A multiplex real-time PCR assay enables simultaneous rapid detection and quantification of bacteria associated with acute oak decline

Acute oak decline (AOD) is a syndrome affecting mature oak trees and is characterized by stem bleeds from vertical fissures on trunks, and inner bark necrosis caused by a polybacterial consortium, in which Gibbsiella quercinecans and Brenneria goodwinii, and to a lesser extent Rahnella victoriana and Lonsdalea britannica, play key roles. Here we report a novel multiplex real-time PCR assay that enables simultaneous and rapid detection and quantification of these four bacterial species from stem bleed swabs. Experiments with axenic cultures were performed to determine specificity and sensitivity of the multiplex quantitative PCR (qPCR). Whilst the primer/probe set for B. goodwinii was species-specific, primer/probe sets for the other three species were able to identify other members of their respective genera. There was no cross detection of genera within the multiplex qPCR, and non-target bacteria were not detected. The multiplex AOD assay had differential sensitivity for each bacterial species. The assay was evaluated on swab samples collected from stem bleeds of declining oak trees at a site in south-east England and was able to detect all four bacterial species. Absolute quantification of the bacteria from swab samples was possible through the inclusion of a standard curve prepared from dilutions of gene copy standards. This diagnostic tool will facilitate rapid detection of AOD-associated bacteria from samples that can easily be taken by non-specialists without specific training, and will also find application in other experimental work such as pathogenicity and control trials.     

Genotype-environment interaction of barley doubled haploids with regard to malting quality

The genotype-environment (GE) interaction of barley doubled haploid (DH) lines was studied in relation to some malting quality characters. DH lines were obtained, using the bulbosum method, from F₁ hybrids between the cultivars ‘Grit’ and ‘Havila’. Thirty DH lines, the parental forms, F₂ and F₃ hybrids of ‘Grit’בHavila’ and the standard cultivar ‘Rudzik’ were studied in a series of field experiments repeated at six environments. Malt protein content, Kolbach index, extract yield and malt fine-coarse difference were measured. The statistical analysis was performed using the computer program SERGEN. For protein content, it was found that most of the lines studied were stable, i.e. their GE interactions were not significant. Among the malt traits, phenotypic values of Kolbach index and fine-coarse difference were most dependent on environmental conditions. For Kolbach index, only one line was found to be stable, while for fine-coarse difference, all lines displayed interaction with the environment.     

Pleistocene extinction of genyornis newtoni: human impact on australian megafauna

More than 85 percent of Australian terrestrial genera with a body mass exceeding 44 kilograms became extinct in the Late Pleistocene. Although most were marsupials, the list includes the large, flightless mihirung Genyornis newtoni. More than 700 dates onGenyornis eggshells from three different climate regions document the continuous presence of Genyornis from more than 100,000 years ago until their sudden disappearance 50,000 years ago, about the same time that humans arrived in Australia. Simultaneous extinction of Genyornis at all sites during an interval of modest climate change implies that human impact, not climate, was responsible.     

Loop‐mediated isothermal amplification (LAMP) assays for rapid detection of Pyrenopeziza brassicae (light leaf spot of brassicas)

Pyrenopeziza brassicae (anamorph Cylindrosporium concentricum) is an ascomycete fungus that causes light leaf spot (LLS) disease of brassicas. It has recently become the most important pathogen of winter oilseed rape (Brassica napus) crops in the UK. The pathogen is spread by both asexual splash‐dispersed conidia and sexual wind‐dispersed ascospores. Such inoculum can be detected with existing qualitative and quantitative PCR diagnostics, but these require time‐consuming laboratory‐based processing. This study describes two loop‐mediated isothermal amplification (LAMP) assays, targeting internal transcribed spacer (ITS) or β‐tubulin DNA sequences, for fast and specific detection of P. brassicae isolates from a broad geographical range (throughout Europe and Oceania) and multiple brassica host species (B. napus, B. oleracea and B. rapa). Neither assay detected closely related Oculimacula or Rhynchosporium isolates, or other commonly occurring oilseed rape fungal pathogens. Both LAMP assays could consistently detect DNA amounts equivalent to 100 P. brassicae conidia per sample within 30 minutes, although the β‐tubulin assay was more rapid. Reproducible standard curves were obtained using a P. brassicae DNA dilution series (100 ng–10 pg), enabling quantitative estimation of amounts of pathogen DNA in environmental samples. In planta application of the β‐tubulin sequence‐based LAMP assay to individual oilseed rape leaves collected from the field found no statistically significant difference in the amount of pathogen DNA present in parts of leaves either with or without visible LLS symptoms. The P. brassicae LAMP assays described here could have multiple applications, including detection of symptomless host infection and automated real‐time monitoring of pathogen inoculum.     

