The successful application of a marker-assisted wheat breeding strategy

A number of useful marker-trait associations have been reported for wheat. However the number of publications detailing the integrated and pragmatic use of molecular markers in wheat breeding is limited. A previous report by some of these authors showed how marker-assisted selection could increase the genetic gain and economic efficiency of a specific breeding strategy. Here, we present a practical validation of that study. The target of this breeding strategy was to produce wheat lines derived from an elite Australian cultivar ‘Stylet’, with superior dough properties and durable rust resistance donated from ‘Annuello’. Molecular markers were used to screen a BC₁F₁ population produced from a cross between the recurrent parent ‘Stylet’ and the donor parent ‘Annuello’ for the presence of rust resistance genes Lr34/Yr18 and Lr46/Yr29. Following this, marker-assisted selection was applied to haploid plants, prior to chromosome doubling with cochicine, for the rust resistance genes Lr24/Sr24, Lr34/Yr18, height reducing genes, and for the grain protein genes Glu-D1 and Glu-A3. In general, results from this study agreed with those of the simulation study. Genetic improvement for rust resistance was greatest when marker selection was applied on BC₁F₁ individuals. Introgression of both the Lr34/Yr18 and Lr46/Yr29 loci into the susceptible recurrent parent background resulted in substantial improvement in leaf rust and stripe rust resistance levels. Selection for favourable glutenin alleles significantly improved dough resistance and dough extensibility. Marker-assisted selection for improved grain yield, through the selection of recurrent parent genome using anonymous markers, only marginally improved grain yield at one of the five sites used for grain yield assessment. In summary, the integration of marker-assisted selection for specific target genes, particularly at the early stages of a breeding programme, is likely to substantially increase genetic improvement in wheat.     

Coexpression of the High Molecular Weight Glutenin Subunit 1Ax1 and Puroindoline Improves Dough Mixing Properties in Durum Wheat (Triticum turgidum L. ssp. durum)

Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS), plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina) and Puroindoline b (Pinb) genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread) and may improve the economic values of durum wheat.     

Molecular mapping of a gene for stripe rust resistance in spring wheat cultivar IDO377s

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. The best strategy to control stripe rust is to grow resistant cultivars. One such cultivar resistant to most races in North America is ‘IDO377s’. To study the genetics of its resistance this spring wheat cultivar was crossed with ‘Avocet Susceptible’ (AvS). Seedlings of the parents, F₂ plants, and F₃ lines were tested under controlled greenhouse conditions with races PST-43 and PST-45 of P. striiformis f. sp. tritici. IDO377s carries a single dominant gene for resistance. Resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the resistance gene. A total of ten markers were identified, two of which flanked the locus at 4.4 and 5.5 cM. These flanking RGAP markers were located on chromosome 2B with nulli-tetrasomic lines of ‘Chinese Spring’. Their presence in the ditelosomic 2BL line localized them to the long arm. The chromosomal location of the resistance gene was further confirmed with two 2BL-specific SSR markers and a sequence tagged site (STS) marker previously mapped to 2BL. Based on the chromosomal location, reactions to various races of the pathogen and tests of allelism, the IDO377s gene is different from all previously designated genes for stripe rust resistance, and is therefore designated Yr43. A total of 108 wheat breeding lines and cultivars with IDO377s or related cultivars in their parentage were assayed to assess the status of the closest flanking markers and to select lines carrying Yr43. The results showed that the flanking markers were reliable for assisting selection of breeding lines carrying the resistance gene. A linked stripe rust resistance gene, previously identified as YrZak, in cultivar Zak was designated Yr44.     

