Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Quantitative assessment of the interaction between the antagonistic fungus Talaromyces flavus and the wilt pathogen Verticillium dahliae on eggplant roots

Quantitative aspects of the interaction between the antagonist Talaromyces flavus, the pathogen Verticillium dahliae and eggplant roots, were studied. When eggplant roots were inoculated with T. flavus, prior to the infection with the pathogen, the population density of T. flavus on V. dahliae-infected roots was at least 3 times higher than on healthy uninfected roots, and the proliferation of T. flavus on diseased eggplant roots was related to the severity of wilt symptoms, in the two levels of application of T. flavus studied. However, in all classes of disease severity tested (disease index, 0–3), the population density of T. Flavus on eggplant roots treated with 10⁶ ascospores g^–1 rooting mixture was significantly (p=0.05) higher than with 10⁵ ascospores g^–1. In roots treated with 10⁵ and 10⁶T. flavus ascospores g^–1 rooting mixture, the population density of V. dahliae was reduced by 51% and 69%, respectively. When testing the relationships between the population density of V. dahliae in the roots and disease severity, no significant (p=0.05) difference was found between disease indexes 2 and 3. However, the density of V. dahliae on roots of plants with disease index 1 was significantly (p=0.05) lower than disease indexes 2 and 3. The positive relationship between the inoculum concentration of V. dahliae and the population density of T. flavus developed on eggplant roots was significant (p=0.001), linear, and highly correlated (r=0.945) on a logarithmic scale. In addition, the analysis of these data revealed a significant (p=0.05), high, negative and linear correlation (r=–0.985) between the log concentration of V. dahliae inoculum and the disease reduction achieved by T. flavus.     

Genetic analysis of the anther-culture response of three spring wheat crosses

Summary Anther-culture response was examined among three spring wheat (Triticum aestivum L.) cultivars to evaluate the genetic component of response and to determine whether androgenetic performance could be improved by selection. The three lines, the three possible F₁'s among the three lines, their F₂'s, and the backcrosses to the parents were evaluated for callus production and regeneration capacity. Significant variation was observed among the generations of the three crosses for callus formation. Genetic variation for regenerability was nonsignificant. Callus production was negatively correlated (-0.24) with regeneration capacity. The random variation in the study was too great to determine whether major-gene differences for antherculture response exist among the three lines by examining population distributions. When the material was evaluated for quantitative gene effects, the estimates for the additive gene effects were generally greater than the estimates for the dominance gene effects for callus formation. Only the Pavon x Chris cross, however, exhibited a significant narrow sense heritability estimate for callusing response (0.94). Due to the large component of random variation and the varying selection potential among crosses for androgenetic performance, improving anther-culture response in wheat by selection could prove difficult unless the anther-culture process itself selects for response traits at the gametic level.     

Line x tester analysis for fixed effect model in cotton (Gossypium hirsutum L.)

Summary The data from an experiment in cotton consisting of three testers and 12 lines selected deliberately have been analysed. The investigation showed higher specific combining ability variance for yield of seed cotton and number of bolls, indicating the predominance of non-additive gene action. Of parental lines, H777 was found to possess high g.c.a. effects for seed cotton yield, number of bolls and number of sympodes. Parent H842 contributed only for boll weight, whereas H655 was good general combiner for number of monopodes. There appeared to be better chances for increasing the yield by exploiting hybrid vigour for the number of bolls and boll weight. The presence of marked non-additive gene effects, in addition to additive gene effects, indicated the need for exploiting both the fixable and non-fixable components of genetic variance for increasing productivity in cotton.     

Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen

Summary Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f₁, F₂, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1–10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.Contribution of the Department of Soil and Crop Sciences and the Texas Agricultural Experiment Station     

Genetic analysis of carbon isotope discrimination and agronomic characters in a bread wheat cross

Carbon isotope discrimination () has been suggested as a selection criterion to improve transpiration efficiency (W) in bread wheat (Triticum aestivum L.). Cultivars Chinese Spring with low A (high W) and Yecora Rojo with high (low W) were crossed to develop F₁, F₂, BC₁, and BC₂ populations for genetic analysis of and other agronomic characters under well-watered (wet) and water-stressed (dry) field conditions. Significant variation was observed among the generations for only under the wet environment. Generation x irrigation interactions were not significant for . Generation means analysis indicated that additive gene action is of primary importance in the expression of under nonstress conditions. Dominance gene action was also detected for , and the direction of dominance was toward higher values of . The broad-sense and the narrow-sense heritabilities for were 61 % and 57% under the wet conditions, but were 48% and 12% under the draughted conditions, respectively. The narrow-sense heritabilities for grain yield, above-ground dry matter, and harvest index were 36%, 39%, and 60% under the wet conditions and 21%, 44%, and 20% under dry conditions, respectively. The significant additive genetic variation and moderate estimate of the narrow-sense heritability observed for indicated that selection under wet environments should be effective in changing in spring bread wheat.     

