Use of plant bioassays in homeopathic basic research - a systematic review

Background: Experimental research on the effects of treatments with homeopathic preparations on plants was last reviewed in 1990. Aims: The objective was to compile a systematic literature review on plant bioassays in homeopathic basic research using predefined criteria. Methods: Literature search was carried out on publications that reported experiments with homeopathic preparations on whole plants, seeds, plant parts or cells from 1920 to 2010, in healthy, abiotically or biotically stressed conditions. Outcomes had to be measured by established state-of-the-art procedures and statistically evaluated. Using a Manuscript Information Score (MIS) those publications were identified that provided sufficient information for proper interpretation (MIS > 5). Further evaluation focused on the use of adequate controls to investigate specific effects of homeopathic preparations and on the use of systematic negative control experiments to ensure proper system performance. Results: A total of 157 publications with plants were identified [1–3]. The 157 publications described a total of 167 experimental studies. 84 studies included statistics and 48 had a MIS > 5 allowing proper interpretation. 29 studies were identified with adequate controls to identify specific effects of homeopathic preparations, reporting significant effects of decimal and centesimal homeopathic potencies, including dilution levels beyond Avogadro’s number. Studies that tested series of consecutive potency levels reported a non-linear and discontinuous relation between effect and potency level. There were many individual studies with diverse methods and very few replication trials. 10 studies reported use of systematic negative control experiments, yielding evidence for the stability of the experimental set-up. Conclusions: Plant models appear to be a useful approach to investigate basic research questions on homeopathic preparations, but more independent replication trials and systematic research are needed. Systematic negative control experiments should be implemented on a routine basis to exclude false-positive and false-negative results.

Development of a biocrystallisation assay for examining effects of homeopathic preparations using cress seedlings

Background: A major challenge of homeopathic basic research is to develop test systems that yield consistent results. Outcome of plant bioassays is usually based on growth parameters (e.g. germination rate, seedling length, leaf area). Aims: We aimed to evaluate the potential of a crystallisation method with additives (“biocrystallisation”) as complementary outcome measure. This method used is based on the crystallographic phenomenon that when crystallising watery solutions of dihydrate CuCl2 in the presence of organic additives (juices/extracts), reproducible dendritic crystal structures are observed. The resulting biocrystallograms can be evaluated visually and/or by computerized image analysis. Methods: Cress seeds (Lepidium sativum L.) germinated and grew in vitro in either Stannum met. 30x or water 30x. Per experiment, six coded (blinded) 30x preparations were applied in randomized order, representing three independent replicates of the two treatments. Seedlings grew for 96 hours in darkness and were subsequently processed into a watery extract. Biocrystallisation was performed on circular glass plates in 6-fold replication per treatment group, yielding 36 biocrystallograms per experiment. A total of 15 independent experiments were performed at two independent laboratories. Biocrystallograms were scanned and analysed by computerized texture image analysis, using 15 second-order parameters as outcome measure. 3-way-ANOVA with the independent parameters treatment (n=2), internal replicate (n=3), and number of experiment (n=15) was used to analyse the data. Results: All 15 texture analysis variables yielded significant or highly significant results for the homeopathic treatment. Two variables yielded differences between the internal replicates, most probably due to a processing order effect. There were only minor differences between the results of the two laboratories. Conclusions: The texture of biocrystallograms of homeopathically treated cress exhibited specific characteristics, differentiating water 30x and Stannum met. 30x. Thus, the biocrystallisation method seems to be a promising complementary outcome measure for plant bioassays investigating effects of homeopathic preparations.

A loop-mediated isothermal DNA amplification (LAMP) assay for detection of the clubroot pathogen Plasmodiophora brassicae

Clubroot caused by Plasmodiophora brassicae is a serious threat to cruciferous crops around the world. The resting spores of P. brassicae are primary source of infection and can survive in soil for many years. Detection of resting spores in soil is essential for forecasting clubroot prevalence. Detection of P. brassicae has been relying on plant bioassays or polymerase chain reaction (PCR)-based methods. The loop-mediated isothermal DNA amplification (LAMP) is a promising approach for microorganism detection with the advantage of high sensitivity, being accurate and convenient to visualize. In this study, we developed a LAMP assay for detection of P. brassicae in soil, roots and seeds. This method can detect P. brassicae at a minimal amount of 1 fg plasmid DNA or 10 resting spores in the soil. Compared to conventional PCR, the LAMP was more sensitive in detection P. brassicae at the lower levels in soil samples. In conclusion, we elaborated a sensitive, accurate and easy-to-use LAMP assay to detect P. brassicae, which will facilitate to plan sustainable clubroot management.

Biochemical and Histological Insights into the Interaction Between the Canker Pathogen Neofusicoccum parvum and Prunus dulcis L.

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis L.), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars (cvs. ‘Butte’, ‘Carmel’, ‘Monterey’ and ‘Nonpareil’), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was only triggered by fungal infection, and not by wounding. A two-dimensional NMR and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared to healthy, non-infected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum mainly colonized the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the CODIT model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.