Mechanisms of Soybean Host-Plant Resistance Against Megacopta cribraria (Hemiptera: Plataspidae).

A number of soybean varieties traditionally bred for resistance to various soybean arthropod pests have been identified as resistant to Megacopta cribraria (F.) (Hemiptera: Plataspidae). However, the mechanisms of host-plant resistance (HPR) in this system are not understood. The goal of this study was to identify the mechanisms of resistance by examining the role of plant volatile organic compounds (VOCs) and free amino acids (FAAs) among 16 soybean varieties. Choice and no-choice cage experiments identified several soybean varieties that demonstrated antixenosis as well as antibiosis. However, resistance varied over time in certain soybean varieties, such as N02-7002 and PI567352B. Mean nymph number from choice experiments had positive correlations with the FAAs asparagine, tryptophan, alanine, phenylanaline, and serine; negative correlation with leucine and threonine. Four plant volatiles, hexanal, 2-pentylfuran, beta-cyclocitral, and cis-9-hexadecenal, were positively correlated with subsequent nymph development, whereas n-hexadecenoic acid was negatively correlated with nymph number only, in adult choice cage experiments. This study contributes to understanding the mechanisms of HPR through associations with plant VOCs and FAAs in relation to M. cribraria development and provides useful knowledge for developing soybean varieties for M. cribraria management.

Soybean Host Plant Resistance to Megacopta cribraria (Hemiptera: Plataspidae) and the Potential Role of Leaf Trichome Density.

Megacopta cribraria (F.) (Hemiptera: Plataspidae) is an invasive pest of soybean that has spread across the southeastern United States since its initial discovery in 2009 in Georgia. Previous studies in the southeastern states have documented both the population dynamics of this pest and host plant resistance (HPR) among soybean varieties, although the specific mechanisms of HPR remain unknown. The objectives of this study were, therefore, to 1) quantify field resistance to M. cribraria in multiple soybean varieties in two states previously affected by severe M. cribraria infestations, North Carolina (NC) and South Carolina (SC); and 2) study the role of soybean trichome density in imparting resistance against M. cribraria. Soybean variety 'Camp' was least attractive to M. cribraria, through time and locations, suggesting consistent resistance. Other varieties showed variable performance among the locations and sampling dates. A significant difference in trichome density was evident. However, there was no correlation between trichome density and M. cribraria infestation. Compared to a previously published study in the same location, when M. cribraria adults emerging from overwintering dispersed into soybeans, in our study only first-generation adults dispersed into soybeans. Considering the current trend of significantly lower M. cribraria infestation rates in North and South Carolina, this pest may be finally succumbing to indigenous natural enemies and should be managed by incorporating integrated pest management tactics, such as HPR, that help conserve natural enemy populations.

Host Plant Resistance to Megacopta cribraria (Hemiptera: Plataspidae) in Diverse Soybean Germplasm Maturity Groups V Through VIII.

Initially discovered in Georgia in 2009, the exotic invasive plataspid, Megacopta cribraria (F.), has become a serious pest of soybean ( Glycine max (L.) Merrill). Managing M. cribraria in soybean typically involves the application of broad-spectrum insecticides. Soybean host plant resistance is an attractive alternative approach; however, no commercial soybean cultivars have been identified as resistant. During 2013 and 2014, we compared 40 and 44 soybean genotypes, respectively, for resistance to M. cribraria in a split-plot design under natural insect infestation in small-plot experiments. Soybean genotypes were selected to maximize diversity with respect to maturity group, pubescence type, leaf shape, seed size, nitrogen fixation, drought tolerance, seed protein content, and pest resistance. Megacopta cribraria egg masses, nymphs, and adults were counted during the growing season to identify potentially resistant soybean genotypes. Soybean seed yield was measured in insecticide-protected and unprotected conditions to determine tolerance to M. cribraria feeding. In both years, a range of host plant resistance was observed. The fewest M. cribraria adults and nymphs were found on narrow-leaf, small-seeded cultivars 'N7103' and 'Vance,' as well as the nonnodulating cultivar 'Nitrasoy.' Additionally, N7103 and Vance were among the least susceptible genotypes to M. cribraria oviposition in the field. Most 'Benning' cultivar insect-resistant near-isogenic breeding lines also displayed moderate levels of resistance to M. cribraria . Seed yields of Vance and N7103 were less affected by M. cribraria in 2013 than most other soybean genotypes. These results may be useful to soybean breeders to develop cultivars with resistance to M. cribraria.

A major QTL conditioning salt tolerance in S-100 soybean and descendent cultivars.

