First Report of Bacterial Leaf Blight on Mustard Greens (Brassica juncea) Caused by Pseudomonas cannabina pv. alisalensis in Mississippi.

In 2010, a brassica leafy greens grower in Sunflower County, MS, observed scattered outbreaks of a leaf blight on mustard greens (Brassica juncea) in a 180-ha field. A severe outbreak of leaf blight occurred on mustard greens and turnip greens (B. rapa) in the same field in 2011 with more than 80 ha affected. The affected field, established in 2010, had no prior history of being cropped to brassica leafy greens. Symptoms appeared on the 6-week-old transplants as brown to tan necrotic spots with faint chlorotic borders and associated water-soaking. Lesions varied from 4 mm to 3 cm in diameter and often coalesced to cover >90% of older leaves. Whole plants of the mustard greens cv. Florida Broadleaf were collected in 2011 from the symptomatic field. Leaves were surface-disinfested with 0.5% NaOCl for 5 min, rinsed twice in sterilized distilled water [(sd)H2O], macerated in sdH2O, then streaked onto nutrient agar (NA), pseudomonas agar F (PAF), and potato dextrose agar (PDA). Little or no bacterial growth was observed on PDA, while on NA and PAF the majority of bacterial growth appeared to be a single colony type. All strains collected (25 total, one per plant) were gram-negative and fluoresced blue-green under UV light after 48 h at 28°C on PAF. All 25 strains were identified as belonging to Pseudomonas group 1a using Lelliot's determinative assay (2). Ten of the 25 strains were tested for pathogenicity on Florida Broadleaf, and turnip greens cv. Alamo. Bacteria were grown on PAF for 48 h, and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants (three plants per cultivar) were sprayed with the appropriate bacterial suspension to runoff, placed at 100% relative humidity for 48 h, and then put in a growth chamber at 28°C with a 16-h diurnal light cycle for 14 days. Additionally, three plants each of Florida Broadleaf and Alamo were either sprayed with H2O or inoculated with Pseudomonas cannabina pv. alisalensis (Pca) pathotype strain BS91 (1). All 10 strains, as well as the Pca pathotype strain, were pathogenic on both cultivars and caused symptoms similar to those observed in the field. Symptoms were not observed on H2O-sprayed plants. Comparative rep-PCR analysis using the BOXA1R primer showed the 10 strains had identical DNA-banding profiles and were identical to that of Pca BS91 (5). Five strains tested using a Pca-specific, 'light-tagged' reporter bacteriophage gave a strong positive reaction, while a negative control strain, P. syringae pv. maculicola, gave no signal (3). From these tests, the isolated bacteria were determined to be Pca. Bacteria re-isolated on PAF from the inoculated Florida Broadleaf plants had identical rep-PCR profiles with those of the strains used for inoculations. Over the past 10 years, Pca has been found in numerous states in the United States, as well as in Europe, Australia, and Japan (4). As brassica leafy greens production expands to new fields and new states, leaf blight caused by Pca appears to become a problem rapidly. Since resistant cultivars and highly effective bactericides are lacking, growers are extremely concerned about the rapid spread of this disease into existing and new brassica leafy greens regions. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. Lelliott. J. Appl. Bacteriol. 29:470, 1066. (3) D. Schofield et al. Appl. Environ. Microbiol. 78:3592, 2012. (4) F. Takahashi et al. J. Gen. Plant Pathol. 79:260, 2013. (5) J. Versalovic et al. Methods Mol. Cell Biol 5:25, 1994.

Expression of Bacterial Blight Resistance in Brassica Leafy Greens Under Field Conditions and Inheritance of Resistance in a Brassica juncea Source.