Photosynthetic Acclimation to Cold as a Potential Physiological Marker of Winter Barley Freezing Tolerance Assessed under Variable Winter Environment

Winter‐hardiness is a complex trait limiting cultivation of winter barley (Hordeum vulgare ssp. vulgare) with respect to the regions of temperate climate. In the present studies, we verified whether inexpensive and fast physiological markers characterizing photosynthetic acclimation to cold may provide robust characteristics of winter barley genotypes for improved frost resistance. Freezing tolerance of 28 winter barley varieties and advanced breeding lines were tested for three winters in field‐laboratory experiment and under fully controlled conditions. To increase the environmental variability of freezing tolerance, a part of the plants were also de‐acclimated under semi‐controlled conditions and re‐acclimated in laboratory before freezing tests. After controlled cold acclimation, apparent quantum yield of photosystem II (F_v/F_m) as well as photochemical (q_P) and non‐photochemical (NPQ) coefficients of chlorophyll fluorescence quenching were studied. Field‐laboratory method assessment of freezing tolerance gives distinct and even opposite results in subsequent years. Also de‐acclimation interacted with growth conditions in the field, giving different rankings of genotypes each year. The results obtained suggest that high level of freezing tolerance measured in laboratory, which is connected with photosynthetic acclimation to cold may be not sufficient for the expression of field resistance, especially when winter conditions are not favourable for cold acclimation.     

The use of statistical methods to evaluate winter oilseed rape doubled haploids for industrial purposes

Doubled haploid (DH) production is valuable technology in modern plant breeding of oilseed rape. Doubled haploids can be produced from in vitro isolated and cultured microspores, which, instead of forming pollen, develop into haploid embryos. Either spontaneously or after artificial doubling of chromosomes, the arising resulting plants are diploid and fertile, and 100% homozygous for all genes. Biotechnological DH production offers various advantages for plant breeders, including the possibility to obtain homozygous lines rapidly, as well as easy selection due to the absence of heterozygosity. A DH line with desirable agronomic characters can be chosen and introduced into a breeding program as a result of selection for a combination of optimum characters. The study presents the application of uni- and multi-variate statistical methods to evaluate winter oilseed rape DH lines studied in a series of six experiments repeated at different localities and over a number of years. These methods made it possible to estimate individual DH lines under various environmental conditions, including the assessment of the content and stability of two different fatty acid compositions. The aim of the study was to classify and select the best oilseed rape genotypes with regard to the requirements of chemical and biodiesel industry concerning fatty acid compositions at simultaneously high seed yield. The paper contains the results of statistical analysis of 37 genotypes (32 DH lines, 2 parental forms, F₂ and F₃ hybrids and standard cultivar) performed on the basis of experiments carried out at 6 environments. A total of 14 genotypes were found satisfactory (11 stable and 3 unstable) for the chemical industry and 10 genotypes satisfactory (6 stable and 4 unstable) for the biodiesel industry. Among them 5 genotypes had very profitable significant and positive estimates of the main effect for seed yield.     