Deployment of salt tolerant cultivars is an effective approach to minimize yield loss in a saline soil. In soybean, Glycine max (L.) Merr., substantial genetic variation exists for salt response. However, breeding for salt tolerance is hampered because no economically viable screening method has been developed for practical breeding. To facilitate the development of an effective screening method for salt tolerance in soybean, the present study was conducted to determine the heritability of salt tolerance and to identify associated quantitative trait loci (QTL). F2:5 lines from the cross of 'S-100' (salt tolerant) x 'Tokyo' (salt sensitive) were evaluated in a saline field in Hyde County, N.C., USA, in 1999 and in a greenhouse located in Raleigh, N.C., USA, in 2001. S-100 and Tokyo are ancestors of popular soybean cultivars released for the southern USA. The visual salt tolerance ratings of the F2:5 lines ranged from 0 (complete death) to 5 (normal healthy appearance). The entry-mean heritability for salt tolerance was 0.85, 0.48, and 0.57 in the field (four replications), greenhouse (two replications), and combined environments, respectively. The genotypic correlation between field and greenhouse ratings was 0.55, indicating reasonably good agreement between the two screening environments. To identify QTL associated with salt tolerance, each line was characterized with RFLP markers and an initial QTL single-factor analysis was completed. These results were used to identify genomic regions associated with the trait and to saturate the selected genomic regions with SSR markers to improve mapping precision. Subsequently, a major QTL for salt tolerance was discovered near the Sat_091 SSR marker on linkage group (LG) N, accounting for 41, 60, and 79% of the total genetic variation for salt tolerance in the field, greenhouse, and combined environments, respectively. The QTL allele associated with tolerance was derived from S-100. Pedigree tracking was used to examine the association between the salt tolerance QTL and flanking SSR marker alleles in U.S. cultivars descended from S-100 or Tokyo through 60 years of breeding. The presence of alleles from S-100 at the Sat_091 and Satt237 marker loci was always associated with salt tolerance in descendants. Alleles from Tokyo for these same markers were generally associated with salt sensitivity in descendent cultivars. The strong relationship between the SSR marker alleles and salt tolerance suggests that these markers could be used for marker-assisted selection in commercial breeding.

Aluminum accumulation at nuclei of cells in the root tip. Fluorescence detection using lumogallion and confocal laser scanning microscopy.

The mechanistic basis for Al toxicity effects on root growth is still a matter of speculation, but it almost certainly involves decreased cell division at the root apex. In this series of experiments, we attempt to determine whether Al enters meristematic cells and binds to nuclei when roots are exposed to a low Al(3+) activity in solution. The methodology involved the use of the Al-sensitive stain lumogallion (3-[2,4 dihydroxyphenylazo]-2-hydroxy-5-chlorobenzene sulfonic acid), the DNA stain 4',6-diamino-phenylindole, and confocal laser scanning microscopy. Soybean (Glycine max L. Merr.) cv Young (Al-sensitive) and PI 416937 (Al-tolerant) genotypes were exposed to 1.45 microM Al(3+) for periods ranging from 30 min to 72 h, and then washed with 10 mM citrate to remove apoplastic Al. Fluorescence images show that within 30 min Al entered cells of the sensitive genotype and accumulated at nuclei in the meristematic region of the root tip. Substantial Al also was present at the cell periphery. The images indicated that the Al-tolerant genotype accumulated lower amounts of Al in meristematic and differentiating cells of the root tip and their cell walls. Collectively, the results support an important role for exclusion in Al tolerance.

RFLP tagging of QTLs conditioning specific leaf weight and leaf size in soybean.

Selection for high specific leaf weight (SLW) in soybean [Glycine max (L) Merr.] may increase apparent photosynthetic rate per unit leaf area (AP), which in turn may improve seed yield. In general, the SLW and leaf size are negatively correlated in soybean. To maximize total photosynthetic performance, and perhaps the seed yield, of a soybean cultivar, it would be necessary to establish a large leaf area rapidly while maintaining a high SLW. The objective of the present study was to identify quantitative trait loci (QTLs) conditioning SLW and leaf size in soybean. One hundred and twenty F4-derived lines from a 'Young'×PI416937 population were evaluated using restriction fragment length polymorphism (RFLP) markers. The genetic map consisted of 155 loci on 33 linkage groups (LGs) covering 973 cM of map distance. The phenotypic data were collected from two different environments - a greenhouse at Athens, Ga. and a field site at Windblow, N.C. The SLW and leaf-size measurements were made on leaves from the 8th and 9th node of soybean plants at the V12 stage of development. Combined over environments, six putative independent RFLP markers were associated with SLW, and four of these loci were consistent across environments. Individually, the six markers each explained between 8 and 18% of the phenotypic variation among lines for SLW. The Young alleles contributed to a greater SLW at four of the six independent marker loci, and transgressive segregation occurred among the progeny for SLW. Three putative independent RFLP markers were associated with leaf size, each explaining between 6 to 11% of the phenotypic variation in the trait, and one of these markers was identified in both environments. There was no correlation between SLW and leaf size in this population. Similarly, none of the six QTLs conditioning SLW were linked to any of the three QTLs for leaf size. In this soybean population, it is possible to select for progeny lines with greater SLW than either parent perhaps without affecting the leaf size. It is feasible to pyramid all of the desirable alleles for greater SLW and large leaf size in a single genetic background.