Brassica leafy greens are one of the most economically important vegetable commodity groups grown in the southeastern United States, and more than 28,000 metric tons of these crops are harvested in the United States annually. Collard and kale (Brassica oleracea Acephala group), mustard green (B. juncea), and turnip green (B. rapa) are the most commonly planted members of the brassica leafy greens group. In the last 10 years, numerous occurrences of bacterial blight on these leafy vegetables have been reported in several states. One of the pathogens responsible for this blight is designated Pseudomonas cannabina pv. alisalensis. Two B. rapa (G30710 and G30499) and two B. juncea (PI418956 and G30988) plant introductions (PIs) that exhibited moderate to high levels of resistance to this pathogen in greenhouse studies were tested for field resistance in comparison with eight commercial cultivar representatives of turnip green, mustard green, collard, and kale. The two B. juncea PIs and one of the B. rapa PIs (G30499) were found to have significantly less disease than all tested cultivars except 'Southern Curled Giant' mustard green (B. juncea) and 'Blue Knight' kale (B. oleracea). Inheritance of resistance studies performed with populations derived from the resistant G30988 and two susceptible PIs provided some evidence that resistance may be controlled by a single recessive gene.

First Report of Bacterial Leaf Blight on Broccoli and Cabbage Caused by Pseudomonas syringae pv. alisalensis in South Carolina.

In May of 2009, leaf spot and leaf blight symptoms were observed on broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata) on several farms in Lexington County, the major brassica-growing region of South Carolina. Affected areas ranged from scattered disease foci within fields to entire fields. Initial infection symptoms on leaves of both crops included circular and irregular-shaped necrotic lesions that were 3 to 10 mm in diameter, often with yellow halos and water soaking. As the disease progressed, the lesions tended to coalesce into a general blight of the entire leaf. Diseased leaves from both broccoli and cabbage were collected from each of four fields at different locations in the county. Leaves were surface disinfested, macerated in sterile distilled water, then aliquots of the suspension were spread on King's medium B (KB) agar. All samples produced large numbers of bacterial colonies that fluoresced blue under UV light after 24 h of growth. In total, 23 isolates (13 from broccoli and 10 from cabbage) were collected. These isolates were gram negative, levan production positive, oxidase negative, pectolytic activity negative, arginine dihydrolase negative, and produced a hypersensitive response on tobacco, thus placing them in the Pseudomonas syringae LOPAT group (2). Two broccoli and two cabbage isolates were selected at random and tested for pathogenicity to cabbage cv. Early Jersey Wakefield, broccoli cv. Decicco, turnip cv. Topper, broccoli raab cv. Spring, collard cv. Hi-Crop, and oat cv. Montezuma in greenhouse tests. Bacteria were grown on KB agar for 24 h and a bacterial suspension was prepared and adjusted to an optical density of 0.1 at 600 nm. Three-week-old plants were spray inoculated to runoff and held at 100% relative humidity for 12 h after inoculation, prior to return to the greenhouse bench (4). P. syringae pv. maculicola strain F18 (4) and the pathotype strain of P. syringae pv. alisalensis BS91 were included as controls, along with a water-inoculated negative control. Plants were evaluated at 14 days postinoculation. The four unknown bacterial isolates and BS91 were pathogenic on all brassica plants tested, as well as on oat. In contrast, the P. syringae pv. maculicola strain F18 was not pathogenic on broccoli raab or oat. Symptoms produced by all isolates and strains tested were similar to those observed in the field. No symptoms were observed on water-inoculated plants. Comparative repetitive sequence-based (rep)-PCR DNA analysis using the BOXA1R primer (3) resulted in a DNA banding pattern of each of the isolates from the South Carolina fields (23 isolates), as well as those reisolated from inoculated plants, that was identical to P. syringae pv. alisalensis BS91 and differed from the P. syringae pv. maculicola F18 strain. On the basis of the rep-PCR assays and the differential host range (1), the current disease outbreak on broccoli and cabbage in South Carolina is caused by the bacterium P. syringae pv. alisalensis. Broccoli is a relatively new, albeit rapidly expanding, production vegetable in South Carolina; this disease may represent a limiting factor to future production. References: (1) N. A. Cintas et al. Plant Dis. 86:992, 2002. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (3) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994. (4) Y. F. Zhao et al. Plant Dis. 84:1015, 2000.

First Report of Severe Outbreaks of Bacterial Leaf Spot of Leafy Brassica Greens Caused by Xanthomonas campestris pv. campestris in South Carolina.

