The Effect of Antifungal Drugs and Fungicides on the Viability and Vigour of Barnyard Millet (Echinochloa Crus-Galli) Seeds
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Bochenek, Giełwanowska, Czermińska and Bojarowska (2019). Seed Science and Technology, 47, 2, 121-130. https://doi.org/10.15258/sst.2019.47.2.01 The effect of antifungal drugs and fungicides on the viability and vigour of barnyard millet (Echinochloa crus-galli) seeds Anna Bochenek*, Irena Giełwanowska, Monika Czermińska and Klaudia Bojarowska Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719 Olsztyn, Poland (E-mail: [email protected]; [email protected]; [email protected]) *Author for correspondence (E-mail: [email protected]) (Submitted January 2019; Accepted April 2019; Published online May 2019) Abstract Disinfectants applied in seed viability and vigour testing should effectively inhibit the development of pathogens while exerting a minimal impact on seed physiology. However, many chemical disinfectants are not highly effective, and they alter metabolic processes in seeds. In this study, selected antifungal drugs were tested on barnyard millet seeds and compared with agricultural fungicides. Seedling roots were analysed microscopically to determine the causes of chemical agents’ adverse effects. Incubation of barnyard millet seeds under continuous exposure to fungicides and nystatin inhibited seedling growth and failed to sterilise seeds to the extent required for laboratory analyses. Fungal infections on the seed surface were most effectively eliminated by 0.5% natamycin suspension applied continuously or for one hour. However, when applied at the above concentration, natamycin also inhibited seedling growth and decreased the vigour index. Incubation of seeds with 0.25% natamycin suspension for one hour was less detrimental to seed physiology and matched the most effective disinfection. The apices of seedling roots treated with 0.25% natamycin for one hour were free of necrotic changes. Prolonged exposure to natamycin at a higher concentration led to necrosis of root apex cells, which decreased the growth rate and vigour of barnyard millet seedlings. Keywords: antifungal drugs, barnyard millet, callose, cell necrosis, natamycin, seed disinfection Introduction In laboratory tests of seed viability or vigour and in analyses of biochemical processes in seeds, the examined material has to be free of bacteria and fungi which can alter seed metabolism. Microorganisms colonising seeds, in particular fungal endophytes, influence seed physiology by secreting toxic chemicals that can inhibit germination and seedling growth or even induce seed necrosis (Abdrassulova et al., 2014; Shen et al., 2014). © 2019 Bochenek et al. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/licenses/by-nc/4.0 121 ANNA BOCHENEK, IRENA GIEŁWANOWSKA, MONIKA CZERMIŃSKA AND KLAUDIA BOJAROWSKA Microbial metabolism can also alter the results of laboratory analyses. Microorganisms colonise all seeds, but they are particularly abundant on the diaspores of wild plants, including weeds, which are not chemically protected (Howell et al., 2002; Ahmad et al., 2012; Raghavendra et al., 2013). Developing methods of seed disinfection for seed testing should contribute to a better understanding of the seed biology of both arable and wild plants (Bochenek et al., 2010, 2016). Barnyard millet (Echinochloa crus-galli (L.) Beauv.) is a ubiquitous and highly problematic weed in Europe and on other continents. The weed grows in temperate and tropical climates, and it invades various types of crop plants, including cereals, root vegetables and tubers. Barnyard millet is also widely encountered on fallow and roadside land (Maun et al., 1986; Holm et al., 1997). Therefore, the development of effective methods for disinfecting barnyard millet seeds for laboratory testing is an important consideration. Seed disinfectants should effectively inhibit the development of pathogens, while exerting a minimal impact on seed physiology. In laboratory practice, seeds are most commonly disinfected with inorganic chemical compounds (sodium hypochlorite, calcium hypochlorite, hydrogen peroxide, mercury chloride) or organic compounds (ethanol, formalin and antibiotics, in particular chloramphenicol, penicillin and streptomycin). Agricultural fungicides are also used for seed disinfection. However, these disinfecting agents are often ineffective, and under the supportive growth conditions of a