Confirming QTLs and Finding Additional Loci Responsible for Resistance to Rice Sheath Blight Disease.

Rice sheath blight disease, caused by Rhizoctonia solani AG1-1A, is one of the most destructive rice diseases worldwide. Utilization of host resistance is the most economical and environmentally sound strategy in managing sheath blight (ShB). Ten ShB quantitative trait loci (QTLs) were previously mapped in a Lemont × Jasmine 85 recombinant inbred line (LJRIL) population using greenhouse inoculation methods at an early vegetative stage. However, confirmation of ShB-resistant QTLs under field conditions is critical for their utilization in marker-assisted selection (MAS) for improving ShB resistance in new cultivars. In the present study, we evaluated ShB resistance using 216 LJRILs under field conditions in Arkansas, Texas, and Louisiana during 2008 and 2009. We confirmed the presence of the major ShB-QTL qShB9-2 based on the field data and also identified one new ShB-QTL between markers RM221 and RM112 on chromosome 2 across all three locations. Based on the field verification of ShB evaluations, the microchamber and mist-chamber assays were simple, effective, and reliable methods to identify major ShB-QTLs like qShB9-2 in the greenhouse at early vegetative stages. The markers RM215 and RM245 were found to be closely linked to qShB9-2 in greenhouse and field assays, indicating that they will be useful for improving ShB resistance in rice breeding programs using MAS.

Determination of Host Responses to Magnaporthe grisea on Detached Rice Leaves Using a Spot Inoculation Method.

Through the use of standard assays, where conidia of the pathogen Magnaporthe grisea are sprayed onto rice, it is impossible to determine the exact number of conidia in any given area and to predict the locations of disease lesions in the rice blast system. To develop a localized, quantitative inoculation of M. grisea, a novel spot method was investigated. Serially diluted Tween 20 was added to M. grisea conidial suspensions in 0.25% (wt/vol) gelatin to promote adherence of conidia on detached rice leaves. Standard assays indicated no deleterious effects of Tween 20 to rice blast development and 0.02% (vol/vol) Tween 20 was necessary for promoting adherence of spore suspensions to the detached leaves. The spot method was evaluated using three well-characterized races of M. grisea and confirmed with standard assays. Disease reactions of rice to four predominant races of M. grisea were tested concurrently using the spot method and standard assays. Successful application of this assay will help identify novel sources of rice blast resistance and evaluate virulence of M. grisea to aid in breeding resistance to rice blast.

Screening Oryza Species Plants for Rice Sheath Blight Resistance.

Rice wild relatives, Oryza species, are one possible source of sheath blight (Rhizoctonia solani) resistance genes. However, Oryza spp. cannot be screened in the field as is done for cultivated rice (O. sativa) because the plant canopy does not favor disease development and many plants drop mature seed. Thus, a growth chamber-greenhouse method of screening Oryza spp. and their early generation progeny is needed. Primary-secondary and ratoon tillers of rice cultivars-germplasm which ranged from moderately resistant to very susceptible were evaluated first for sheath blight susceptibility. Plants were inoculated by placing R. solani-colonized toothpicks at the leaf collar, then incubating plants in a growth chamber. After 7 days, plants were visually rated for sheath blight severity, and the lesion length of each leaf was measured. Ranking of cultivar-germplasm susceptibility by visual rating of primary-secondary tillers corresponded to the ranking from field ratings. Visual ratings correlated best with combined lesion length of the second and third leaves. For ratoon tillers, visual ratings correlated best with second-leaf lesion length. Next, this method was used with ratoon tillers to evaluate sheath blight susceptibility of 21 Oryza spp. accessions and F1 progeny from crosses between 17 accessions and cultivated rice. This method proved useful on a limited scale for screening germplasm that could not be evaluated under field conditions.

Free fatty acids induce peripheral insulin resistance without increasing muscle hexosamine pathway product levels in rats.