Severe outbreaks of leaf spot disease of leafy vegetable brassica crops have occurred from early spring to late fall for at least the past 7 years in Lexington County, South Carolina, the major growing region for leafy greens in the state. Significant economic losses to this disease totaling $1.7 million have been incurred by large and small growers. In 2005, Pseudomonas syringae pv. maculicola was reported as one of the causal organisms of leaf spot disease in South Carolina (2). Investigations during 2006 and 2007 have led to the isolation of another bacterium causing leaf spotting of brassica crops. Symptoms in the field were nearly identical to symptoms caused by P syringae pv. maculicola, i.e., small, brown necrotic spots, often with chlorotic halos that expand and coalesce to cover the leaves. Colonies recovered from diseased tissues were xanthomonad like, nonfluorescent on Pseudomonas Agar F, mucoid on yeast extract dextrose chalk medium, grew at 35°C, hydrolyzed starch, positive for protein digestion, alkaline in litmus milk, and produce acid from arabinose. Sequence data from the 16S rDNA and fatty acid methyl ester analysis gave the best homology to Xanthomonas campestris pv. campestris with a similarity score index of >0.98 and >0.70, respectively, confirming genus and species. Excised-cotyledon assays, used to differentiate between pathovars campestris and armoraciae, confirmed the pathovar as campestris (1). Pathogenicity assays with spray inoculations (1 × 107 CFU/ml) (3) on eight plants each of 'Topper' and 'Alamo' turnip, 'Early Jersey Wakefield' cabbage, and 'Money maker' tomato produced leaf-spot symptoms within 10 days in the greenhouse and growth chamber on the turnip and cabbage plants, but not the tomato. X. campestris pv. campestris, which is common throughout the world, also is the causal agent of black rot in brassica. Typical black rot symptoms are seen often in Lexington County fields in summer and are quite different from the leaf spot symptoms observed. Leaf-spotting X. campestris pv. campestris (LS) strains and black rot (BR) strains, recovered from black rot-symptomatic plants lacking leaf spots, from the same fields were compared in greenhouse pathogenicity assays on six plants each of 'Topper' turnip and 'Early Jersey Wakefield' cabbage. Spray inoculations with 20 individual LS strains and 10 individual BR strains, collected from 2005 to 2007, produced symptoms unique to each group. These symptoms included chlorotic 'V'-shaped lesions initiating from the leaf margins with black veining when plants were inoculated with BR strains, versus rapid and severe leaf spotting followed by chlorotic 'V'-shaped lesions typically lacking black-veining 10 to 16 days postinoculation associated with LS strains. Additional inoculation tests gave similar results. To our knowledge, this is the first report of a severe leaf spotting disease of field-grown brassica leafy greens caused by X. campestris pv. campestris in South Carolina. These findings may have importance in differentiation of bacterial leaf spot pathogens in brassica crops. References: (1) A. M. Alvarez et al. Phytopathology 84:1449, 1994. (2) A. P. Keinath et al. Plant Dis. 90:683, 2006. (3) W. P. Wechter et al. Hortic Sci. 42:1140, 2007.

Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two multiplex RT-PCR assays.

A total of 360 type A swine influenza virus-positive samples including cell culture isolates, nasal swabs or lung tissues along with 30 virus-negative samples were tested for the detection and subtyping of H1N1, H1N2 or H3N2 by two multiplex reverse transcription (RT)-PCR assays. The positive samples had been collected between 1999 and 2001 from pigs with respiratory diseases, and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI) test at the Minnesota Veterinary Diagnostic Laboratory (MVDL). Two multiplex RT-PCR assays specific for H1 and H3, and N1 and N2 were developed. RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced, and the sequences were demonstrated to be specific for H1N1, H1N2 or H3N2. Genomic RNAs or DNAs from 12 common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets. Positive amplification reaction could be visualized with RNA extracted from up to 10(-5) dilution of each reference virus with original infectivity titer of 10(5) TCID(50)/ml. Of the 360 samples tested, swine influenza virus H1N1, H1N2 and H3N2 were identified in 200, 13 and 139 samples, respectively. The remaining eight samples were positive for both H1N1 and H3N2 viruses. The results of multiplex RT-PCR were 100% in agreement with those of virus isolation. These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes. The results also indicate an increased occurrence of H1N2 in US swine population.

