The Efficacy of Ultramolecular Aqueous Dilutions on a Wheat Germination Model as a Function of Heat and Aging-Time

This study evaluates the effects of temperature and aging on the efficacy of As2O3 at the 45th decimal potency in a wheat germination model, compared against a control and potentized H2O 45×. Each treatment-temperature combination was tested on seeds (Triticum aestivum L.) of Pandas variety, using six Petri dishes (33 seeds/dish) per trial, performing eight trials. Seeds were pre-treated by poisoning with 0.1% As2O3 solution to reduce germination, to allow a better evaluation of homeopathic treatment effects. The outcome variable was the number of non-germinated seeds after 96 h. Temperature effect was investigated by heating each treatment in a water bath for 30 min (at 20, 40 or 70°C), or for 5 min (at 100°C), and that of aging by dividing experimental data, collected over a period of nearly five months, into two groups: early and late experiments. Results seem to show that the efficacy of As2O3 45× is unaltered at 20 and 40°C, increases at 70°C and decreases at 100°C. As regards aging, a notable difference was found between early trials, with no significant efficacy, and late trials, where As2O3 45× exhibits a repeated significant effect versus control, except at 100°C. A reduction in variability was observed for As2O3 45× at 20°C versus control, confirming the findings of previous work. The main conclusion suggested by this experiment is that the efficacy of As2O3 45× on wheat germination may be influenced by heating degree and seems to have an increasing trend as a function of aging

Droplet evaporation method applied to test the efficacy of Zincum metallicum 30c on stressed and non-stressed wheat seeds

Background: The droplet evaporation method (DEM) has been recently proposed as a possible tool to test the efficacy of ultra high dilutions (UHDs) [1]. Here we applied the same methodology consisting in the evaporation of droplets from leakages obtained by placing wheat seeds in UHDs to test whether DEM patterns vary in function of the tested treatment: Zincum metallicum 30c (ZM), lactose 30c as dynamized control (L), and unsuccussed, undiluted water as negative control (C). Since our previous study [1] showed that there is a significant increase in the UHD action in the stressed model, with respect to the non-stressed one, also in the present experiment we tested ZM, L and C on both stressed (s-seeds) and non-stressed wheat seeds (ns-seeds). Aims: The aim of the experiment was to test whether treatments ZM, L, and C applied on ns- and s-seeds influence the DEM pattern characteristics. Materials and methods: Whole, undamaged wheat seeds (Triticum aestivum cv. Inallettabile, harvesting year 2010) were used for the experiment, following the experimental protocol described in [1]. The distinction between s- and ns-seeds is based upon different storing conditions from the harvest until the analysis day: the ns-seeds were kept in controlled conditions at 5°C and in the dark, whereas the s-seeds were kept in lab at varying temperature, humidity and light conditions. As far as treatment preparation is concerned, ZM and L were obtained from triturations 3c (received from the Federal University of Rio de Janeiro) by vertical mechanical succussions and following dilutions; negative control (C) was ultra-pure water from the same water batch used for dilutions. The experiment was repeated on 6 days within one month; each seed lot (ns- and s-seeds) was analyzed on 3 different days (s-seeds on 10th, 11th and 24th February 2014 and ns-seeds on 12th, 26th February and 3th March 2014), with three treatment-replicates for ZM and L and two for C, three repetitions for each treatment-replicate and 5 droplets per repetition (360 patterns for each seed lot). The complexity degree of polycrystalline structures obtained from the evaporating droplets was measured by means of the ImageJ software [2] by calculating their local connected fractal dimensions (LCFD). The data on moon phase, moon position and moon distance from Earth at the beginning of each evaporation process were collected from the online tool virtuelles Telescope [3]. All data were processed by a three-way ANOVA at a significance level of p ≤ 0.05. Correlations between the moon data and LCFD were evaluated by r Pearson coefficient. Results: The effect of the treatment on LCFD values of DEM patterns was significant only in the stressed model: ZM showed a crystallization inhibiting action vs. C on all 3 analysis days, whereas a significant difference between ZM and L could be observed only on first and third analysis day (Fig 1a). ANOVA analysis showed that the overall LCFD means for the s-seed lot differed significantly between each other: 1.33 (a) for C, 1.26 (b) for L, and 1.19 (c) for ZM. Moreover, the LCFD values of both seed lots showed a strong day factor influence. A possible explanation of this finding might be the influence of the moon (position, phase, and distance from Earth) on the crystallization process: strong correlation between the LCFD of the patterns and moon data were found (r values were from -0.72 to -0.97). Figure 1: Graphical representation of the mean local connected fractal dimension values (LCFD) of droplet evaporation patterns obtained from stressed and non-stressed wheat seeds following treatments with Zincum metallicum 30c (ZM), lactose 30c (L), and unsuccussed/undiluted water (C). Discussion: The present study confirms a pre-sensitization effect towards UHD action in stressed models: a significant inhibiting effect of ZM 30c was found in all experimentation day. Furthermore, the strong correlations observed between the LCFD values and moon data indicate that the complexity of polycrystalline structures from evaporating droplets of wheat seed leakages might be affected by tidal forces. Since DEM needs to be standardized, these correlations can be considered only simultaneous and not causal, however their strength gives good reasons for further studies. Conclusions: The results of the present pilot-study seem to encourage further DEM experiments on s-seeds following UHD treatments. For further confirmation of the inhibiting effect of ZM on s-seeds, germination tests should be planned. Finally, the performance of DEM experimentations during days and hours with equal tidal influence on gravity might be helpful for the reduction of the day factor. Keywords:Ultra high dilutions, droplet evaporation method, patterns, Zincum metallicu