RFLP loci associated with soybean seed protein and oil content across populations and locations.

Molecular markers provide the opportunity to identify marker-quantitative trait locus (QTL) associations in different environments and populations. Two soybean [Glycine max (L.) Merr.] populations, 'Young' x PI 416 937 and PI 97100 x 'Coker 237', were evaluated with restriction fragment length polymorphism (RFLP) markers to identify additional QTLs related to seed protein and oil. For the Young x PI 416937 population, 120 F4-derived lines were secored for segregation at 155 RFLP loci. The F4-derived lines and two parents were grown at Plains, G.a., and Windblow and Plymouth, N.C. in 1994, and evaluated for seed protein and oil. For the PI 97100 x Coker 237 population, 111 F2-derived lines were evaluated for segregation at 153 RFLP loci. Phenotypic data for seed protein and oil were obtained in two different locations (Athens, G.a., and Blackville, S.C.) in 1994. Based on single-factor analysis of variance (ANOVA) for the Young x PI 416937 population, five of seven independent markers associated with seed protein, and all four independent markers associated with seed oil in the combined analysis over locations were detected at all three locations. For the PI 97 100 x Coker 237 population, both single-factor ANOVA and interval mapping were used to detect QTLs. Using single-factor ANOVA, three of four independent markers for seed protein and two of three independent markers for seed oil were detected at both locations. In both populations, singlefactor ANOVA, revealed the consistency of QTLs across locations, which might be due to the high heritability and the relatively few QTLs with large effects conditioning these traits. However, interval mapping of the PI 97100 x Coker 237 population indicated that QTLs identified at Athens for seed protein and oil were different from those at Blackville. This might result from the power of QTL mapping being dependent on the level of saturation of the genetic map. Increased seed protein was associated with decreased seed oil in the PI 97100 x Coker 237 population (r = -0.61). There were various common markers (P[Symbol: see text]0.05) on linkage groups (LG) E, G,H,K, and UNK2 identified for both seed protein and oil. One QTL on LG E was associated with seed protein in both populations. The other QTLs for protein and oil were population specific.

Molecular markers associated with seed weight in two soybean populations.

Seed weight (SW) is a component of soybean, Glycine max (L.) Merr., seed yield, as well as an important trait for food-type soybeans. Two soybean populations, 120 F4-derived lines of 'Young'xPI416937 (Pop1) and 111 F2-derived lines of PI97100x'Coker 237' (Pop2), were mapped with RFLP makers to identify quantitative trait loci (QTLs) conditioning SW across environments and populations. The genetic map of Pop1 consisted of 155 loci covering 973 cM, whereas Pop2 involved 153 loci and covered 1600 cM of map distance. For Pop1, the phenotypic data were collected from Plains, GA., Windblow, N.C., and Plymouth, N.C., in 1994. For Pop2, data were collected from Athens, GA., in 1994 and 1995, and Blackville, S.C., in 1995. Based on single-factor analysis of variance (ANOVA), seven and nine independent loci were associated with SW in Pop1 and Pop2, respectively. Together the loci explained 73% of the variability in SW in Pop1 and 74% in Pop2. Transgressive segregation occurred among the progeny in both populations. The marker loci associated with SW were highly consistent across environments and years. Two QTLs on linkage group (LG) F and K were located at similar genomic regions in both populations. The high consistency of QTLs across environments indicates that effective marker-assisted selection is feasible for soybean SW.

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast.

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll](-1) s(-1). Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I](-1). In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll](-1) s(-1), respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I](-1). No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b6f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid.

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23 alpha. These components had RF values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbent assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.

Ethanol embolization of vascular malformations.