Comparative Gene Expression Analysis of Susceptible and Resistant Near-Isogenic Lines in Common Wheat Infected by Puccinia triticina

Gene expression after leaf rust infection was compared in near-isogenic wheat lines differing in the Lr10 leaf rust resistance gene. RNA from susceptible and resistant plants was used for cDNA library construction. In total, 55 008 ESTs were sequenced from the two libraries, then combined and assembled into 14 268 unigenes for further analysis. Of these ESTs, 89% encoded proteins similar to (E value of ≤10⁻⁵) characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions, cellular localization and biological processes based on gene ontology classification. Further, the unigenes were classified into susceptible and resistant classes based on the EST members assembled from the respective libraries. Several genes from the resistant sample (14-3-3 protein, wali5 protein, actin-depolymerization factor and ADP-ribosylation factor) and the susceptible sample (brown plant hopper resistance protein, caffeic acid O-methyltransferase, pathogenesis-related protein and senescence-associated protein) were selected and their differential expression in the resistant and susceptible samples collected at different time points after leaf rust infection was confirmed by RT–PCR analysis. The molecular pathogenicity of leaf rust in wheat was studied and the EST data generated made a foundation for future studies.     

Internalization of Flax Rust Avirulence Proteins into Flax and Tobacco Cells Can Occur in the Absence of the Pathogen

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.     

TaAbc1, a Member of Abc1-Like Family Involved in Hypersensitive Response against the Stripe Rust Fungal Pathogen in Wheat

To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.     

Molecular Characterization of Glutenins in Wheat Varieties Differing in Chapati Quality Characteristics

Five wheat (Triticum aestivum) varieties differing in chapati quality characteristics viz. C-306, K-68, HD-2745 and HD-2735 with good and Sonalika with poor chapati quality characteristics, were selected for the characterization or distribution of glutenin genes. Polymorphism was observed when genomic DNA of wheat varieties was hybridized with a HMW glutenin probe [glutenin subunit 10 (Dy10)]. No hybridization was observed in Sonalika. PCR amplification of genomic DNA with the LMW glutenin gene-specific primers did not show any polymorphism. However, with HMW glutenin gene-specific primers a single band of ～ 650 by was obtained in all the good chapati characteristic wheat varieties.The amplified fragment was sequenced and found to have sequence homology with HMW glutenin subunit Dx5.The deduced protein structure analysis showed that the peptide was made up of N-terminally placed (x-helices and centrally placed repetitive β-turns.     

Comparative field performance over 3 years and two sites of transgenic wheat lines expressing HMW subunit transgenes

A series of transgenic wheat lines expressing additional high molecular weight (HMW) subunit genes and the corresponding control lines were grown in replicate field trials at two UK sites (Rothamsted Research, approximately 50 km north of London and Long Ashton, near Bristol) over 3 years (1998, 1999, 2000), with successive generations of the transgenic lines (T₃, T₄, T₅) being planted. Four plots from each site were used to determine grain dry weight, grain nitrogen, dough strength (measured as peak resistance by Mixograph analysis) and the expression levels of the endogenous and “added” subunits. Detailed statistical analyses showed that the transgenic and non-transgenic lines did not differ in terms of stability of HMW subunit gene expression or in stability of grain nitrogen, dry weight or dough strength, either between the 3 years or between sites and plots. These results indicate that the transgenic and control lines can be regarded as substantially equivalent in terms of stability of gene expression between generations and environments.     