Preliminary Study on Force-Like Effects between As45x, Water, and Wheat Seeds Performed by Means of the Droplet Evaporation Method

Background: The droplet evaporation method (DEM) is based on pattern formation in evaporating droplets. It has been shown that the local connected fractal dimension (LCFD; a complexity measure) of crystalline structures grown in evaporating droplets of wheat seed leakages depended upon whether the seeds were treated with an ultra-high dilution (UHD) or water control.1, 2 In homeopathy it is usually assumed, that UHD's need to come in direct (physical) contact with the organism to be treated to exhibit their action; however, recently, it has been demonstrated on a wheat seed germination model that UHD's may influence organisms also at a distance, i.e. without any physical contact. 3 In this case the UHD's effectiveness decreases with increasing distance. Here we test whether DEM might serve also as a tool to study such force-like influences. In a series of three DEM experiments (E1-3) we studied (i) force-like effects occurring between As2O3 45x treatment (As45x) and undiluted, unsuccussed, ultrapure water (W), (ii) As45x and wheat seeds, as also (iii) whether force-like effects may be shielded by means of aluminum foil, commonly applied for such purposes in homeopathic research

From Kolisko to nowadays: progresses and discoveries in agro-homeopathy.

The use of ultra-diluted preparations method in agriculture was introduced with agro-homeopathy, which allows to influence biological processes of plants by either accelerating or delaying growth. Moreover, it can contribute to the control of plagues and diseases, directly promoting an increase of the yield and an improvement of product qualitative traits. Since the pioneering works of Kolisko on wheat germination [1] and Junker on growth of microorganisms (paramecium, yeast, fungi) [2], in the last 30 years work has flourished from independent researchers from worldwide (Americas, Europe and Australasia). The international research works on agro-homeopathy can be conceptually divided in two main groups: effects of ultra-diluted preparations on crop growth and applicability for crop disease control. The first type of investigations usually are carried out on both healthy organisms for determining the growth stimulation of treatments and on abiotically stressed plants (i.e. heavy metal over-exposition, salt excess, water and nutrients deficiency) for determining the re-growth stimulation of ultra-dilutions [3,4]. The second type of investigations are usually performed on artificially diseased organisms (i.e. fungal and viral pathogens or nematode infection), which may react more markedly to homeopathic treatments than healthy ones [5]. Unfortunately, on the basis of the extensive critical review of published papers, there is little firm evidence to support the reliability of the reported results. Except for a limited number of publications, the most common drawbacks of agro-homeopathy researches are the poor experimental methodology and the inadequate statistical analysis. Moreover, since there is no agricultural homeopathic pharmacopoeia, much work is required to find suitable remedies, potencies and dose levels. Considering the criticism on the practical applicability of ultra-diluted preparations, in order to be accepted as a valid part of agricultural practices a well structured research strategy for agro-homeopathy is needed. This is often hampered by methodological problems as well as by the general underinvestment on the academic and nonacademic research structures. Fundamental researches based on collaborative approaches (i.e. ring tests on selected crop models) and on common experimental protocols (i.e. statistical robustness) are the keys for determining the worldwide acceptability of agro-homeopathy as a sustainable agro-technique. Statement of conflict of interest Authors declare there is no conflict of interest. Statement of financial support Authors declare that this study received no funding. Bibliography 1. Kolisko L. Physiologischer und physikalischer Nachweis der Wirksamkeit kleinster Entitäten. 1923; Stuttgart, Verlag Der Kommende Tag AG. 2. Junker H.. Die Wirkung extremer Potenzverdünnungen auf Organismen. Pflugers Arch ges Phys 1928; 219B, 5/6, 647-672. 3. Jäger T, Scherr C, Shah D, Majewsky V, Betti L, Trebbi G, Bonamin L, Simões-Wüst AP, Wolf U, Simon M, Heusser P, Baumgartner S. Use of homeopathic preparations in experimental studies with abiotically stressed plants. Homeopathy 2011; 100: 275-287 4. Majewsky V, Heuwieser, Shah D, Scherr C, Jaeger Tim, Betti L, Trebbi G, Bonamin L, Klocke P, Baumpartner S. Use of homeopathic preparations in experimental studies with healthy plants. Homeopathy 2009; 98: 228-243. 5. Betti L, Trebbi G, Majewsky V, Scherr C, Shah-Rossi D, Jäger T, Baumgartner S. Use of homeopathic preparations in phytopathological models and in field trials: a critical review. Homeopathy 2009; 98: 244-266

