Survey of indigenous bacterial endophytes from cotton and sweet corn

The genotypic diversity of indigenous bacterial endophytes within stems and roots of sweet corn (Zea mays L.) and cotton (Gossypium hirsutum L.) was determined in field trials throughout one growing season. Strains were isolated from surface-disinfested tissues and identified by fatty acid analysis. Gram-negative bacteria comprised 70.5% of the endophytic bacteria and 27 of the 36 genera identified. The most frequently isolated groups from sweet corn roots, were Burkholderia pickettii and Enterobacter spp.; from sweet corn stems, Bacillus megaterium. Bacterial genera present in sweet corn roots were also generally present in sweet corn stems. However, Burkholderia gladioli, Burkholderia solanacearum and Enterobacter cloacae were isolated much more frequently from sweet corn roots than stems, whereas Methylobacterium spp. were found more frequently in sweet corn stems than roots. Agrobacterium radiobacter, Serratia spp. and Burkholderia solanacearum, were the most frequently isolated groups from cotton roots; and Bacillus megaterium and Bacillus pumilus from cotton stems. Acinetobacter baumannii and Arthrobacter spp. were present in cotton stems but not in cotton roots. There were 14 taxonomic groups present in cotton roots that were not in cotton stems; all but one were Gram-negative. These included, Agrobacterium radiobacter, Bacillus megaterium, Bacillus pumilus, Enterobacter asburiae, Pseudomonas chlororaphis, Serratia spp. and Staphylococcus spp. Rhizobium japonicum and Variovorax paradoxus were isolated, almost exclusively, from the roots of both crops. Bacterial taxa present in both sweet corn and cotton early in the season were generally present late in the season. The diversity of bacteria was greater in roots than stems for each crop.     

Effects of ultraviolet-B radiation on cotton (Gossypium hirsutum L.) morphology and anatomy

Cotton (Gossypium hirsutum L.) crop, cultivated between 40 degrees N and 40 degrees S, is currently experiencing 2-11 kJ m-2 d-1 of UV-B radiation. This is predicted to increase in the near future. An experiment was conducted to study the effect of enhanced UV-B radiation on vegetative and reproductive morphology and leaf anatomy of cotton in sunlit, controlled environment chambers. From emergence to harvest, cotton plants were exposed to 0, 8 or 16 kJ m-2 d-1 of UV-B in a square wave approach for 8 h from 0800 to 1600 h. Changes in plant height, internode and branch length, mainstem node number, leaf area, length and area of petals and bracts, and anther number per flower were recorded. Epidermal cell and stomatal density, stomatal index, leaf thickness, and epidermal, palisade and mesophyll tissue thickness were also measured. Initial chlorotic symptoms on leaves turned into necrotic patches on continued exposure to enhanced UV-B. Exposure to high UV-B reduced both vegetative and reproductive parameters and resulted in a smaller canopy indicating sensitivity of cotton to UV-B radiation. Enhanced UV-B radiation increased epicuticular wax content on adaxial leaf surfaces, and stomatal index on both adaxial and abaxial leaf surfaces. Leaf thickness was reduced following exposure to UV-B owing to a decrease in thickness of both the palisade and mesophyll tissue, while the epidermal thickness remained unchanged. The vegetative parameters studied were affected only by high levels of UV-B (16 kJ m-2 d-1), whereas the reproductive parameters were reduced at both ambient (8 kJ m-2 d-1) and high UV-B levels. The study shows that cotton plants are sensitive to UV-B at both the whole plant and anatomical level.     

Indirect somatic embryogenesis and plant recovery from cotton (Gossypium hirsutum L.)

Summary We describe a tissue culture procedure for somatic embryogenesis and plantlet regeneration in cotton (Gossypium hirsutum L. cv. Coker 312). Callused explants or individual globular embryos were transferred to basal media to induce somatic embryogenesis. To determine characteristic early indicators of successful germination and conversion, we identified six types of embryos that developed on basal media. Two of the six embryo types, designated as tulip-shaped and trumpet-shaped, could undergo conversion in preliminary tests, whereas the others had little or no developmental potential. Several media treatments designed to enhance the maturation of globular somatic embryos failed to increase the fraction of embryos which matured to form recoverable types. In efforts to improve plantlet recovery, tulip-shaped embryos were used in limited trials to contrast the effects of chemical and physical desiccation treatments on germination and conversion. The selective use of tulip-shaped somatic embryos, coupled with partial desiccation, seems to have augmented plant recovery. Growth habit, flowering, seed set, and lint production of most of the regenerated plants were comparable to seed-derived plants grown under the same conditions. Partial research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.     

Measuring gene flow in the cultivation of transgenic cotton (Gossypium hirsutum L.)