Effects of planting pattern of collards on resistance to whiteflies (Homoptera: Aleyrodidae) and on parasitoid abundance.

Fourteen collard entries, Brassica oleraceae L., Acephala group, were evaluated for resistance to natural populations of Bemisia argentifolii Bellows & Perring in replicated field plots in Charleston, SC. Glossy-leaf phenotypes ('SC Glaze', 'SC Landrace,' 'Green Glaze') were the most resistant collard entries and had fewer whiteflies than the nonglossy, open-pollinated cultivars. Also, two F1 hybrid cultivars with normal, nonglossy leaves ('Blue Max' and 'Top Bunch') were resistant. In laboratory experiments, there were no differences in the intrinsic rate of growth (rs) of B. argentifolii populations on either glossy or nonglossy collard phenotypes. Over a 2-yr period, there were no differences in the abundance of whiteflies on the glossy phenotype of Green Glaze when it was planted in solid 20-plant plots or when it was alternated (every other plant) with the nonglossy phenotype of Green Glaze. In a similarly designed experiment, there was no difference in the resistance of Blue Max in either solid or mixed planting scheme compared with the susceptible 'Morris Heading'. Higher numbers of whiteflies and parasitoids (primarily Eretmocerus spp.) were collected on yellow sticky cards in the solid plantings of the nonglossy phenotype of Green Glaze than were collected in the solid plantings of the glossy Green Glaze phenotype. Counts on sticky cards in the mixed plots were intermediate. These data show that planting pattern of collard entries is relatively unimportant in the deployment of these sources of host plant resistance. The data also suggest that nonpreference is the primary mode of resistance to whiteflies for certain collard entries.

Improved phytonutrient content through plant genetic improvement.

During the twentieth century, plant breeding and genetics improved the nutritive value of horticultural and agronomic crops, particularly of macronutrients and fiber. Current research focuses more on micronutrients. Successful development of phytonutrient-enriched crop plants will be bolstered by interdisciplinary collaborative research, analytical and biotechnology advances, and public education. Although the melding of plant and nutrition research holds great promise, the genetic enhancement of crop plants for improved phytonutrient content will be challenging. This paper reviews the knowledge base on which genetic enhancement may be based, identifies gaps in scientific knowledge and technical capacities, and suggests a role for the federal government in research.

Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels.

Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C(4) species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.

Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression.

The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F1 and S1 progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.

Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species.

Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species.

Aspartate Aminotransferase in Alfalfa Root Nodules : II. Immunological Distinction between Two Forms of the Enzyme.

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.

Temporal variation in the VT-TI relationship in humans.

The variation with time of the relationship between tidal volume (VT) and inspiratory duration (TI) was assessed by analysis of 34 breathing sequences, nominally of 300 breath duration, during eupnea and hypercapnic hyperpnea in 6 human subjects. Two approaches were used: (1) each sequence was divided into consecutive 50-breath blocks and standard regression techniques used to characterize VT as a function of TI; and (2) a piecewise linear regression technique was applied to cluster consecutive breaths into regression regimes. Analysis of covariance was used with the results of both approaches to determine the likelihood that a single regression line was adequate to describe the entire data set. Similar results obtained regardless of the approach used. In only 4 of the 34 experiments would the hypothesis of regression slope homogeneity be accepted (P greater than 0.05) using the clustering approach; in 27 experiments, it was indicated that the regression slope estimates of VT on TI should not be considered homogeneous. Changes in the VT-TI slope were not correlated with changes in mean levels of VT, TI, minute ventilation (V), mean inspiratory flow (VT/TI), nor alveolar PCO2 (PACO2). Thus it is apparent that the VT-TI relation cannot be considered temporally invariant, but changes with time over periods ranging from less than 20 to more than 100 breaths.