Raffinose family oligosaccharides in seeds of <Emphasis Type="Italic">Pisum</Emphasis> wild taxa,type lines for seed genes,domesticated and advanced breeding materials

The content of raffinose family oligosaccharides (RFOs) in pea seeds constrains their usage in feeding humans and animals. In our research, the content of soluble carbohydrates—particularly α-d-galactosides of sucrose (RFOs)—was analyzed. The materials were as follows: 248 accessions from the Polish Pisum Genebank including representatives of taxa (from species to convarietas), type lines for genes controlling seed characters, and breeding materials and cultivars. Accessions were divided into homogeneous groups considering content of total soluble carbohydrates, total RFOs and individual RFOs: raffinose, stachyose and verbascose. The highest content of total soluble carbohydrates and total RFOs were stated for accessions with wrinkled seeds (r and rb genes) and the lowest content for seeds of the wild species P. fulvum Sibth. et Sm. Accessions valuable for breeding (for further decreasing of anti-nutritional compounds) were found among domesticated taxa (P. sativum subsp. sativum convar. vulgare Alef. and speciosum (Dierb.) Alef.), breeding lines, and some wild taxa. Accessions with decreased content of a total RFOs and verbascose are particularly valuable. It was found that the content of total RFOs was the most highly, frequently, and positively correlated with a stachyose and verbascose content. However, in P. fulvum seeds with the lowest content of RFOs and verbascose, a high correlation between the content of total RFOs and stachyose was revealed. Contents of all oligosaccharides were substantially lower in lines with dominant alleles of main pea seed genes (R, A, and I). It can be assumed that wild, primitive peas were characterized by not-all-to-high (rather not high) content of oligosaccharides; then recessive mutations in mentioned genes resulted in an increased content of RFOs. It seems to be an interesting observation from an evolutionary point of view.     

Quantitative and qualitative analysis of alkaloids composition in the seeds of a white lupin (<Emphasis Type="Italic">Lupinus albus</Emphasis> L.) collection

White lupin (Lupinus albus L.) has unexploited potential as a crop plant due to its high seed yield as well as protein and oil content in seeds. Well-characterized collections of gene resources are very important for breeding as a source of genetic variation. This paper presents the results of analyses for total content and qualitative composition of alkaloids in seeds of 367 L. albus accessions from the Polish Genebank. Accessions were divided into four classes of origin: wild collected material, land races, breeding lines, and cultivars. Apart from the expected broad variation as well as strong differentiation in the alkaloid content, a clear influence of domestication was observed. This was shown as an apparent decrease in the alkaloid content in breeding lines and cultivars classes. The total alkaloid content varies from 0.02 to 12.73% of the seed dry weight. Six major alkaloids (abundance >1%) were revealed: lupanine (28.22–94.49%, mean 76.06% in total content), 13-hydroxylupanine (0.10–32.78%, mean 8.23%), multiflorine (0.00–21.67%, mean 5.52%), albine (0.00–18.55%, mean 4.48%), angustifoline (0.24–12.14%, mean 2.07%), 11,12-seco-12,13-didehydromultiflorine (0.00–12.28%, mean 1.74%). Owing to its abundance, lupanine was found to be the most closely correlated to the total alkaloid content.     

Relationship between phenotypic and genetic diversity of parental genotypes and the specific combining ability and heterosis effects in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The purpose of the study was to evaluate the relationship between the genetic distances (GD) and phenotypic distances (PD) of parents and the specific combining ability (SCA) and heterosis effects. The experiment comprised 18 parental genotypes of wheat (Triticum aestivum L.) and 76 F₂ hybrids, obtained after crossing in a line × tester scheme. Parents and hybrids were examined in a field experiment conducted in a block design with three replications. SCA as well as mid-parent heterosis effects were estimated for selected morphological and technological traits. PDs and GDs were investigated between pairs of parental genotypes. GD between parental genotypes was evaluated by using randomly amplified polymorphic DNA markers. Heterosis was observed in all hybrids, and protein content exhibited the highest heterosis among the seven examined traits. The relationship between PD and GD as well as the SCA and mid-parent heterosis effects were evaluated using correlation coefficient. The correlation between PD, SCA and heterosis were low and not significant for the examined traits, whereas the correlation between SCA, heterosis and GDs were significant for protein content and rheological properties. The results indicate that GDs between parents can be used to predict performance of hybrids for selected technological traits.