Functional ultrasound imaging of the human spinal cord.

Utilizing the first in-human functional ultrasound imaging (fUSI) of the spinal cord, we demonstrate the integration of spinal functional responses to electrical stimulation. We record and characterize the hemodynamic responses of the spinal cord to a neuromodulatory intervention commonly used for treating pain and increasingly used for the restoration of sensorimotor and autonomic function. We found that the hemodynamic response to stimulation reflects a spatiotemporal modulation of the spinal cord circuitry not previously recognized. Our analytical capability offers a mechanism to assess blood flow changes with a new level of spatial and temporal precision in vivo and demonstrates that fUSI can decode the functional state of spinal networks in a single trial, which is of fundamental importance for developing real-time closed-loop neuromodulation systems. This work is a critical step toward developing a vital technique to study spinal cord function and effects of clinical neuromodulation.

Comparison of Severity Assessment Methods for Predicting Yield Loss to Rhizoctonia Foliar Blight in Soybean.

Rhizoctonia foliar blight, caused by Rhizoctonia solani Kühn anastomosis group 1, causes rapid and severe destruction of soybean foliage and pods during hot, wet weather. The objectives of this study were to determine the yield components affected by this disease and whether rating pod damage or foliar damage provides a more reliable method of assessing disease severity relative to impact on yield. Disease severity in the moderately resistant cv. NK S57-11 and the susceptible cv. Buckshot 723 was assessed in field plots in 1996 and 1997 using foliar and pod ratings (0-to-10 scale corresponding to 0 to 100% of tissue affected). Based on results from regression analysis, pod number, seed number, and seed weight per plot decreased as disease severity increased, whereas the proportion of partially filled pods and the weight of 100 seed were not affected. Yield loss appeared to be due primarily to loss of entire pods. Foliar and pod assessments of disease severity correlated positively in 1996 (r = 0.8343) and 1997 (r = 0.5958) for both cultivars, which suggests that either method can be used to identify relative differences among cultivars. However, pod assessments accounted for more variability than foliar assessments under low-disease conditions. Plants exhibiting moderate to severe symptoms of Rhizoctonia foliar blight also retained green stems and pods at harvest, which was evidence of delayed maturity.

Factors Affecting Soybean Root Colonization by Calonectria ilicicola and Development of Red Crown Rot Following Delayed Planting.

Field studies were conducted in 1994, 1995, and 1996 to determine the effects of planting date, cultivar susceptibility, and soil pathogen population on soybean root colonization by Calonectria ilicicola and subsequent development of red crown rot. Early season colonization of roots was important for red crown rot symptom development. Symptom development in the more susceptible cultivar, Sharkey, was reduced following delayed planting and remained low in the less susceptible cultivar, Cajun, regardless of planting date. Taproot colonization was positively correlated with inoculum density during all three growing seasons but was strongest in 1994. Also, lateral root colonization correlated positively with inoculum density in 1994, the only year in which foliar symptoms were detected. A substantial decrease in inoculum density in 1995, along with reduced soybean root colonization, were attributed to high soil temperatures and probably low rainfall recorded during that summer. The effect of soybean plant age on root colonization was examined by exposing plants to the pathogen at different ages. Soybean plants were most susceptible to C. ilicicola during the first week after seedling emergence. By the second week, susceptibility was reduced by nearly half, and it remained near that level for the next several weeks.

Effects of Soil Temperature on Microsclerotia of Calonectria ilicicola and Soybean Root Colonization by this Fungus.

