Interference of Copper Sulphate in Growth of Erwinia amylovora

To understand the toxicity of copper salts on Erwinia amylovora, which are used in the control of fire blight, bacterial growth and cell metabolism was assayed with copper sulphate in the presence or absence of complex-forming compounds such as various amino acids or citrate. In minimal medium without amino acids copper sulphate strongly interfered with the growth of E. amylovora. A concentration of 15 μm CuSO₄ resulted in about 50% growth inhibition. In contrast to a strong effect of streptomycin, copper ions barely killed the cells when incubated in minimal medium for 1 h. The addition of 4 g asparagine per litre relieved a‘bacteriostatic’effect of copper ions and allowed growth of the bacteria at 2 mm CuSO₄. Other amino acids had a similar effect in the protection of E. amylovora against copper ions. This was in contrast to glycine betain, which was unable to suppress growth inhibition by CuSO₄. Presumably, the free ammonium groups of amino acids participated in the protective effect. The addition of citrate, exceeding the amount of copper-ions, was also protective. Bioluminescence of E. amylovora cells was expressed via a constitutive promoter from the lux-operon of Vibrio fischeri. The light emission is dependent on active cell metabolism. In a novel approach to determine the immediate response of E. amylovora after the addition of copper sulphate, the change of bioluminescence was determined. Addition of copper ions to MM3 medium strongly affected the bioluminescence, but no change in light production was noticed, when citrate or asparagine were present in addition to copper sulphate. A decrease of bioluminescence to 50% was observed for 50 μm CuSO₄ in the absence of amino acids.     

Detection of trait donors and QTL boundaries for early blight resistance using local ancestry inference in a library of genomic sequences for tomato

By conducting hierarchical clustering along a sliding window, we generated haplotypes across hundreds of re-sequenced genomes in a few hours. We leveraged our method to define cryptic introgressions underlying disease resistance in tomato (Solanum lycopersicum L.) and to discover resistant germplasm in the tomato seed bank. The genomes of 9 accessions with early blight (Alternaria linariae) disease resistance were newly sequenced and analyzed together with published sequences for 770 tomato and wild species accessions, most of which are available in germplasm collections. Identification of common ancestral haplotypes among resistant germplasm enabled rapid fine mapping of recently discovered quantitative trait loci (QTL) conferring resistance and the identification of possible causal variants. The source of the early blight QTL EB-9 was traced to a vintage tomato named ‘Devon Surprise’. Another QTL, EB-5, as well as resistance to bacterial spot disease (Xanthomonas spp.), was traced to Hawaii 7998. A genomic survey of all accessions forecasted EB-9-derived resistance in several heirloom tomatoes, accessions of S. lycopersicum var. cerasiforme, and S. pimpinellifolium PI 37009. Our haplotype-based predictions were validated by screening the accessions against the causal pathogen. There was little evidence of EB-5 prevalence in surveyed contemporary germplasm, presenting an opportunity to bolster tomato disease resistance by adding this rare locus. Our work demonstrates practical insights that can be derived from the efficient processing of large genome-scale datasets, including rapid functional prediction of disease resistance QTL in diverse genetic backgrounds. Finally, our work finds more efficient ways to leverage public genetic resources for crop improvement.     

Efficient somatic embryogenesis and plant regeneration from shoot apex explants of different Indian genotypes of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

An efficient somatic embryogenesis and plant regeneration system was developed from shoot apex explants of finger millet, Eleusine coracana. Eight genotypes, CO 7, CO 9, CO 13, CO 14, GPU 26, GPU 28, GPU 45, and GPU 48, were assessed in this study. The maximum somatic embryogenic induction, at 98.6%, was obtained from explants cultured on Murashige and Skoog medium supplemented with 18.0 μM dichlorophenoxyacetic acid and 2.3 μM kinetin. The highest number of shoot induction (26) was observed after transfer of embryonic callus to regeneration medium supplemented with 4.5 μM thidiazuran and 4.6 μM kinetin. Significant differences were observed between genotypes for somatic embryogenesis and plant regeneration. GPU 45 gave the best response, while CO 7 was the least responsive under the culture conditions tested in this study. Regenerated plants were successfully rooted and grown to maturity after hardening in soil.     

HPLC-DAD analysis of arbutin produced from hydroquinone in a biotransformation process in Origanum majorana L. shoot culture

The hydroquinone glucoside arbutin is a plant derived compound medically applied due to its uroantiseptic activity. It also has skin whitening properties and thus is widely used in dermatology and cosmetology. Origanum majorana L. (Lamiaceae) is known to produce arbutin, however the content of the compound in cultivated plants is very variable and low. Since plant cell and tissue cultures are capable to perform specific biotransformation reactions including glucosylation, this investigation targeted the formation of arbutin from hydroquinone in agitated O. majorana shoot cultures. For this purpose different doses of hydroquinone (96, 144, 192, 288 and 384 mg/L of medium) were added to the culture flasks in one, two or three portions. Arbutin was qualitatively and quantitatively determined in methanol extracts from dry biomass and lyophilized media using HPLC-DAD. Cells of O. majorana shoot cultures efficiently converted hydroquinone into arbutin. The product was accumulated in the biomass and was not observed (or in trace amounts) in the medium samples. Different doses as well as portioning of the precursor had a significant impact on the biotransformation process. Arbutin accumulation increased from 0.23 ± 0.03 mg/g DW up to 52.6 ± 4.8 mg/g DW in the biomass. The highest product content was observed after the addition of 192 mg/L hydroquinone in three portions. The highest efficiency of the biotransformation process, i.e. 67.5 ± 5.2% was calculated for a dose of 96 mg/L precursor divided into three portions. After further optimization of the biotransformation process, O. majorana shoot cultures could serve as a rich source of arbutin.     