Ultrastructural changes and location of beta-1, 3-glucanase in resistant and susceptible cotton callus cells in response to treatment with toxin of Verticillium dahliae and salicylic acid

Calli from two cotton cultivars susceptible and resistant to Verticillium wilt, were treated with a crude toxin of Verticillium dahliae (VD-toxin) plus salicylic acid (SA). Cells treated with VD-toxin showed distinct ultrastructural changes. Cells from the susceptible cultivar displayed damage to plasma membrane and cytoplasm. The deleterious effect on cells of the resistant cultivar, with an accumulation of electron-dense precipitate in the vacuoles, was less noticeable. Exogenous SA protected callus cells from VD-toxin. We also report the localization of β-1,3-glucanase in callus cells with immunofluorescence labeling. Stronger fluorescence was observed in the extra-cellular space in resistant than in susceptible cotton; strongest in resistant cotton after 5 days of treatment with VD-toxin plus SA. The findings reported here indicate an important role of exogenous salicylic acid in the induction of resistance to VD-toxin in cotton. Coupled with an increase in β-1,3-glucanase, cellular integrity is maintained and damage to cell wall and plasma membrane is avoided.     

ATG-anchored AFLP (ATG-AFLP) analysis in cotton

Amplified fragment length polymorphism (AFLP) marker system has had broad applications in biology. However, the anonymous AFLP markers are mainly amplified from non-coding regions, limiting their usefulness as a functional marker system. To take advantages of the traditional AFLP techniques, we propose substitution of a restriction enzyme that recognizes a restriction site containing ATG, called ATG-anchored AFLP (ATG-AFLP) analysis. In this study, we chose NsiI (recognizing ATGCAT) to replace EcoRI in combination with MseI to completely digest genomic DNA. One specific adaptor, one pre-selective primer and six selective amplification primers for the NsiI site were designed for ligation and PCR. Six NsiI and eight MseI primers generated a total of 1,780 ATG-AFLP fragments, of which 750 (42%) were polymorphic among four genotypes from two cultivated cotton species (Upland cotton, Gossypium hirsutum and Pima cotton, G. barbadense). The number of ATG-AFLP markers was sufficient to separate the four genotypes into two groups, consistent with their evolutionary and breeding history. Our results also showed that ATG-AFLP generated less number of total and polymorphic fragments per primer combination (2-3 vs. 4-5) than conventional AFLP within Upland cotton. Using a recombination inbred line (RIL) population, 62 polymorphic ATG-AFLP markers were mapped to 19 linkage groups with known chromosome anchored simple sequence repeat (SSR) markers. Of the nine ATG-AFLP fragments randomly chosen, three were found to be highly homologous to cotton cDNA sequences. An in-silico analysis of cotton and Arabidopsis cDNA confirmed that the ATG-anchored enzyme combination NsiI/MseI did generate more fragments than the EcoRI/MseI combination.     

Stable genetic transformation of cotton plants using polybrene-spermidine treatment

Polybrene and/or spermidine treatments were used to deliver plasmid DNA into cotton suspension culture obtained from cotyledon-induced callus. The transforming plasmid (pBI221.23) contained the selectablehpt gene for hygromycin resistance and the screenablegus gene. Primary transformant cotton plants were regenerated and analyzed by DNA hybridization and β-glucuronidase assay. The combination polybrene-spermidine treatment greatly enhanced the uptake and expression of DNA and the recovery of nonchimeric germ-line transgenic cotton plants.     