Cool-season whole-plant gas exchange of exotic and native semiarid bunchgrasses

The success of invasive aridland plants may depend on their utilization of precipitation not fully exploited by native species, which could lead to seasonally altered ecosystem carbon and water fluxes. We measured volumetric soil water across 25-cm profiles (??_25cm) and springtime whole-plant water- and carbon-fluxes of the exotic Lehmann lovegrass (Eragrostis lehmanniana) and a native bunchgrass, bush muhly (Muhlenbergia porteri), following typical (55?mm in 2009) and El Ni?o-enhanced accumulations (154?mm in 2010) in a SE Arizona savanna. Across both years, ??_25cm was higher under lovegrass plots, with similar evapotranspiration (ET) between lovegrass and bush muhly plots. However, in 2010 transpiration (T) was higher in bush muhly than lovegrass, implying higher soil evaporation in lovegrass plots maintained similar ET. Net ecosystem carbon dioxide exchange (NEE) was similar between lovegrass and bush muhly plots in 2009, but was more negative in bush muhly plots following El Ni?o, indicating greater CO₂ assimilation. Ecosystem respiration (R _eco) and gross ecosystem photosynthesis (GEP) were similar between lovegrass and bush muhly plots in 2009, but were higher in bush muhly plots in 2010. As a result, lovegrass plots reduced ecosystem water-use efficiency (WUE_e?=?NEE/ET), while bush muhly WUE_e remained constant between 2009 and 2010. Concurrent whole-plant WUE (WUE_p?=?GEP/T) did not change in lovegrass plots, but increased in bush muhly plots between these years. We concluded that cool-season precipitation use is not a component of Lehmann lovegrass invasive success, but that the change in ET partitioning and attendant shifts in cool-season WUE_e may increase interannual variation in ecosystem water- and carbon-exchange dynamics in the water-limited systems it dominates.     

Swedish Spring Wheat Varieties with the Rare High Grain Protein Allele of NAM-B1 Differ in Leaf Senescence and Grain Mineral Content

Some Swedish spring wheat varieties have recently been shown to carry a rare wildtype (wt) allele of the gene NAM-B1, known to affect leaf senescence and nutrient retranslocation to the grain. The wt allele is believed to increase grain protein concentration and has attracted interest from breeders since it could contribute to higher grain quality and more nitrogen-efficient varieties. This study investigated whether Swedish varieties with the wt allele differ from varieties with one of the more common, non-functional alleles in order to examine the effect of the gene in a wide genetic background, and possibly explain why the allele has been retained in Swedish varieties. Forty varieties of spring wheat differing in NAM-B1 allele type were cultivated under controlled conditions. Senescence was monitored and grains were harvested and analyzed for mineral nutrient concentration. Varieties with the wt allele reached anthesis earlier and completed senescence faster than varieties with the non-functional allele. The wt varieties also had more ears, lighter grains and higher yields of P and K. Contrary to previous information on effects of the wt allele, our wt varieties did not have increased grain N concentration or grain N yield. In addition, temporal studies showed that straw length has decreased but grain N yield has remained unaffected over a century of Swedish spring wheat breeding. The faster development of wt varieties supports the hypothesis of NAM-B1 being preserved in Fennoscandia, with its short growing season, because of accelerated development conferred by the NAM-B1 wt allele. Although the possible effects of other gene actions were impossible to distinguish, the genetic resource of Fennoscandian spring wheats with the wt NAM-B1 allele is interesting to investigate further for breeding purposes.     

Stability of puroindoline peptides and effects on wheat rust

Peptides modelled on the tryptophan rich domain of puroindolines and the related grain softness protein-1 have a broad range of antibacterial and antifungal activities. With the aims of further investigating the activities of these antimicrobial peptides we studied their activity against wheat rust diseases and environmental stability. PINA-based peptides were found to have high pH and thermal stability in addition to being stable over long periods at room temperature. These properties could make them excellent candidates as preservatives in food. PuroA, Pina-R39G and PuroB peptides adversely affected the morphology of the stripe rust spores (Puccinia striiformis f. sp. tritici), while PuroA and PuroB showed moderate inhibition of their germination. Additionally, GSP-5D reduced the germination of leaf rust spores (P. triticina). PuroA and PuroB sprayed onto stripe rust infected plants effected a moderate reduction in the number of stripe rust uredinia on wheat seedlings, as did PuroB sprayed onto the seedlings and allowed to coat the leaves for 5 day prior to spore infection. The results suggest that the presence of the PIN-based peptides may lower frequency of initial infection foci.     

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message

“To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.”

Abstract

Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F_3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.     