To evaluate the role of the hexosamine biosynthesis pathway (HBP) in fat-induced insulin resistance, we examined whether fat-induced insulin resistance is additive to that induced by increased HBP flux via glucosamine infusion and, if so, whether such additive effects correlate with muscle HBP product levels. Prolonged hyperinsulinemic (approximately 550 pmol/l) euglycemic clamps were conducted in conscious overnight-fasted rats. After the initial 150 min to attain steady-state insulin action, rats received an additional infusion of saline, Intralipid, glucosamine, or Intralipid and glucosamine (n = 8 or 9 for each) for 330 min. At the conclusion of clamps, skeletal muscles (soleus, extensor digitorum longus, and tibialis anterior) were taken for the measurement of HBP product levels. Intralipid and glucosamine infusions decreased insulin-stimulated glucose uptake (Rd) by 38 and 28%, respectively. When the infusions were combined, insulin-stimulated Rd decreased 47%, significantly more than with Intralipid or glucosamine alone (P < 0.05). The glucosamine-induced insulin resistance was associated with four- to fivefold increases in muscle HBP product levels. In contrast, the Intralipid-induced insulin resistance was accompanied by absolutely no increase in HBP product levels in all of the muscles examined. Also, when infused with glucosamine, Intralipid decreased insulin action below that with glucosamine alone without changing HBP product levels. In a separate study, short-term (50 and 180 min) Intralipid infusion also failed to increase muscle HBP product levels. In conclusion, increased availability of plasma free fatty acids induces peripheral insulin resistance without increasing HBP product levels in skeletal muscle.

Rice Blast Epidemics Initiated by Infested Rice Grain on the Soil Surface.

Field experiments were conducted in 1996 and 1997 with a marked strain of Pyricularia grisea to determine if inoculum from infested rice grain could cause primary infections and sustain a rice blast epidemic during the growing season by giving rise to leaf, collar, and neck symptoms. The marked strain, a sulfate nonutilizing (sul) mutant of P. grisea, was grown on autoclaved rice seed for 7 days at 25°C. Infested rice grains were applied to the soil surface at the time of plant emergence (approximately 10 days after planting) at densities of 0, 0.5, 5, 25, and 50 grains per 0.1 m2 in plots planted to the blast susceptible cv. M-201. Leaf blast symptoms were first detected in the plots containing infested grain 35 days after plant emergence in both 1996 and 1997. The sul mutant was isolated from more than 90% of the lesions sampled from rice seedlings 35 to 45 days after plant emergence. Leaf blast increased more rapidly in plots with 25 and 50 infested grains per 0.1 m2 than in plots with less inoculum pressure (0.5 and 25 infested grains per 0.1 m2), although in 1996, leaf blast incidence recorded at midseason in plots containing 0.5 and 5 infested grains per 0.1 m2 was 41 and 55%, respectively. At the end of both seasons, the sul mutant was recovered from over 90% of the leaf, collar, and neck blast lesions except for one sample date in 1996. Rice blast was not detected in the control plots (no infested grain) in 1997 and not until 65 days after planting in 1996. Comparisons of disease progress on leaves between the marked strain and the parental wild-type strain under field conditions indicated that development of disease caused by the sul mutant was similar to disease caused by the wild-type strain.

Regional Population Diversity of Pyricularia grisea in Arkansas and the Influence of Host Selection.

MGR586 DNA fingerprinting has been widely used to characterize population diversity of the rice blast pathogen, Pyricularia grisea. However, the frequency and distribution of particular haplotypes (individuals) within MGR-delimited lineages has not been examined in the United States. MGR586 DNA fingerprinting, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLPs), and virulence phenotyping were used to examine genetic diversity of P. grisea in Arkansas. A total of 470 monoconidial isolates were recovered from eight rice cultivars in 18 commercial fields in nine counties in Arkansas. All isolates were examined for nuclear DNA RFLPs with the MGR586 DNA fingerprint probe, and both the MGR lineage (isolates with >80% similarity) and the haplotype frequencies were determined. Four distinct MGR586 DNA fingerprint lineages (designated A, B, C, and D) were identified among the 470 field isolates. All four lineages were found in 9 of the 18 locations. Three lineages were found in four locations, two lineages in three locations, and only a single lineage was found at two locations. In all, 10, 19, 16, and 13 haplotypes (isolates which had MGR586 DNA fingerprints which differed by 1 to 20%) were identified within lineages A, B, C, and D, respectively, among the 470 isolates examined. Within each lineage, a single haplotype (clone) predominated, representing 51 to 71% of the isolates collected for each of the four lineages. Overall, 60% of the 470 isolates belonged to one of only four haplotypes (A1, B1, C1, and D1) and these four predominant haplotypes were recovered from between 7 and 14 of the 18 locations sampled, indicating a widespread distribution of these four clones. These data indicate an exceptionally low level of genetic diversity in the regional rice blast pathogen population in Arkansas relative to several other populations of P. grisea examined from tropical environments. In addition, no mtDNA RFLPs were detected among representative haplotypes within each of the lineages, indicating a single mtDNA haplotype was present in the population. Examination of virulence indicated that two races predominated in the regional collection. All 30 isolates in lineages A and C tested had an IB-49 virulence phenotype. Out of 30 isolates in lineages B and D, 29 had an IC-17 virulence phenotype. One isolate in lineage B, isolated from a highly susceptible cultivar (L201), had an IG-1 virulence phenotype. The frequencies of the four lineages varied among the locations sampled and may have been due, in part, to the cultivar from which isolates were recovered. A single lineage was recovered from two cultivars, Mars and Millie. Although only a single field of each of these cultivars was sampled, the data indicate that certain cultivars grown in Arkansas may serve as a "bottleneck", selecting out specific lineages in the regional population. To test this hypothesis, an additional 283 isolates were recovered from replicated plots of cvs. M204 and Mars located within commercial rice fields at two locations during two seasons. All four MGR586 lineages were recovered from each location. However, there was a strong bias for lineage B on cv. M204 (79% of all isolates) and a strong bias for lineage A on cv. Mars (95% of all isolates), indicating some cultivars were effective in excluding certain lineages.