Self-Organized Crystallization Patterns from Evaporating Droplets of Common Wheat Grain Leakages as a Potential Tool for Quality Analysis

We studied the evaporation-induced pattern formation in droplets of common wheat kernel leakages prepared out of ancient and modern wheat cultivars as a possible tool for wheat quality analysis. The experiments showed that the substances which passed into the water during the soaking of the kernels created crystalline structures with different degrees of complexity while the droplets were evaporating. The forms ranged from spots and simple structures with single ramifications, through dendrites, up to highly organized hexagonal shapes and fractal-like structures. The patterns were observed and photographed using dark field microscopy in small magnifications. The evaluation of the patterns was performed both visually and by means of the fractal dimension analysis. From the results, it can be inferred that the wheat cultivars differed in their pattern-forming capacities. Two of the analyzed wheat cultivars showed poor pattern formation, whereas another two created well-formed and complex patterns. Additionally, the wheat cultivars were analyzed for their vigor by means of the germination test and measurement of the electrical conductivity of the grain leakages. The results showed that the more vigorous cultivars also created more complex patterns, whereas the weaker cultivars created predominantly poor forms. This observation suggests a correlation between the wheat seed quality and droplet evaporation patterns

Spatial allocation effects within a potentization basic research model – evidence for field-like effects of homeopathic preparations?

Background: The mode of action of ultramolecular homeopathic preparations is still unknown. Interactions between objects or entities can be grouped in four main general scientific categories: material, force-/field-like, entanglement-like or informational. Should homeopathic preparations have a field-like mode of action, there is greater probability of cross-contamination as long as the means to "shield" objects from each other is unknown. A field-like interaction would also lead to treatment at a distance effects that are distance-dependent. Aims: We analysed a set of experiments with Arsenicum album 45x treated wheat seedlings regarding a possible distance-dependent cross-contamination. Materials and Methods: We performed an a posteriori analysis of a set of 17 independent experiments [1,2] with wheat seedlings pre-treated with 1‰ arsenic. Three treatments were applied (Arsenicum album 45x, water 45x, or unpotentized water) with 150 seedlings in each treatment group per experiment. Seedlings were arranged in hanging plastic bags side-by-side in identically treated blocks of 10 seedlings. The 3x15 blocks were coded and randomly allocated to the three treatments. Wheat shoot length was measured after 7 days. Treatment effects were analysed as function of the position (1–10) within the blocks of 10 seedlings. Results: Analyzing all data, Arsenicum album 45x exerted an inhibiting effect (–3.2%, p=0.01) compared to both water and water 45x. When restricting the analysis to the outermost seedlings of all subgroups (pos. 1, 10), the treatment effect vanished (0.3%, p=0.92). In contrast, the innermost seedlings of all subgroups (pos. 5, 6) showed a treatment effect of –5.6% (p=0.02). Intermediate pairs of positions (pos. 2–4, 7–9) showed intermediate effects. Regarding shoot length, dependency on spatial position was observed for the plants of the water control groups, but not for the plants treated with Arsenicum album 45x. Conclusions: Whilst the effect of Arsenicum album 45x on wheat-shoot growth was not dependent on the spatial position within the subgroup, the water-control plants became smaller the closer they were to Arsenicum album 45x-treated seedlings. This observation is compatible with the existence of a field-like effect of homeopathic dilutions. Another possible explanation that cannot be ruled out by the present experiments, is contamination through the gas-phase. Future investigations of ultramolecular homeopathic preparations should control any such effects since they may mask treatment effects, leading to false-negative results. Closer investigation of the nature of this distance-dependent effect might contribute to identification of the mode of action of ultramolecular homeopathic preparations

ultra high diluted arsenic reduces spore germination of alternaria brassicicola and dark leaf spot in cauliflower