Improving the baking quality of bread wheat by genomic selection in early generations

Key message

Genomic selection shows great promise for pre-selecting lines with superior bread baking quality in early generations, 3 years ahead of labour-intensive, time-consuming, and costly quality analysis.

Abstract

The genetic improvement of baking quality is one of the grand challenges in wheat breeding as the assessment of the associated traits often involves time-consuming, labour-intensive, and costly testing forcing breeders to postpone sophisticated quality tests to the very last phases of variety development. The prospect of genomic selection for complex traits like grain yield has been shown in numerous studies, and might thus be also an interesting method to select for baking quality traits. Hence, we focused in this study on the accuracy of genomic selection for laborious and expensive to phenotype quality traits as well as its selection response in comparison with phenotypic selection. More than 400 genotyped wheat lines were, therefore, phenotyped for protein content, dough viscoelastic and mixing properties related to baking quality in multi-environment trials 2009–2016. The average prediction accuracy across three independent validation populations was r = 0.39 and could be increased to r = 0.47 by modelling major QTL as fixed effects as well as employing multi-trait prediction models, which resulted in an acceptable prediction accuracy for all dough rheological traits (r = 0.38–0.63). Genomic selection can furthermore be applied 2–3 years earlier than direct phenotypic selection, and the estimated selection response was nearly twice as high in comparison with indirect selection by protein content for baking quality related traits. This considerable advantage of genomic selection could accordingly support breeders in their selection decisions and aid in efficiently combining superior baking quality with grain yield in newly developed wheat varieties.

     

Rising atmospheric CO<Subscript>2</Subscript> concentration may imply higher risk of <Emphasis Type="Italic">Fusarium</Emphasis> mycotoxin contamination of wheat grains

Increasing atmospheric CO₂ concentration not only has a direct impact on plants but also affects plant–pathogen interactions. Due to economic and health-related problems, special concern was given thus in the present work to the effect of elevated CO₂ (750 μmol mol^?1) level on the Fusarium culmorum infection and mycotoxin contamination of wheat. Despite the fact that disease severity was found to be not or little affected by elevated CO₂ in most varieties, as the spread of Fusarium increased only in one variety, spike grain number and/or grain weight decreased significantly at elevated CO₂ in all the varieties, indicating that Fusarium infection generally had a more dramatic impact on the grain yield at elevated CO₂ than at the ambient level. Likewise, grain deoxynivalenol (DON) content was usually considerably higher at elevated CO₂ than at the ambient level in the single-floret inoculation treatment, suggesting that the toxin content is not in direct relation to the level of Fusarium infection. In the whole-spike inoculation, DON production did not change, decreased or increased depending on the variety × experiment interaction. Cooler (18 °C) conditions delayed rachis penetration while 20 °C maximum temperature caused striking increases in the mycotoxin contents, resulting in extremely high DON values and also in a dramatic triggering of the grain zearalenone contamination at elevated CO₂. The results indicate that future environmental conditions, such as rising CO₂ levels, may increase the threat of grain mycotoxin contamination.     

Identification and mapping in spring wheat of genetic factors controlling stem rust resistance and the study of their epistatic interactions across multiple environments