Photosynthetic electron transport in thylakoids from cotton cultivars (Gossypium sp.) differing in tolerance to prometryn

A method to determine photosynthetic electron transport in thylakoid membranes is described for Gossypium barbadense (cv. Pima S-7) and G. hirsutum (cv. DP 5415). These cultivars differed markedly in tolerance to prometryn, a PS II inhibitor. The rates of photosynthetic electron transport obtained were 245 mole oxygen mg^–1 chl h¹. Plant age and leaf size influenced the activity of the thylakoid preparations. Thylakoids from leaves of plants 24 to 37 d and 50–70 mm in diameter had the highest activities; thylakoids from cotyledons, fully expanded leaves and young leaves had low activity. Thylakoids from both species had similar photosynthetic activities and I₅₀'s for prometryn, atrazine and diuron. Thus, tolerance to prometryn was not due to differential binding at D1 protein.Abbreviations PSII photosystem II - DAP day after planting - DQ duroquinone - DBMIB dibromothymoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - I₅₀ concentration to inhibit reaction by 50% - Q_A quinone A - Q_B quinone B     

Studies of pollen maturation in cotton: the storage reserve accumulation phase

Summary This study follows the maturation of the pollen grain of cotton (Gossypium hirsutum L.), particularly the development of the vegetative cytoplasm and the various storage products formed. CTEM, HVEM, stereoscopy, and cyto-histochemistry were used to examine the events occurring during the 9 days before anthesis. Starch began to accumulate in plastids at anthesis minus 9 days and reached a peak concentration shortly before anthesis; lipid deposition followed a similar pattern, but started at 6 days before anthesis. Lipid bodies were always seen closely oppressed to the endoplasmic reticulum (ER). Dictyosomes appear active during the entire 9 days; first producing vesicles involved in the formation of the intine and, later, producing vesicles stored in the pollen grain. The dictyosome vesicles appear to contain polysaccharides and concentrate in layers around the lipid bodies. Ribosomes increase in number from 6 days before anthesis and are particularly numerous in the mature pollen. From anthesis minus 6 days until anthesis, the ER cisternae become increasingly inflated and, in the hours immediately before pollen release, form pockets filled with lipid bodies and dictysosome vesicles. The mature pollen has a core region filled with ER pockets and a peripheral cytoplasm in which such pockets are generally lacking.This research was supported in part by NSF Grant BMS575-22-23 and Grant N.RR-00592 from the Division of Research Resources, National Institutes of Health     

Integrative placement and orientation of non-redundant SSR loci in cotton linkage groups by deficiency analysis

A combination of previously mapped and unmapped non-redundant SSR loci, using 381 primer pairs were chromosomally and sub-chromosomally localized by deficiency analysis of two sets of quasi-isogenic interspecific Gossypium hirsutum L. hypoaneuploid F₁ hybrids involving Gossypium barbadense L. and Gossypium tomentosum (Nuttall ex Seemann). Polymorphisms were detected for 369 SSR primer pairs. A total of 318 SSR loci were rendered deficient by the available hypoaneuploid stocks, which included primary monosomics (2n = 51), monotelodisomics and duplication-deficient (segmental trisomic–monosomic) (2n = 52) types. Chromosomal associations were newly determined for 123 SSR loci, of which 90, 106 and 73 were polymorphic in G. tomentosum, G. barbadense, and both sets, respectively. The deficiency tests independently confirmed the recent identifications of linkage groups (LG) A01, A02, A03 and D08 to be chromosome (Chr)-13, Chr-8, Chr-11 and Chr-19, respectively, and collectively delimited LG D02 and D03 to Chr-21 and 24, and their homeologs to Chr-8 and 11. Segmental homeology was detected between Chr-2 and Chr-17 loci, adding to evidence of segmental homeology between Chr-2 and 3 versus Chr-14 and 17. The 318 non-redundant SSR loci localized in this study will enhance the construction of linkage maps and QTL identification in molecular marker assisted selection since the confirmed and newly discovered SSR loci can serve as anchor loci for their respective chromosomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Linkage Arrangement of RFLP loci in progenies from crosses between doubled haploid Asparagus officinalis L. clones

A preliminary genetic map of the dioecious species Asparagus officinalis L. (2n = 20) has been constructed on the basis of restriction fragment length polymorphism (RFLP) and isozyme marker data. With DNA samples digested with either EcoRI or HindIII 61 out of 148 probes (41%) identified RFLPs in six families of doubled haploid lines obtained through anther culture. A higher level of polymorphism (65%) was observed when a single family was screened for RFLPs using six distinct restriction enzymes. Segregation analysis of the BC progenies (40–80 individuals) resulted in a 418-cM extended map comprising 43 markers: 39 RFLPs, three isozymes and one morphological (sex). These markers are clustered in 12 linkage groups and four of them exhibited significant deviations from the expected 11 ratio. One isozyme and three RFLP markers were assigned to the sex chromosome.     