Field soil artificially infested with laboratory-produced microsclerotia of Calonectria ilicicola was incubated for 1, 2, 3, or 6 weeks at 20, 25, 30, 35, and 40°C. These temperatures approximate soil temperatures that were measured in soybean fields during the growing season in south Louisiana. Germinable microsclerotia were enumerated after incubation at different temperatures, and soybean seeds were planted in these soils. After 8 weeks, percent root colonization was determined as a measure of infectivity of microsclerotia. Results showed that soil temperature is a critical factor in survival of microsclerotia. The optimal soil temperature range for survival of microsclerotia was 20 to 30°C, and the maximum soil temperature limit was 35°C, above which microsclerotia did not survive. Effects of temperature on soybean root colonization were examined in growth chambers by growing soybean plants in soil infested with laboratory-grown microsclerotia for 4 weeks after seed germination. Maximum infection of young soybean roots by C. ilicicola occurred between 25 and 30°C but decreased with increasing temperatures and was negligible at 40°C. According to these results, soil temperature is a critical environmental factor controlling the development of red crown rot in soybeans in Louisiana. These findings suggest that, if red crown rot is a threat, soybean planting time should be based on soil temperature rather than calendar dates.

Response of Soybean Sudden Death Syndrome to Subsoil Tillage.

The soilborne pathogen Fusarium solani f. sp. glycines causes sudden death syndrome (SDS) of soybean. Previous research indicated that soil compaction related directly to disease foliar symptoms. Therefore, we hypothesized that decreasing soil compaction would increase soil porosity and provide a more aerated root zone that would hinder root infection by the fungus and decrease SDS foliar symptom severity. Two experimental areas (110 by 120 m) were established to evaluate the relationship between soil variables and SDS. Across the experimental area, strips (9.14 m wide) were subsoiled perpendicular to soybean rows to a depth of 40 to 45 cm, which alternated with strips that were not tilled. In both 1999 and 2000, subsoiling dramatically reduced foliar symptoms of SDS. Compared with no-till plots, subsoiled plots had lower soil bulk density, greater soil porosity, and less soil moisture. In areas where SDS occurs and soil compaction exists, the use of subsoiling can be used to reduce severity of foliar symptoms of SDS.

Comparison of constitutive and inducible maize kernel proteins of genotypes resistant or susceptible to aflatoxin production.

Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared for differences in both constitutive and inducible proteins. Five additional constitutive proteins were found to be associated with resistance in over 8 of the 10 genotypes examined. Among these, the 58- and 46-kDa proteins were identified as globulin-1 and globulin-2, respectively. Differences in the ability to induce specific antifungal proteins, such as the higher synthesis of the 22-kDa zeamatin in resistant genotypes, were also observed between resistant and susceptible kernels incubated under germinating conditions (31 degrees C, 100% humidity). Both constitutive and inducible proteins appear to be necessary for kernel resistance. Embryo-killed kernels (unable to synthesize new proteins) supported the highest level of aflatoxins, whereas imbibed kernels (to hasten protein induction) supported the lowest among all treatments. This suggests that the synthesis of new proteins by the embryo plays an important role in conferring resistance. However, significantly lower levels of aflatoxin production in embryo-killed resistant kernels than in susceptible ones suggest that, in reality, high levels of constitutive antifungal proteins are indispensable to kernel resistance.

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina.

ABSTRACT To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.

Delayed maturity associated with southern green stink bug (Heteroptera: Pentatomidae) injury at various soybean phenological stages.

Delayed maturity in soybean, Glycine max (L.) Merr., occurred in response to infestation by southern green stink bug, Nezara viridula (L.), in 4 yr of field studies. Maturity delays followed stink bug infestation that occurred only during the pod set and filling stages (R3-R5.5), and infestations at R3-4 and R5 resulted in delayed maturity more consistently than did infestation at R5.5. Infestation levels of six stink bugs per 0.3 m of row for 7-14 d generally were required to delay soybean maturity. The greatest impact on seed yield and quality parameters followed stink bug infestations that occurred during R3-R5.5, which corresponded closely with the periods of infestation that resulted in delayed maturity. If both delayed maturity and yield reduction are considered, the pod elongation through late pod filling stages were most critical for protecting soybeans from southern green stink bugs.