ABSTRACT A major problem in cauliflower crop is the fungus Alternaria brassicicola, which causes dark leaf spot on Brassicaceae family. The current use of copper salts in agriculture is questioned. In fact, these products present some disadvantages, connected mainly with their deposits in the soil and toxicity on plants. This work investigated the effects of arsenic treatments, in ultra high diluted form (UHD), prepared by a process of repeated dilution and succussion (shaking), through: 1) in vitro germination experiments, where spores of A. brassicicola were suspended in the treatments; 2) in planta experiments and 3) a field trial, where cauliflower plants infected by the fungus were sprayed with treatments. The results showed that ultra high dilutions of arsenic (where no more molecules of this substance are present) were effective in all the experiments, inhibiting spore germination by 60.0%, controlling fungal disease in in planta experiments (relative efficacy of 42.1%), and, in field trial, decreasing the mean infection level in cauliflower heads by 45.7% and 41.6% in artificially inoculated and naturally infected plants, respectively. This is the first study to demonstrate that ultra high dilutions effectively reduce in vitro spore germination and infection of A. brassicicola in cauliflower plants, both under controlled conditions and in the field. Our research is still very experimental, however, in light of the significant results obtained with ultra-diluted arsenic, and given that its extreme high dilution level precludes any toxicity or accumulation in the environment, the use of UHDs could be considered a potential and reliable approach for sustainable agriculture

Ultra high diluted arsenic reduces spore germination of Alternaria brassicicola and dark leaf spot in cauliflower

ABSTRACT A major problem in cauliflower crop is the fungus Alternaria brassicicola, which causes dark leaf spot on Brassicaceae family. The current use of copper salts in agriculture is questioned. In fact, these products present some disadvantages, connected mainly with their deposits in the soil and toxicity on plants. This work investigated the effects of arsenic treatments, in ultra high diluted form (UHD), prepared by a process of repeated dilution and succussion (shaking), through: 1) in vitro germination experiments, where spores of A. brassicicola were suspended in the treatments; 2) in planta experiments and 3) a field trial, where cauliflower plants infected by the fungus were sprayed with treatments. The results showed that ultra high dilutions of arsenic (where no more molecules of this substance are present) were effective in all the experiments, inhibiting spore germination by 60.0%, controlling fungal disease in in planta experiments (relative efficacy of 42.1%), and, in field trial, decreasing the mean infection level in cauliflower heads by 45.7% and 41.6% in artificially inoculated and naturally infected plants, respectively. This is the first study to demonstrate that ultra high dilutions effectively reduce in vitro spore germination and infection of A. brassicicola in cauliflower plants, both under controlled conditions and in the field. Our research is still very experimental, however, in light of the significant results obtained with ultra-diluted arsenic, and given that its extreme high dilution level precludes any toxicity or accumulation in the environment, the use of UHDs could be considered a potential and reliable approach for sustainable agriculture

Statistical tools for alternative research in plant experiments

In this paper we describe the set of statistical tools applied for evaluating a series of experiments concerning the effects of homeopathic treatments in simple plant models, involving wheat seeds and seedlings and tobacco plants. The homeopathic treatment used in our experiments is arsenic trioxide, diluted in a decimal scale and dynamized k times (with k varying from 5 to 45). Since the most significant results have been achieved with As 45dH (45-th decimal potency of As2O3), we report a brief summary of these results. The statistical analysis was performed by using parametric and non parametric tests, and Poisson distribution has an essential role when dealing with germination experiments. Finally, we describe some interesting results related to the changes in variability, which seems to be a primary target of homeopathic treatment effect

Analisi esplorativa dell'effetto di soluzioni ultramolecolari di triossido di arsenico sullo sviluppo vegetativo in vitro di plantule di grano

This paper deals with an experiment in which wheat seeds were stressed with a material dose of As2 O3, and then treated with ultra-molecolar dilutions of the same substance, with and without potentization. We focused our analysis on seedling stem length during 7 days of treatment, and tried to give a thorough statistical description of the data. We applied simple nonparametric tests for comparing treated and control groups: it seems to be a significant stimulating effect when seeds are treated with the 45th decimal potency of As2 O3 . A similar effect was obtained in a previous experiment (Betti et al., 1997), and shows that our model allows a proper reproducibility of results