Genetic control of Lucilia cuprina: analysis of field trial data using simulation techniques

Summary An analytical version of the genetic control simulation program GENCON has been used to further analyze the data obtained during field trials of genetic control of the sheep blowfly, Lucilia cuprina, in 1976–79. In the simulations, population trends from a nonrelease area were used as an estimate of the rates of increase that would have occurred in the target population if there had been no releases. Genetic data from the target area (frequencies of matings by released males) were used to predict the frequencies of descendants of released males, the resulting genetic death, and the effects of this on population trends. In simulations that assumed no migration and full survival and competitiveness of all field-reared descendants of released males (translocation-bearing males and males and females heterozygous for deleterious mutations), neither the predicted genetic changes nor the predicted population trends agreed well with the observed data. Further simulations suggested that reduced survival or competitiveness of field-reared descendants did not account for this disagreement, but that immigration of wild flies into the test areas was probably a major contributor to the failure to achieve suppression. However, immigration alone was not sufficient to explain all the differences between observed and expected results. Other plausible contributors to this failure were: (1) lower survival of translocation males due to the effects of a dieldrin resistance allele carried on the translocation, and (2) increased survival of immature stages of L. cuprina at low population densities.     

Genetic mapping of EST-derived microsatellites from the diploid<Emphasis Type="Italic"> Gossypium arboreum</Emphasis> in allotetraploid cotton

To increase the numbers of microsatellites available for use in constructing a genetic map, and facilitate the use of functional genomics to elucidate fiber development and breeding in cotton, we sampled microsatellite sequences from expressed sequence tags (ESTs) transcribed during fiber elongation in the A-genome species Gossypium arboreum to evaluate their frequency of occurrence, level of polymorphism and distribution in the At and Dt subgenomes of tetraploid cotton. From among ESTs derived from G. arboreum fibers at 7–10 days post anthesis (dpa), 931 ESTs were found to contain simple sequence repeats (SSRs); 544 (58.4%) EST-SSR primer pairs were developed, and 468 (86%) amplified PCR products from allotetraploid cotton ( G. hirsutum cv. TM-1 and G. barbadense cv. Hai7124). However, only 99 (18.2%) of these were found to be polymorphic and segregating in our interspecific BC₁ mapping population [(TM-1×Hai7124)×TM-1]. In these amplified and informative EST-SSRs, hexa- and tri-nucleotide repeat motifs were the most frequent, representing 40.1 and 30%, respectively, of the total. A total of 111 loci detected with these 99 EST-SSRs were integrated into our backbone map including 511 SSR loci. The distribution of the EST-SSRs appeared to be non-random, since 72 loci were anchored to the At and 37 to the Dt subgenome of allotetraploid cotton based on linkage tests. Interestingly, out of the 10 pairs of duplicate loci amplified, seven were mapped to the corresponding homeologous linkage groups and/or chromosomes. BLASTX analysis revealed that 69 of the 99 ESTs showed significant similarities to known genes. Some genes important for fiber development, such as sucrose synthase, were mapped to corresponding chromosomes. These EST-SSRs provide structural and functional genomic information that will be useful for understanding cotton fiber development.Communicated by R. Hagemann     

Effectiveness of three Glomus species in protecting pepper (Capsicum annuum L.) against verticillium wilt

The objective of this work was to study the influence of three Glomus species—Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, Glomus intraradices (Schenck and Smith) and Glomus deserticola (Trappe, Bloss, and Menge)—on the development of Verticillium-induced wilt in Capsicum annuum cv. Piquillo. Results showed that the effectiveness of arbuscular mycorrhizal fungi (AMF) as biocontrol agents varied among different Glomus species. In pepper colonized by G. intraradices the severity of the disease was even higher than that observed in non-mycorrhizal plants in terms of plant growth and pepper yield. On the other hand, the high effectiveness exhibited by G. mosseae in improving plant growth and the early beginning of the reproductive stage in these plants was not associated with great plant protection and high pepper yield in diseased plants. Only plants associated with G. deserticola had greater yield than non-mycorrhizal ones despite the lower P fertilization applied to the mycorrhizal treatment and this fact was observed in both healthy and diseased plants. It is suggested that the higher specific phosphorus uptake in Verticillium-inoculated plants associated with G. deserticola could contribute to diminish the deleterious effect of pathogen on yield. On the other hand, the possible influence of endogenous phenolics in roots on the tolerance or resistance of pepper against wilt induced by Verticillium dahliae remains unclear.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid