Optimizing wheat seed treatment with entomopathogenic fungi for improving plant growth at early development stages

Aim of study: Entomopathogenic fungi (EPF) are biocontrol agents, plant growth promoters, and increase tolerance to biotic-abiotic stresses. In this study we investigated the factors associated to the application method, which are crucial for the interaction between the fungus and the host plant at initial crop growth stages. Area of study: The study was performed in Cordoba (Spain) Material and methods: Three experiments were performed to investigate: (i) the effect of different concentrations of the surfactant Tween® 80 (0, 0.5, 1, 5, and 10%) on wheat seed coating with conidia of Metarhizium brunneum and seed and conidia viability; (ii) the performance of wheat seedlings at first growth stages after their inoculation with Beauveria bassiana or M. brunneum via seed coating or soil drenching; and (iii) the role of soil sterilization and seed disinfection on leaf concentration of chlorophyll (SPAD) and B. bassiana or M. brunneum colonization. Main results: Tween® 80 concentration linearly improved seed coating (up to 127%) without altering wheat seeds and fungal conidia germination. Seedling length of inoculated plants was significantly increased with B. bassiana and M. brunneum (67% and 46%, respectively) via seed coating. Seed disinfection was key to achieve an enhancement in wheat SPAD (10-18%) with B. bassiana or M. brunneum concerning Control, that combined with sterilization of soil showed the highest endophyte colonization rates (up to 83.3% with both fungi) Research highlights: The surfactant concentration, application method, seed disinfection, and soil sterilization are key parameters to improve the potential benefits on the EPF-plant relationship.

In Vitro Regeneration of The Endangered Cactus Turbincarpus Mombergeri Riha, A Hybrid of T. Laui x T. Pseudopectinatus

Turbinicarpus mombergeri is a cacti species formed by a hybridization process between Turbinicarpus laui and Turbinicarpus pseudopectinatus. Under natural conditions, it is very difficult for two species be genetically compatible for hybridization, and to produce flowers at the same time. Thus, T. mombergeri is a very interesting and a rare species. Unfortunately, the current populations are decreasing and now it is considered critically endangered. The aim of this research was to develop a successful protocol for propagating T. mombergeri using the in vitro culture techniques. Seed disinfection was performed with Plant Preservative Mixture, and 80% of germination occurred at day 45 in Murashige-Skoog medium. The shoots were cut longitudinally, and the segments were transferred to media containing 2.22 or 4.44 µM benzyladenine to induce shooting. The generated shoots were highly hydrated, and presented abundant callus. The hyperhydricity was controlled by reducing salt medium concentration, by increasing calcium levels and by using polyethylenglycol. The reduction of callus was attained by adding tri-iodo benzoic acid. Vigorous and thick shoots were generated in medium containing urea, and rooting improved in the presence of 0.5 µM indoleacetic acid. Plantlets with normal morphology were obtained, and the survival rate of the plants in soil was 80%. The methodology developed represents an alternative for propagation of T. mombergeri under controlled conditions for commercial or conservation purposes.

Mechanisms of Plasma-Seed Treatments as a Potential Seed Processing Technology

Plasma treatments are currently being assessed as a seed processing technology for agricultural purposes where seeds are typically subjected to pre-sowing treatments to improve the likelihood of timely and uniform germination. The aim of this review is to summarize the hypotheses and present the evidence to date of how plasma treatments affect seeds, considering that there is difficulty in standardizing the methodology in this interdisciplinary field given the plethora of variables in the experimental setup of the plasma device and handling of biological samples. The ever increasing interest for plasma agriculture drives the need for a review dedicated to seeds, which is understandable to an interdisciplinary audience of biologists and plasma physicists. Seeds are the first step of the agricultural cycle and at this stage, the plant can be given the highest probability of establishment, despite environmental conditions, to exploit the genetic potential of the seed. Furthermore, seedlings seem to be too sensitive to the oxidation of plasma and therefore, seeds seem to be the ideal target. This review intentionally does not include seed disinfection and sterilization due to already existing reviews. Instead, a summary of the mechanisms of how plasma may be affecting the seed and its germination and developmental properties will be provided and discussed.

Analysis of Biofilm Formation on the Surface of Organic Mung Bean Seeds, Sprouts and in the Germination Environment

This study aimed to analyse the impact of sanitation methods on the formation of bacterial biofilms after disinfection and during the germination process of mung bean on seeds and in the germination environment. Moreover, the influence of Lactobacillus plantarum 299v on the growth of the tested pathogenic bacteria was evaluated. Three strains of Salmonella and E. coli were used for the study. The colony forming units (CFU), the crystal violet (CV), the LIVE/DEAD and the gram fluorescent staining, the light and the scanning electron microscopy (SEM) methods were used. The tested microorganisms survive in a small number. During germination after disinfection D2 (20 min H2O at 60 °C, then 15 min in a disinfecting mixture consisting of H2O, H2O2 and CH₃COOH), the biofilms grew most after day 2, but with the DP2 method (D2 + L. plantarum 299v during germination) after the fourth day. Depending on the method used, the second or fourth day could be a time for the introduction of an additional growth-limiting factor. Moreover, despite the use of seed disinfection, their germination environment could be favourable for the development of bacteria and, consequently, the formation of biofilms. The appropriate combination of seed disinfection methods and growth inhibition methods at the germination stage will lead to the complete elimination of the development of unwanted microflora and their biofilms.