Soybean Yield Loss to Sudden Death Syndrome in Relation to Symptom Expression and Root Colonization by Fusarium solani f. sp. glycines.

Field studies were conducted to determine the relationships between soybean yield and foliar disease index (FDX) of sudden death syndrome (SDS) as well as root colonization by Fusarium solani f. sp. glycines, the causal agent. Single-row plots in a soybean field with relatively uniform SDS incidence and severity were identified at growth stage R6 on cultivar Pioneer 9492 in DeSoto, IL, in 1997 and 1998. For each plot, foliar disease index (FDX), yield, and yield components were determined. In 1997, linear relationships between yield (Y, grams per meter of row) and FDX were obtained from the wide-row (Y = 207.84 - 1.09 FDX) and narrow-row (Y = 126.66 - 0.745 FDX) plots, respectively. A linear relationship (Y = 124.23 - 1.11 FDX) also was observed in 1998. Increase in each FDX unit caused yield loss from 18 to 29 kg/ha (0.7 - 1.1 g/m of row). FDX was negatively correlated with seed weight (grams per plant) in both years and with seed size (grams per 100 seeds) in 1997. A no-tilled field at Southern Illinois University planted to soybean cultivar Asgrow 5403 was divided into 25 plots in 1997 and 40 plots in 1998. Root samples were taken from each plot at five or six sampling times during the seasons. Roots were used to isolate and enumerate F. solani f. sp. glycines on a selective medium to obtain the CFU. FDX was assessed and soybean yield was obtained from each plot. Soybean yield correlated negatively with FDX in both years. Both yield and FDX correlated significantly with CFU from slightly before growth stage R1 to R2 in both years, and with area under the pathogen population curve (AUPC) in 1997. An increase in one unit of AUPC or CFU per gram of root at R6 was associated with yield loss of 0.19 or 0.014%, respectively.

Temperature Effects on Race Determination in Heterodera glycines.

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 degrees C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 degrees C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 degrees C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.

Nitrogen fertilizer influence on aflatoxin contamination of corn in Louisiana.

Studies were conducted in 1997 and 1998 on a Gigger silt loam at the Macon Ridge Research Station at Winnsboro, LA, to determine the influence of nitrogen (N) rate, timing, and starter nitrogen fertilizer on aflatoxin contamination in corn. Fertilizer N (0, 50, 100, 150, 200, and 250 lb of N/acre), two timings (at planting and six-leaf stage), and starter N fertilizer (a control and 10 lb of N/acre applied in furrow) were evaluated. Application of starter, N rates, and the interaction of starter with N timing and N rates significantly affected aflatoxin levels. Rates of 50-250 lb of N/acre were 34-43% lower in aflatoxin contamination than plots receiving no N. The application of 10 lb of N/acre starter reduced the aflatoxin levels by 20% compared to the no-starter control.

Distribution of antifungal proteins in maize kernel tissues using immunochemistry.

This study examined the distribution of two antifungal proteins, ribosome-inactivating protein (RIP) and zeamatin, in maize kernel tissues. Proteins were extracted from endosperm (including aleurone layer) and embryo tissues of imbibed maize kernels. Western blot analyses revealed that RIP-like protein was present at higher levels in endosperm than in embryo tissues, whereas zeamatin-like protein was more concentrated in embryo tissues than in endosperm tissues. However, there were three protein bands in the endosperm and two bands in the embryo that reacted to anti-RIP antibody in Western blot analyses. Tissue prints were conducted to localize the antifungal proteins. Imbibed kernels were cut longitudinally and transversely and blotted onto nitrocellulose membranes. Using antibodies against maize RIP and zeamatin, RIP was found primarily in the aleurone layer of the endosperm and glandular layer of scutellum, whereas zeamatin was located mainly in the kernel embryo. These results provide insight into the potential functions of these antifungal proteins, especially since the presence of RIP and zeamatin within maize kernels uniquely protects kernels from pathogens.

Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli.