Absolute ethanol was used to treat 20 patients with symptomatic vascular malformations (SVMs) (ie, venous malformations, arteriovenous malformations, and congenital and posttraumatic arteriovenous fistulas) in whom previous surgery or standard embolotherapy had failed or who were not candidates for surgery. All large complex lesions required multiple embolizations as staged procedures. Immediate thrombosis was achieved in all patients; complications (13% of cases) were generally minor and were treated conservatively. Follow-up studies, performed in 19 of 20 patients, showed persistent occlusion of the SVM in all cases. Ethanol embolization of SVMs, performed according to strict techniques, has proved efficacious in SVM management and is emerging as a definitive form of therapy.

Image-directed percutaneous biopsies with a biopsy gun.

Core tissue for histologic study is believed by many pathologists to be more diagnostic than material from needle aspiration. Recently, a biopsy "gun" has been introduced, which simplifies core biopsies. With this device, 182 biopsies of multiple anatomic sites were performed with ultrasonic, computed tomographic, and fluoroscopic guidance and 18-gauge needles. High-quality histopathologic specimens were obtained in 177 of the biopsies, and diagnostic target tissue was obtained in 167. Only three significant complications occurred: one bleeding complication that required transfusion and two cases of pneumothorax that necessitated placement of chest tubes. The biopsy gun eliminated the disjointed movements of conventional "skinny" needle biopsies, and none of the samples demonstrated significant "crush" artifact or obscuring blood, problems that are commonly associated with manual biopsy techniques. Patient discomfort was decreased with this system compared with that of manual biopsies, and the total procedure time was reduced. Because of these distinct advantages, the authors now use the biopsy gun exclusively for all percutaneous biopsies and recommend that other institutions consider the use of this biopsy method.

Symptomatic vascular malformations: ethanol embolotherapy.

Absolute ethanol was used to perform nine transcatheter embolizations and 21 direct percutaneous puncture embolizations in eight patients with unresectable vascular malformations. Six patients had arteriovenous malformations and two patients had hemangiomas. Four of these patients had undergone unsuccessful surgery, and the other four were not considered candidates for operation. All large complex symptomatic vascular malformations (SVMs) required multiple embolizations that were staged procedures. Ethanol embolotherapy, performed according to strict techniques, has proved efficacious in the management of SVMs.

Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes.

Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.

Bovine brain calmodulin-dependent protein phosphatase. Regulation of subunit A activity by calmodulin and subunit B.

Calmodulin-dependent protein phosphatase isolated from bovine brain consists of a catalytic subunit A (Mr = 60,000) and a regulatory subunit B (Mr = 19,000) present in equal molar ratios. The two subunits were dissociated by gel filtration in 6 M urea and reconstituted to investigate the role of calmodulin and subunit B in regulating the phosphatase activity of subunit A. The activity of subunit A was stimulated 2-fold by calmodulin, 13-fold by subunit B, and 21-fold by both, indicating that the effects of both were synergistic. Maximum stimulation by calmodulin was observed at a calmodulin to subunit A molar ratio of 2:1 in the presence or absence of subunit B, whereas that by subunit B was observed at a B to A molar ratio of 3:1 in the presence or absence of calmodulin. Calmodulin and subunit B increased the Vmax of subunit A 2- and 5-fold, respectively, but had little effect on the Km for casein. The specific activity of the phosphatase reconstituted from subunits A and B reached 86% that of the native enzyme, whereas that of the holoenzyme reached 90%. Subunit B, even though similar to calmodulin in many respects, did not stimulate the activity of native phosphatase, suggesting that it cannot substitute for calmodulin. Limited trypsinization of subunit A increased its catalytic activity to the level observed with calmodulin; and this activity was further stimulated by subunit B but not by calmodulin. These results indicate that subunit A of phosphatase contains one catalytic domain and two distinct regulatory domains, one for calmodulin, and another for subunit B, that these two proteins do not substitute for one another and that they stimulate subunit A synergistically.

Subunit A of calmodulin-dependent protein phosphatase requires Mn2+ for activity.

Bovine brain calmodulin-dependent protein phosphatase comprises a catalytic subunit A (Mr 60,000) and a regulatory subunit B (Mr 19,000). The native enzyme was active with Ca2+ or Mn2+. Upon resolution into its subunits in 6 M urea and 15 mM EDTA, subunit A was active with Mn2+; Co2+ and Ni2+ partially substituted for Mn2+, but Ca2+, Mg2+ and Zn2+ were ineffective. The stimulating effect of Mn2+ was not easily reversed by EGTA. Like the native phosphatase, subunit A was markedly stimulated by calmodulin or by controlled trypsinization. Unlike the native enzyme, however, trypsinized subunit A still required Mn2+ for activity. These findings provide evidence that the catalytic subunit of phosphatase may be a metallo (possibly Mn2+) enzyme.