Georgia Mountains are the southernmost region along the United States East Coast where European wine grapes (Vitis vinifera) are grown commercially. Epidemics of downy mildew, caused by Plasmopara viticola, are frequent and severe, but little is known about the epidemiology and population biology of the pathogen in this region. Disease monitoring in an experimental vineyard from 2015 to 2017 indicated that times of disease onset and progress rates were highly variable across years and cultivars. Oospores were observed microscopically, and simulation with a process-based model indicated presence of conditions favourable for oospore germination in the spring and early summer each year. A total of 409 P. viticola isolates collected over three  years were genotyped with seven microsatellite markers, revealing very high genotypic diversity, which when combined with the observation of oospores is indicative of a sexually reproducing population. Among the 409 isolates, 225 multilocus genotypes (MLGs) were identified, of which 164 were detected only once and 61 were repeated (clonal). Eight MLGs (represented by 28 isolates) were detected across years, suggesting the possibility of asexual overwintering of P. viticola in this region. Across sampling dates, the percentage of isolates belonging to nonrepeated (unique) MLGs ranged from 27.3% to 63.2%. Even towards the end of the annual epidemic, the percentage of isolates in nonrepeated MLGs was still relatively high, around 30%. These MLGs may have originated from oospores germinating late during the growing season, although incomplete sampling at earlier dates and contribution by immigration cannot be fully excluded.     

Identifying and exploring significant genomic regions associated with soybean yield,seed fatty acids,protein and oil

Soybean [Glycine max (L.) Merrill] yield and seed fatty acids, protein, and oil content are important traits for which an improved understanding of significant genomic regions would be useful. To accomplish this, a soybean population consisting of 203 F5 derived recombinant inbred lines (RILs) was developed and genotyped with 11,633 polymorphic single nucleotide polymorphisms (SNPs). Each RIL was grown in a single plot at Knoxville, TN in 2010; followed by replicated, multi-location field trials in 2013 and 2014. The data from 2010, 2013, and 2014 were analyzed together in order to detect quantitative trait loci (QTL) for these traits, and 30 total QTLs were detected. Five QTLs are candidates for confirmed status and one QTL is a candidate for positional confirmation. Many of the genes with mutations in close proximity to the fatty acid QTLs are involved in biological processes for fatty acids and/or lipids and could be considered possible candidate genes. Similarly, genes with mutations in genomic regions near yield, protein, and oil QTLs were plentiful and may contribute to the variation observed in these traits. Except for yield and stearic acid, each trait displayed pleiotropic effects with other traits in this study. Notable are the pleiotropic effects for oleic and linolenic acid on chromosomes 9, 13, and 19. Overall, the findings from this research contribute new information to the genetic understanding of soybean yield and seed fatty acids, protein and oil content. This understanding will be useful in making trait improvements.