Stem rust (Puccinia graminis f. sp. tritici) is responsible for major production losses in hexaploid wheat (Triticum aestivum L.) around the world. The spread of stem rust race Ug99 and variants is a threat to worldwide wheat production and efforts are ongoing to identify and incorporate resistance. The objectives of this research were to identify quantitative trait loci (QTL) and to study their epistatic interactions for stem rust resistance in a population derived from the Canadian wheat cultivars AC Cadillac and Carberry. A doubled haploid (DH) population was developed and genotyped with DArT^® and SSR markers. The parents and DH lines were phenotyped for stem rust severity and infection response to Ug99 and variant races in 2009, 2010 and 2011 in field rust nurseries near Njoro, Kenya, and to North American races in 2011 and 2012 near Swift Current, SK, Canada. Seedling infection type to race TTKSK was assessed in a bio-containment facility in 2009 and 2012 near Morden, MB. Eight QTL for stem rust resistance and three QTL for pseudo-black chaff on nine wheat chromosomes were identified. The phenotypic variance (PV) explained by the stem rust resistance QTL ranged from 2.4 to 48.8 %. AC Cadillac contributed stem rust resistance QTL on chromosomes 2B, 3B, 5B, 6D, 7B and 7D. Carberry contributed resistance QTL on 4B and 5A. Epistatic interactions were observed between loci on 4B and 5B, 4B and 7B, 6D and 3B, 6D and 5B, and 6D and 7B. The stem rust resistance locus on 6D interacted synergistically with 5B to improve the disease resistance through both crossover and non-crossover interactions depending on the environment. Results from this study will assist in planning breeding for stem rust resistance by maximizing QTL main effects and epistatic interactions.     

Specificity of Activated CryIA Proteins from Bacillus thuringiensis subsp. kurstaki HD-1 for Defoliating Forest Lepidoptera

The insecticidal activity of the CryIA(a), CryIA(b), and CryIA(c) toxins from Bacillus thuringiensis subsp. kurstaki HD-1 was determined in force-feeding experiments with larvae of Choristoneura fumiferana, C. occidentalis, C. pinus, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, and Actebia fennica. The toxins were obtained from cloned protoxin genes expressed in Escherichia coli. The protoxins were activated with gut juice from Bombyx mori larvae. Biological activity of the individual gene products as well as the native HD-1 toxin was assessed as the dose which prevented 50% of the insects from producing frass within 3 days (frass failure dose [FFD₅₀]). The three toxins were about equally active against M. disstria. In the Choristoneura species, CryIA(a) and CryIA(b) were up to fivefold more toxic than CryIA(c). In the lymantriid species, CryIA(a) and CryIA(b) were up to 100-fold more toxic than CryIA(c). The toxicity of HD-1 was similar to that of the individual CryIA(a) or CryIA(b) toxins in all of these species. None of the CryIA toxins or HD-1 exhibited and toxicity towards A. fennica. Comparison of the observed FFD₅₀ of HD-1 with the FFD₅₀ expected on the basis of its crystal composition suggested a possible synergistic effect of the toxins in the two lymantriid species. Our results further illustrate the diversity of activity spectra of these highly related proteins and provide a data base for studies with forest insects to elucidate the molecular basis of toxin specificity.     

Genetic dissection of grain yield in bread wheat. II. QTL-by-environment interaction

The grain yield of wheat is influenced by genotype, environment and genotype-by-environment interaction. A mapping population consisting of 182 doubled haploid progeny derived from a cross between the southern Australian varieties ‘Trident’ and ‘Molineux’, was used to characterise the interaction of previously mapped grain yield quantitative trait locus (QTL) with specific environmental covariables. Environments (17) used for grain yield assessment were characterised for latitude, rainfall, various temperature-based variables and stripe rust infection severity. The number of days in the growing season in which the maximum temperature exceeded 30°C was identified as the variable with the largest effect on site mean grain yield. However, the greatest QTL-by-environmental covariable interactions were observed with the severity of stripe rust infection. The rust resistance allele at the Lr37/Sr38/Yr17 locus had the greatest positive effect on grain yield when an environment experienced a combination of high-stripe rust infection and cool days. The grain yield QTL, QGyld.agt-4D, showed a very similar QTL-by-environment covariable interaction pattern to the Lr37/Sr38/Yr17 locus, suggesting a possible role in rust resistance or tolerance. Another putative grain yield per se QTL, QGyld.agt-1B, displayed interactions with the quantity of winter and spring rainfall, the number of days in which the maximum temperature exceeded 30°C, and the number of days with a minimum temperature below 10°C. However, no cross-over interaction effect was observed for this locus, and the ‘Molineux’ allele remained associated with higher grain yield in response to all environmental covariables. The results presented here confirm that QGyld.agt-1B may be a prime candidate for marker-assisted selection for improved grain yield and wide adaptation in wheat. The benefit of analysing the interaction of QTL and environmental covariables, such as employed here, is discussed.     