Transgenic Bt cotton NewCott 33B and transgenic tfd A cotton TFD were chosen to evaluate pollen dispersal frequency and distance of transgenic cotton (Gossypium hirsutum L.) in the Huanghe Valley Cotton-producing Zone, China. The objective was to evaluate the efficacy of biosafety procedures used to reduce pollen movement. A field test plot of transgenic cotton (6×6 m) was planted in the middle of a nontransgenic field measuring 210×210 m. The results indicated that the pollen of Bt cotton or tfd A cotton could be dispersed into the environment. Out-crossing was highest within the central test plot where progeny from nontransgenic plants, immediately adjacent to transgenic plants, had resistant plant progeny at frequencies up to 10.48%. Dispersal frequency decreased significantly and exponentially as dispersal distance increased. The flow frequency and distance of tfd A and Bt genes were similar, but the pollen-mediated gene flow of tfd A cotton was higher and further to the transgenic block than that of Bt cotton (χ² = 11.712, 1 degree of freedom, p<0.001). For the tfd A gene, out-crossing ranged from 10.13% at 1 m to 0.04% at 50 m from the transgenic plants. For the Bt gene, out-crossing ranged from 8.16% at 1 m to 0.08% at 20 m from the transgenic plants. These data were fit to a power curve model: y=10.1321x ^−1.4133 with a correlation coefficient of 0.999, and y=8.0031x ^−1.483 with a correlation coefficient of 0.998, respectively. In this experiment, the farthest distance of pollen dispersal from transgenic cotton was 50 m. These results indicate that a 60-m buffer zone would serve to limit dispersal of transgenic pollen from small-scale field tests.     

Effects of ecological environment and host genotype on the phyllosphere bacterial communities of cigar tobacco (Nicotiana tabacum L.)

Microorganisms of plant phyllosphere play an important role in plant health and productivity and are influenced by abiotic and biotic factors. In this study, we investigated the phyllosphere bacterial communities of three cigar tobacco varieties cultivated in Guangcun (GC) and Wuzhishan (WZS), Hainan, China. Metagenomic DNA was extracted from tobacco leaf samples and sequenced by 16S rDNA amplicon sequencing. Our results showed that bacterial communities of cigar tobacco phyllosphere in GC exhibited remarkably higher alpha diversity than that in WZS. There was slight effect of tobacco genotype variations on the alpha diversity in both cultivation sites, and beta diversity and structure of bacterial community were not influenced significantly by the cultivation sites and tobacco varieties. Statistical analyses of species diversity unraveled that the dominant species in bacterial communities of cigar tobacco phyllosphere among all these samples were phylogenetically affiliated to Proteobacteria and Cyanobacteria. At the genus level, the most abundant microorganism was Limnobacter, followed by Brevundimonas, unidentified_Cyanobacteria, and Pseudomonas. Additionally, environmental conditions except for humidity were negatively correlated with the relative abundance of bacterial genera. Further analyses revealed that influence of site‐specific factors on tobacco bacterial community was relatively higher than genotype‐specific factors. In short, this study may contribute to the knowledge base of practical applications of bacterial inoculants for tobacco leaf production.     

Effects of fusicoccin on intact cotton plants (Gossypium hirsutum L.)

Fusicoccin (FC) was applied as a spray to shoots of intact field- and glasshouse-grown cotton plants. Distortions of shoot morphology resulted. Stems and petioles of FC-treated plants were irregular in diameter and twisted, whereas leaf laminae were curled and crinkled. Shoot elongation was inhibited by FC; the effect was dependent upon the concentration and timing of the applications.Abbreviation FC fusicoccin     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Changes in oxidative stress enzymes during artificial ageing in cotton (Gossypium hirsutum L.) seeds

The present study was carried out to elucidate the mechanism of seed deterioration in two cotton (Gossypium hirsutum L.) cultivars (HS6 and H1098). The seeds were artificially aged at 40 +/- 1 degree C and 100% relative humidity for 4 days. In both cultivars, germinability decreased, whereas membrane deterioration, as assayed by electrical conductivity of the seed leachates, increased progressively with artificial ageing. The decrease in germinability was well correlated with increased accumulation of total peroxide and malondialdehyde content and decreased activities of antioxidant enzymes peroxidase, catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase. Hydropriming for 2 h and ascorbic acid priming for 12 h partially maintained germination and the activities of various antioxidant enzymes under artificial ageing and the accumulation of peroxide and MDA content was decreased. The results suggest that cotton seed deterioration during accelerated ageing is closely related to a decrease in activities of various peroxide scavenging enzymes and to lipid peroxidation.     