The possibility of coriander seed disinfection with the essential oil of peppermint

Seed health testing aims to ensure a healthy crop and to prevent the spread of plant diseases. The need to study and apply both essential oils and plant extracts against seed diseases is growing and becoming more important. Coriander (Coriandrum sativum L.) is an annual herbaceous, aromatic, spicy and medicinal plant from the Apiaceae family, known for its seeds with a characteristic and pleasant odour. In human medicine, coriander is a part of various medicines for the treatment of digestive organs. Phytopathogenic fungi and bacteria are the most common and significant causes of coriander seed disease. The aim of this study was to assess coriander seed health and investigate the possibility of seed disinfection using peppermint (Mentha x piperita) essential oil. The health of the seeds was examined by the filter paper laboratory standard method. Different concentrations (1, 2, 5 and 10%) of peppermint essential oil were used for seed disinfection. The composition of the essential oil was determined by gas chromatographic analysis. The results of seed health analysis showed the presence of a fungus after seven days of incubation in a humid chamber at room temperature. The fungus was identified as Alternaria sp. (sect. Alternaria) based on the symptoms and macroscopic and microscopic features of the fungus (on seed and potato dextrose agar). Immersion treatment of infected seed in peppermint essential oil reduced the presence of Alternaria sp. on coriander seeds. The highest disease inhibition, 69,33%, namely 58,93% without the influence of the emulsifier, was shown by oil solution of 5%. Stagnation in inhibition was observed with a further increase in oil concentration. The results of this study show the possibility of disinfecting coriander seeds with peppermint essential oil to reduce infection with Alternaria sp.

IMPROVEMENT OF DIRECT REGENERATION OF MEXICAN SOYBEAN FROM COTYLEDONARY NODES

Plant tissue culture provides an alternative approach to improve the quality of soybean (Glycine max (L.) Merrill) cultivars. This study was undertaken to analyze the susceptibility of Mexican soybean for direct shoot regeneration and to determine the critical factors that affect in vitro performance. Our hypothesis was that Mexican soybean is suitable for in vitro regeneration using a cotyledonary node as explant. The effects of the seed disinfection procedure, soaking pretreatment before germination, soybean variety, as well as the culture medium composition of the shoot induction medium, were evaluated by two split-plot statistical designs. According to the statistical analysis, the seed disinfection procedure, the soaking pretreatment before germination, and the soybean genotype were the factors that brought about a significant effect (p£0.01), while the hormones composition of the shoot induction medium did not have a significant effect. The best response for multiple shoot formation was observed using a chlorine gas seed disinfection method, 3% hydrogen peroxide soaking pretreatment, and Huasteca-100, Nainari and Suaqui soybean genotypes. A robust protocol was developed, and under these selected conditions, it is possible to obtain more than 10 shoots per explant. Well-developed plantlets were obtained after 60 d of in vitro culture.

Tomato Brown Rugose Fruit Virus: Seed Transmission Rate and Efficacy of Different Seed Disinfection Treatments

Tomato brown rugose fruit virus (ToBRFV) is a highly infectious virus, that is becoming a threat to tomato production worldwide. In this work we evaluated the localization of ToBRFV particles in tomato seeds, its seed transmission rate and efficacy of disinfection, and the effects of different thermal- and chemical-based treatments on ToBRFV-infected seeds’ germination. Analyses demonstrated that ToBRFV was located in the seed coat, sometime in the endosperm, but never in the embryo; its transmission from infected seeds to plantlets occurs by micro-lesions during the germination. The ToBRFV seed transmission rate was 2.8% in cotyledons and 1.8% in the third true leaf. Regarding the different disinfection treatments, they returned 100% of germination at 14 days post-treatment (dpt), except for the treatment with 2% hydrochloric acid +1.5% sodium hypochlorite for 24 h, for which no seed germinated after 14 dpt. All treatments have the ability to inactivate ToBRFV, but in six out of seven treatments ToBRFV was still detectable by RT-qPCR. These results raise many questions about the correct way to carry out diagnosis at customs. To our knowledge, this is the first study on the effective localization of ToBRFV particles in seeds.

Effect of sequential presoaking and chlorine dioxide treatment on the inactivation of pathogenic Escherichia coli and Salmonella spp. on sprout seeds

This study was conducted to evaluate the effect of sequential presoaking and chlorine dioxide (ClO2) on the reduction of pathogenic Escherichia coli and Salmonella spp. in alfalfa. When unsoaked and presoaked alfalfa were exposed to 200 ppm ClO2 for 15 min, the population of E. coli and Salmonella spp. on presoaked seeds reduced more than those on unsoaked seeds by 2.07 and 1.43 log CFU g−1 (p < 0.05), respectively. To determine the optimal concentration and treatment time to reduce pathogenic E. coli and Salmonella spp. in alfalfa seeds immersed in water for 5 h, presoaked seeds were exposed to four different concentrations of ClO2 (50, 100, 150, and 200 ppm) for 15, 30, 45, and 60 min. The most effective condition to eliminate E. coli and Salmonella spp. from alfalfa seeds was sequential immersion in water for 5 h and 200 ppm ClO2 treatment for 1 h. After the optimal condition was applied to eight kinds of sprout seeds, the pathogens were completely inactivated in all seeds, except radish seeds. Growth of pathogenic E. coli and Salmonella spp. during sprouting after ClO2 treatment of alfalfa seeds was also completely inactivated. However, the germination rate of seeds did not significantly decrease after ClO2 treatment. In addition, ClO2 residues were not present in any sprout during 3 days of cultivation. These results demonstrated that sequential presoaking and 200 ppm ClO2 treatment is the optimal seed disinfection treatment to prevent foodborne diseases associated with sprout consumption.