Effect of Nitrogen Fertilization on Disease Progress of Rice Blast on Susceptible and Resistant Cultivars.

The effects of three nitrogen fertilization treatments on the development of rice blast were studied on eight cultivars under field conditions in Arkansas in 1995 and 1996. The eight cultivars (Kaybonnet, Cypress, Lacassine, Mars, Adair, Alan, Newbonnet, and RT7015) ranged from resistant to susceptible to blast according to previous field observations. The recommended nitrogen levels for the eight cultivars varied from 123 to 168 kg/ha/year. Three treatments, consisting of different rates and timing of nitrogen applications, were tested over 2 years at one location. The first treatment consisted of a single nitrogen (N) application applied to plots at the recommended rate at preflood during the midtillering stage. The second treatment consisted of applying nitrogen as a single preflood application but at 1.5 times the recommended N rate used in treatment one. The third treatment (control) consisted of applying the recommended amount of nitrogen fertilizer used in treatment one, but in a three-way-split application with 56 to 100 kg/ha (depending on the cultivar) of urea applied at preflood followed by the application of 34 kg/ha of N applied approximately 10 and 20 days after the panicle differentiation (PD) growth stage. Inoculated spreader plots were used to initiate rice blast epidemics in the test plots. The results indicate that the disease progress for rice blast, regardless of N treatments, followed a unimodal curve, whereby disease incidence and total lesion area per plant reached a maximum near midseason (PD growth stage) and then gradually declined. This decline in disease was attributed to adult resistance, leaf senescence, and the formation of new (noninfected) leaves. Application of nitrogen above the recommended rate for any given cultivar significantly increased disease incidence and total lesion area per plant on all cultivars except Kaybonnet, a highly resistant cultivar. Furthermore, a differential cultivar response to nitrogen was observed when measuring both disease incidence and total lesion area per plant. Leaf blast was significantly more severe on the susceptible and very susceptible cultivars when N fertilizer was applied as a single application at preflood than in the split application treatment. Nitrogen treatments did not significantly affect the incidence of collar rot or neck blast.

Characterization of Pyricularia grisea in the United States Using Independent Genetic and Molecular Markers.

ABSTRACT A total of 540 isolates of Pyricularia grisea from rice in the United States were examined for vegetative compatibility, MGR586 DNA fingerprint diversity, and mating type based on hybridization with the mat1-1 and mat1-2 sexual mating type alleles. The collections contained both archived and contemporary field isolates representative of the known MGR586 lineages and races that occur throughout the United States. Complementary nitrate nonutilizing (nit) or sulfate nonutilizing (sul) mutants were used to assess vegetative compatibility in P. grisea. There was a complete correspondence between vegetative compatibility groups (VCGs), MGR586 lineage, and mating type among 527 contemporary isolates (collected between 1991 and 1997) from Arkansas, Louisiana, Missouri, Mississippi, and Texas; all isolates in MGR586 lineages A, B, C, and D belonged to VCGs US-01, US-02, US-03, and US-04, respectively. In addition, all isolates tested in VCGs US-01 and US-04 had the mat1-1 mating type allele whereas those in VCGs US-02 and US-03 had the mat1-2 allele. The strict association of independent markers during this sample period was consistent with a strictly asexual mode of reproduction. However, examination of archived isolates collected in the 1970s and 1980s and contemporary isolates revealed an incongruent relationship between the independent markers. MGR586 C and E isolates were vegetatively compatible which indicated that multiple robust MGR586 delineated lineages could be nested within certain VCGs. Although isolates in lineages C and E were vegetatively compatible, they were of opposite mating type. Several hypotheses, including recombination, could account for the incongruence between the various markers. Among the eight MGR586 lineages (A through H) that occur in the United States, all isolates in lineages A, D, E, G, and H had the mat1-1 allele, whereas isolates in lineages B, C, and F had the mat1-2 allele. Nit mutants can be recovered relatively easy from P. grisea and should allow large numbers of individuals within a population to be assessed for vegetative compatibility. VCGs may prove to be an effective multilocus marker in P. grisea. Thus, VCGs should be a useful means for characterizing genetic structure in populations of the rice blast fungus worldwide, provide a useful genetic framework to assist in interpreting molecular population data, and may provide insight into potential sexual or asexual recombination events.