The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species. It also inhibited A. flavus and F. moniliforme simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops.

A Corn Trypsin Inhibitor with Antifungal Activity Inhibits Aspergillus flavus alpha-Amylase.

ABSTRACT In this study, we found that the inhibition of fungal growth in potato dextrose broth (PDB) medium by the 14-kDa corn trypsin inhibitor (TI) protein, previously found to be associated with host resistance to aflatoxin production and active against various fungi, was relieved when exogenous alpha-amylase was added along with TI. No inhibitory effect of TI on fungal growth was observed when Aspergillus flavus was grown on a medium containing either 5% glucose or 1% gelatin as a carbon source. Further investigation found that TI not only inhibited fungal production of extracellular alpha-amylase when A. flavus was grown in PDB medium containing TI at 100 mug ml(-1) but also reduced the enzymatic activity of A. flavus alpha-amylase by 27%. At a higher concentration, however, TI stimulated the production of alpha-amylase. The effect of TI on the production of amyloglucosidase, another enzyme involved in starch metabolism by the fungus, was quite different. It stimulated the production of this enzyme during the first 10 h at all concentrations studied. These studies suggest that the resistance of certain corn genotypes to A. flavus infection may be partially due to the ability of TI to reduce the production of extracellular fungal alpha-amylase and its activity, thereby limiting the availability of simple sugars for fungal growth. However, further investigation of the relationship between TI levels and fungal alpha-amylase expression in vivo is needed.

Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus.

ABSTRACT Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce beta-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.

Yield Loss Caused by Bacterial Streak in Winter Wheat.

The relationship between severity of bacterial streak and yield in winter wheat was studied in field plots and using a single-tiller method. Regression analysis from single-tiller studies showed that the grain weight per spike decreased as bacterial streak severity increased in cvs. Florida 304 and Savannah. The number of kernels per spike decreased as bacterial streak severity increased in Savannah but not in Florida 304. There was no difference in slope of the regression line between different years, locations, or cultivars for grain weight per spike. However, grain weight per spike at 0% bacterial streak (intercept) was different for different years, locations, and cultivars. The average reduction in grain weight per spike was 0.012 g for every 1% increase in bacterial streak severity. Using this relationship for cv. Savannah, average bacterial streak severity of 10% would result in about a 9% reduction in the grain weight per spike. In Florida 304, bacterial streak severity of 10% would result in about a 7% reduction in the grain weight per spike. During 1993-94, the largest difference in bacterial streak severity between inoculated and noninoculated plots was 4% in cv. Pioneer 2548, and the smallest difference was less than 1% in cvs. Terral 101 and Florida 304. There were no yield differences between inoculated and noninoculated treatments for any genotype. In field plot studies at two locations during 1989-90, bacterial streak severity did not differ between inoculated and noninoculated plots in Alexandria, Louisiana; but in Winnsboro, Louisiana, bacterial streak severity was 18 to 40% in inoculated plots and less than 5% in noninoculated plots. Differences in yield between inoculated and noninoculated plots ranged from 1,370 kg/ha (24% loss) to -121 kg/ha in Winns-boro. During the three seasons in which these studies were conducted, bacterial streak severity averaged about 10% or less in susceptible cultivars in all experiments except one. Based on the relationships derived from single-tiller studies, this suggests that yield loss is likely to be low most years. As indicated by the experiment in Winnsboro, however, more severe yield reductions could occur in a susceptible cultivar if weather conditions are favorable for disease development.

Analysis of Bacterial Leaf Streak Epidemics on Winter Wheat in Louisiana.

Studies were conducted to characterize spatial and temporal progress of bacterial leaf streak disease (Xanthomonas translucens pv. translucens) on susceptible (Florida 304) and moderately resistant (Terral 101) winter wheat cultivars. Epidemics were initiated with rifampicin-resistant strain 88-14rif of X. translucens pv. translucens by establishing point sources of inoculum in plot centers. Incidence of bacterial leaf streak was assessed five times in 1995 and three times in 1996, starting from the first observation of leaf streak symptoms. Rainfall, temperature, and wind speed were significantly related to disease incidence, but relative humidity was not. The Gompertz model gave the best statistical fit for the progression of disease incidence over time. Average rates of disease progress (k) obtained from the regression of bacterial leaf streak incidence against time provided a good method of comparing the cultivars Florida 304 and Terral 101 and were consistent across locations. Bacterial leaf streak disease gradients were best described by the negative exponential model. Bacterial leaf streak incidence decreased with distance from inoculum source for both cultivars. Disease incidence on Terral 101 was near 0% at 2 m from the source, and disease incidence close to the source was consistently lower on Terral 101 than on Florida 304 at all growth stages sampled. This was not unexpected because the two cultivars differed in susceptibility. Disease incidence data were more useful than severity data in providing a good estimate of disease spread away from the source.

Protein profiles and antifungal activities of kernel extracts from corn genotypes resistant and susceptible to Aspergillus flavus.

Mechanisms of resistance to infection by the fungus Aspergillus flavus and accumulation of aflatoxin were studied in kernels of resistant (GT-MAS:gk, Mp420) and susceptible ( Pioneer 3154, Deltapine G-4666) corn genotypes. Proteins from kernel extracts of corn genotypes were analyzed by several methods of polyacrylamide gel electrophoresis. Consistent differences in protein profiles were detected among genotypes. Several proteins were unique to or present in greater concentration in resistant genotypes, whereas others were present only in susceptible genotypes. Extracts of resistant kernels showed markedly greater antifungal activity against A. flavus than did susceptible kernel extracts. Results from the present study suggest a role for kernel proteins in resistance to A. flavus infection and aflatoxin contamination in corn genotypes.

Resistance to Aspergillus flavus in Corn Kernels Is Associated with a 14-kDa Protein.

ABSTRACT Corn genotypes resistant or susceptible to Aspergillus flavus were extracted for protein analysis using a pH 2.8 buffer. The profile of protein extracts revealed that a 14-kDa protein is present in relatively high concentration in kernels of seven resistant corn genotypes, but is absent or present only in low concentration in kernels of six susceptible ones. The N-terminal sequence of this 14-kDa protein showed 100% homology to a corn trypsin inhibitor. The 14-kDa protein purified from resistant varieties also demonstrated in vitro inhibition of both trypsin activity and the growth of A. flavus. This is the first demonstration of antifungal activity of a corn 14-kDa trypsin inhibitor protein. The expression of this protein among tested genotypes may be related to their difference in resistance to A. flavus infection and subsequent aflatoxin contamination.

Competition Between Tylenchorhynchus annulatus and Mesocriconema xenoplax on Grain Sorghum as Influenced by Macrophomina phaseolina.

Greenhouse experiments were conducted to examine competition between Tylenchorhynchus annulatus and Mesocriconema xenoplax on grain sorghum roots that were colonized by the fungus Macrophomina phaseolina or free from fungus colonization. An incomplete factorial treatment design consisted of two levels of M. phaseolina (0 or 10 colony-forming units/g soil) and 12 T. annulatus:M. xenoplax ratios: 1,000:0; 750:0; 500:0; 250:0; 0:0; 0:250; 0:500; 0:750; 0:1,000; 750:250; 500:500; and 250:750. Plants were harvested after 105 days. Despite similar feeding habits, competition between these ectoparasitic nematode species was limited. Tylenchorhynchus annulatus was more susceptible to antagonism by M. xenoplax than the reverse, but susceptibility depended on initial inoculum ratio. Root colonization by M. phaseolina reduced competitive effects of T. annulatus on M. xenoplax but not the reverse. Both nematode species reduced shoot dry weight but only T. annulatus reduced root dry weight. Both plant weight parameters were reduced by M. phaseolina.