Identification and expression analysis of components involved in rice Xa21‐mediated disease resistance signalling

Rice Xa21 gene encodes a receptor‐like kinase that confers broad‐spectrum resistance against Xanthomonas oryzae pv. oryzae (Xoo). Recently, a number of genes involved in the Xa21‐mediated disease resistance pathway have been identified. Based on our previous data and the literature, we chose 16 candidate proteins and made corresponding antibodies. Using Western blotting, we systematically investigated the expression profile of the proteins in Xa21‐mediated disease resistance response. We found nine proteins with altered expression. We further compared their expression in resistance, susceptible and mock responses, and found that GST expression was up‐regulated during the resistance process, indicating GST is a positive regulator in resistance responses. ATPsB expression was down‐regulated during both the resistance and susceptible response processes, although it was higher in the resistance response than that in the susceptible response. The total amount of MYB, GAPDH, CatB, Trx and NB‐ARC proteins was lower in the resistance than in the susceptible response, but their abundance per unit bacteria in the resistance response was still higher than in the susceptible response, suggesting that these proteins might be positive regulators in the resistance response. In addition, expression of another ERF was induced by inoculation with bacterial blight pathogen, and expression of Zf‐LSD1 was activated by wounding stress alone. Interestingly, most proteins showed similar altered expression patterns in the resistance and susceptible responses, but differed to some extents, implying that both responses might share common molecular mechanisms. This study revealed evidence of resistance‐related protein expression, providing a foundation for better understanding of their functions.     

Genotypic diversity and migration patterns of Phytophthora infestans in the Nordic countries

In this study we investigated the genotypic diversity and the migration patterns of Phytophthora infestans in the Nordic countries. Isolates of P. infestans from outbreaks in 43 fields sampled in 2008 were collected using stratified sampling with country, field, and disease foci as the different strata. Microsatellites were used as markers to determine the genotypic variation in the sampled material. The results show a high genotypic variation of P. infestans in the Nordic countries with most of the genotypes found only once among the collected isolates. The major part of the genotypic variation was observed within the fields, with low differentiation between the fields. The observed low association of alleles among loci is consistent with frequent sexual reproduction of P. infestans in the Nordic countries. Coalescence analyses did not support a single common population for the four countries, thus indicating some degree of geographic differentiation. The analyses of migration patterns showed differing levels of gene flow among the Nordic countries. No correlation between migration rates and geographical distance could be seen. This could be explained by different degrees of genetic similarity between the pathogen populations in the different countries.     

Genetic diversity analysis of a world-wide collection of <Emphasis Type="Italic">Ascochyta rabiei</Emphasis> isolates using sequence tagged microsatellite markers

A previously characterized compound microsatellite locus ARMS1, containing penta- and decameric repeat units, has been reported to reveal genetic diversity in Ascochyta rabiei (Pass.) Labr. isolates. Therefore, 37 isolates of Ascochyta rabiei collected from different states of India and 38 isolates from fifteen other countries in the world were examined for their diversity at this locus. Twenty-six alleles on the basis of size (228--451 base pairs) were detected in the world isolates examined, while 15 alleles (287--418 base pairs) were observed in isolates from the Indian subcontinent. To the best of our knowledge, this study is the first to demonstrate diversity in representative Ascochyta rabiei isolates from different parts of the world at the ARMS1 locus.     

Bacterial leaf blight resistance genes (Xa21, xa13 and xa5) pyramiding through molecular marker assisted selection into rice cultivars

Marker assisted selection was employed to pyramid three bacterial blight resistance genes Xa21, xa13 and xa5 into high yielding susceptible rice cultivars ADT43 and ADT47. With the assistance of PCR markers, homozygous and heterozygous genotypes were identified in F₂ generation of two crosses (ADT43 × IRBB60 and ADT47 × IRBB60) and goodness of fit was tested. Eighty nine plants from F₃ generation of ADT43 × IRBB60 were also screened for resistance genes. The genotypes carrying resistance genes in different combinations were identified. The pyramided lines showed a wider spectrum and higher level of resistance against two Xoo isolates under field conditions.     

Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat

Wheat ears were inoculated with conidia of Fusarium spp. at different growth stages between ear emergence and harvest and moist conditions were maintained for up to 7 days subsequently by mist irrigation. Of the fungi tested (Fusarium culmorum, F. avenaceum, F. tricinctum, F. sporotrichioides and Microdochium nivale), only F. culmorum produced ear blight symptoms and grain samples were found subsequently to contain deoxynivalenol. Most ear infection and deoxynivalenol formation occurred following inoculation at about mid-anthesis. Small amounts of deoxynivalenol were formed and some F. culmorum was isolated even in the absence of ear blight symptoms. An overnight wet period was sufficient to initiate infection and deoxynivalenol formation but both were increased by extending the wet period up to at least 3 days. Recovery of Fusarium spp. from harvested grain was usually possible whether or not symptoms developed. F. culmorum usually persisted and often increased to moderately high levels after storage for 7 wk in a range of moisture conditions.