Intraocular pressure variation following retrobulbar anaesthesia among the different sex, age and ethnic groups in Malaysia.

A total of 114 patients (48 Chinese, 34 Malay and 32 Indian) undergoing extracapsular cataract extraction (ECCE) with intraocular lens implantation, were enrolled. All were given 3 ml of local anaesthetic (combination of equal amounts of lignocaine 2% and bupivacaine 0.5%) using retrobulbar technique. Intraocular pressure (IOP) was measured at different time intervals; before, immediately after and 5 minutes after injection with Honan balloon compression. Mean IOP increased by 5.0 mmHg immediately after injection (p < 0.001) and reduced to baseline level after 5 minutes of external compression. Although there were no statistical difference in terms of IOP variation between sex and age groups, Chinese patients demonstrated the highest IOP rise following retrobulbar anaesthesia. This is the first study to demonstrate the influence of race in the IOP response with Chinese subjects having the highest IOP rise.

Genotype distribution of hepatitis C virus infection in Taiwan.

To investigate the prevalence of genotype distribution of hepatitis C virus (HCV) infection in Taiwan, genotypes were identified in 122 (36 anti-HCV-positive blood donors, 44 anti-HCV-positive aborigines, 28 hemodialysis patients, and 14 patients with chronic liver diseases) of 280 subjects, using polymerase chain reaction by Okamoto's type-specific primer method. Type II was the dominant (66.7%) type among anti-HCV-positive blood donors, followed by type III and type IV with the same percentages (16.7%), while none of type I was detected. The prevalence of genotype distribution were 75.0%, 81.1%, and 64.3% for type II, 4.6%, 17.9%, and 21.4% for type III, 13.6%, 0%, and 7.1% for type IV, for the aborigines, hemodialysis, and chronic liver diseases groups, respectively. Four subjects revealed mixed infections by two different genotypes: two cases of II and III; and each one case of II and IV, and III and IV. Diverse genotype distributions in two hemodialysis groups disclose the existence of obvious regional differences even within a region. The results reveal the highest prevalence of type II as in Japan. However, there is a higher prevalence rate of type IV than in Japan.

The effect of recombinant human erythropoietin on hemostatic status in chronic uremic patients.

The hemostatic effects of recombinant human erythropoietin (rHuEP) were investigated in 20 patients with end-stage renal disease (thirteen on hemodialysis, seven without dialysis) receiving this hormone. We studied their hemograms and coagulation profiles before and at 1 month after initiation of rHuEP therapy. One month after rHuEP administration, improvement in anemia (16/20, 80%) and shortening of bleeding time (17/19, 89.5%) were observed. Shortening or correction of bleeding time was achieved in three patients without any increase of the hemoglobin level. This means that factors other than the increased hematocrit level might contribute to shortening bleeding time in uremic patients receiving rHuEP treatment. The platelet count, prothrombin time, partial thromboplastin time, and fibrinogen level did not change over the course of rHuEP therapy. Thrombosis of vascular access was not observed, and heparin doses were not increased in this short-term period. A significant decrease was found in the plasminogen level, from 108.5% to 88.2% (p less than 0.05), in uremic patients on hemodialysis. The antithrombin III level also decreased, from 98.8% to 89.8% (p less than 0.05), and its level dropped to below normal ranges in six of thirteen patients (46%) on hemodialysis after treatment with rHuEP. No significant change was noted in the levels of antithrombin III, plasminogen, and alpha 2-antiplasmin in uremic patients not receiving dialysis. These results suggest that rHuEP administration induces increased extracorporeal dialyzer clotting and consumption coagulopathy, and that this extracorporeal consumption coagulopathy may play a role in the genesis of thrombotic complications.