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 g/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Aph IV aminoglycoside phosphotransferase type IV Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 354-89     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Farmer knowledge,attitude and practice on cotton (Gossypium hirsutum L.) pest resistance management strategies in Zimbabwe

Cotton is an important cash crop and a means of survival for Zimbabwe’s smallholder farmers who are located in the semi-arid areas. However, it is plagued by a wide variety of pests. The cotton industry in Zimbabwe came up with sustainable pest management strategies which include within the season rotation of bollworm pesticides, a closed season and acaricide rotation scheme. The land reform programme brought new players in the cotton industry and it was critical to determine their knowledge on the pest management strategies. A survey was conducted to determine farmer knowledge, practice and perception on the resistance management strategy. Responses indicated that both communal and new farmers based their spray on egg counts. Communal farmers knew when to use pyrethroids compared to new farmers. Farmers observed the acaricide rotation scheme although they use some non acaricide pesticides. More than half of the new farmers could not say which acaricides were in the acaricide scheme. The majority of the farmers reported that they adhered to the closed season although field observation revealed otherwise. A significantly higher number of new farmers knew when to slash cotton compared to communal farmers. Knowledge of acaricides was not common among farmers. Use of pyrethroids was not limited to 1 February onwards as stipulated in the cotton pest resistance management strategy. The results suggest the need for training among the cotton farmers especially the new farmers or refresher courses among the communal farmers.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

Wide-cross whole-genome radiation hybrid mapping of cotton (Gossypium hirsutum L.)

We report the development and characterization of a "wide-cross whole-genome radiation hybrid" (WWRH) panel from cotton (Gossypium hirsutum L.). Chromosomes were segmented by gamma-irradiation of G. hirsutum (n = 26) pollen, and segmented chromosomes were rescued after in vivo fertilization of G. barbadense egg cells (n = 26). A 5-krad gamma-ray WWRH mapping panel (N = 93) was constructed and genotyped at 102 SSR loci. SSR marker retention frequencies were higher than those for animal systems and marker retention patterns were informative. Using the program RHMAP, 52 of 102 SSR markers were mapped into 16 syntenic groups. Linkage group 9 (LG 9) SSR markers BNL0625 and BNL2805 had been colocalized by linkage analysis, but their order was resolved by differential retention among WWRH plants. Two linkage groups, LG 13 and LG 9, were combined into one syntenic group, and the chromosome 1 linkage group marker BNL4053 was reassigned to chromosome 9. Analyses of cytogenetic stocks supported synteny of LG 9 and LG 13 and localized them to the short arm of chromosome 17. They also supported reassignment of marker BNL4053 to the long arm of chromosome 9. A WWRH map of the syntenic group composed of linkage groups 9 and 13 was constructed by maximum-likelihood analysis under the general retention model. The results demonstrate not only the feasibility of WWRH panel construction and mapping, but also complementarity to traditional linkage mapping and cytogenetic methods.     

Influence of explants, genotypes and culture vessels on sprouting and proliferation of pre-existing meristems of cotton (Gossypium hirsutum L. and Gossypium arboreum L.)

Summary A simple and efficient method for multiple shoot induction and proliferation was achieved in six Indian cotton cultivars from the pre-existing meristems of 21-d-old in vitro-grown seedlings. Combinations of naphthalene acetic acid (0.3–10.7 μM) and 6-benzylaminopurine (BA; 2.2 or 4.4 μM) were used for induction of shoots. The shoots proliferated and were maintained on MS (Murashige and Skoog) medium supplemented with 4.4 μM BA. Simultaneous elongation of shoots was obtained in the same medium. Optimum multiplication was observed in cv. LRK-516 (19.7±4.6), in cotyledonary nodes isolated from the adjoining node and cultured individually in 250 ml flasks. This indicates lateral inhibition of adjoining meristems. A positive influence of culture flasks as opposed to test tubes on the proliferation of multiple shoots was observed in all six cultivars tested. The morphogenic response varied with genotype and the nature of explants. Rooting of elongated shoots was achieved on MS medium devoid of growth regulators. The plantlets were transferred to the field after hardening in the greenhouse. All plants flowered and formed bolls on maturity.     

A study of heterosis in upland cotton (Gossypium hirsutum L.)

Summary Heterosis (over mid parent) and useful heterosis (over commercial variety H14) estimates were obtained from a line x tester analysis of crosses involving thirteen diverse female parents with two locally adapted varieties H14 (local standard) and J34. Marked heterosis was observed for seed cotton yield, boll number and halo length. The values of positive heterosis and useful heterosis for seed cotton yield ranged from 28.1 to 87.0% and 20.1 to 45.5%, respectively. The overall study of heterosis revealed that female parents PRS-72 (USSR), 5904F (USSR) and MCU-5 (Madras Cambodian Uganda Selection, Coimbatore) were among the top three females, showing considerable heterosis in crosses with H14 and J34 for seed cotton yield and fibre properties. The practical difficulties in exploiting the phenomenon of heterosis and possible experimental approaches in upland cotton are discussed.