Quenching of the fluorescence of proteins by silver nitrate

The mechanisms by which Ag⁺ may quench protein tryptophanyl fluorescence have been studied. A 1:1 Ag⁺-tryptophan complex was detected spectrophotometrically and shown to have a k_a = 6.5 × 10³ M^?1. The complex was nonfluorescent. Ag⁺ and NO₃^? each caused collisional quenching which proceeded at nearly diffusion-controlled rates in a series of indole-containing compounds. Analysis of the rates by means of Stern-Volmer plots and lifetime measurements showed also that charge and the presence of salt influence the quenching rate constants.The fluorescence of nonsulfhydryl proteins was quenched by AgNO₃ only in concentrations needed for Stern-Volmer quenching of simple indole model compounds. However, the plots for protein quenching were generally nonlinear, a reflection of the heterogeneity of tryptophanyl residues. AgNO₃ quenching increased the polarization of protein fluorescence and decreased the lifetime. Rotational relaxation times were determined from Perrin plots of reciprocal polarization vs fluorescence intensity in the presence of various amounts of AgNO₃.The fluorescence of the sulfhydryl proteins ovalbumin, yeast, and equine liver alcohol dehydrogenases was strongly quenched by AgNO₃ in parallel with the formation of Ag⁺-mercaptide bonds. The quenching of fluorescence of sulfhydryl proteins was exhibited even in 8 m urea, thus ruling out conformational change as a major basis for the quenching. It was found that Ag⁺ mercaptide bond formation was accompanied by development of an ultraviolet absorption band. The reaction of Ag⁺ with cysteine, for example, could be followed spectrophotometrically. The uv absorption of different silver mercaptides varied with the compound and pH.Since the uv absorption of Ag⁺-mercaptides extended up to 340 nm, and was also found in Ag⁺-treated sulfhydryl proteins, energy transfer from excited tryptophans seemed a reasonable basis for the observed fluorescence quenching. This possibility was confirmed by calculation of Förster critical transfer distances for a variety of donor-acceptor (Ag⁺-mercaptide) pairs.The lifetime of sulfhydryl protein fluorescence was decreased by AgNO₃, but the emission spectrum was relatively little affected, in contrast to previously reported quenching by Hg²⁺. Additional mechanisms of fluorescence alteration by Ag⁺ in proteins (e.g., “heavy atom” effect, conformational changes, enhancement of sulfhydryl quenching) are also considered.The spectral effects of Ag⁺ interaction with proteins have the following practical applications:determination of —SH groups; probe of accessibility of binding sites and tryptophan-sulfhydryl distances; determination of rotational relaxation times by Perrin plots of reciprocal polarization vs lifetime; kinetic studies of Ag⁺ interaction with proteins.     

Insecticidal activity of spore-free mutants of Bacillus thuringiensis against the Indian meal moth and almond moth

Three oligosporogenic mutants of Bacillus thuringiensis were assayed for toxicity against larvae of the Indian meal moth, Plodia interpunctella, and the almond moth, Ephestia cautella. The results were compared with insecticidal activity obtained from the parent strain (HD-1) and two standard B. thuringiensis formulations (HD-1-S-1971 and HD-1-S-1980) against the same insect species. The toxicity of the sporeless mutant preparations was significantly diminished against the Indian meal moth (10- to 26-fold increase in LC₅₀) but exceeded the toxicity of the standards against the almond moth. The toxicities of the B. thuringiensis preparations toward the Indian meal moth were consistent with the number of spores in the test samples, but spores did not contribute to toxicity to E. cautella larvae. A rationale for basing dosage on soluble protein was demonstrated for use in situations where spores are not a contributing factor in toxicity.