laboratory (optimal temperature, high moisture content), some seeds are colonised by fungi. Chemical disinfectants are also capable of inducing various changes in seed metabolism (Chun et al., 1997; Van der Berg et al., 2002; Allen et al., 2004; Barampuram et al., 2014; Buts et al., 2014; Haque et al., 2014; Ma et al., 2015). In this study, selected antifungal drugs that are widely used in medicine were tested and their effectiveness compared with popular agricultural fungicides to identify effective methods for seed disinfection in laboratory analyses. These criteria were met by natamycin, which effectively disinfected seeds and was least detrimental to seed vigour. A microscopic analysis of radicles emerging from germinating seeds was performed to determine the causes of natamycin’s inhibitory effects on seedling growth, which varied with natamycin concentration and exposure time. Materials and methods Mature barnyard millet seeds were collected in arable fields in the vicinity of Olsztyn (north-eastern Poland) at the end of July and beginning of August 2013 and 2017. After harvest, the seeds were dried for several days at room temperature (22-23°C), manually cleaned and stored at 4°C in paper bags for two or three months. Two experiments were performed. In the first experiment, seeds were germinated in 90 mm-diameter glass Petri dishes (three replicates of 50 seeds each), on two layers of filter paper (Whatman No. 1) soaked with deionised water or aqueous solutions or suspensions of: – Captan 0.1 or 0.2% (Organika-Azot SA, Poland) – Funaben-T 0.1 or 0.2% (Organika-Azot SA, Poland) 122 ANTIFUNGAL EFFECTS ON BARNYARD MILLET SEEDS – Dithane M-45 0.1 or 0.2% (Indofil Industries Ltd., India) – nystatin 0.45 or 0.90% (Teva Pharmaceuticals, Poland) – natamycin 0.25 or 0.50% (Unia, Poland) – natamycin 0.25 or 0.50% for one hour, followed by deionised water for ten days Petri dishes were placed at 20°C with 12 hours light / 12 hours dark, for ten days in an incubator (Heraeus BK 6160). The concentrations at which the tested disinfecting agents did not induce significant changes in the final germination percentage of seeds were determined in a preliminary experiment. The germination criterion was visible radicle protrusion. Ungerminated seeds were considered viable if they were firm when squeezed with forceps. Tetrazolium tests carried out with 1% (w/v) 2,3,5-triphenyl tetrazolium chloride solution (Moore, 1973) confirmed that firm embryos were viable, but soft ones were not. The results are expressed as the percentage of germinated viable seeds. The final germination percentage (%), percentage of infected seeds (%), mean length of seedlings (mm) and vigour index (mm%) were calculated (Abdul-Baki and Anderson, 1973). Seeds with visible fungal (mycelial) growth on the surface were regarded as infected. Data were analysed by one-way ANOVA, and significant differences were determined in Tukey’s test at P < 0.05. The analysed values were normally distributed. In the second experiment, seeds were germinated in deionised water or natamycin suspensions under identical conditions. When radical protrusions reached a length of five mm, they were cut off and fixed in Carnoy fixative (ethanol, acetic acid; 3:1) for 12 hours. Fixed radicles were placed in 70% ethanol solution. Twenty-four hours before microscopic analysis, root apices were transferred to distilled water for rinsing. The specimens were stained with 0.05% aqueous solution of aniline blue and examined under the Nikon Eclipse 80i fluorescence microscope (UV ʎ 400 nm) with Nikon DS Fi2 camera to detect callose (Clark, 1981). Results Experiment 1 The final germination of seeds in the presence of various antifungal agents did not differ significantly from the control and ranged from 89.6% (0.5% natamycin) to 98.7% (0.1% Dithane M-45) (figure 1A). Significant differences in the number of infected seeds were observed (figure 1B). The 0.5% natamycin suspension was the most potent disinfectant, and none of the treated seeds were colonised by fungi after 10 days of incubation. Only 1.3% of seeds sterilised with 0.5% natamycin suspension were infected after one- hour incubation and during successive germination in the presence of deionised water. The 0.25% natamycin suspension and nystatin at both concentrations were relatively effective disinfectants. Agricultural fungicides, in particular Funaben-T, were clearly less effective in eliminating fungal infections on seeds (figure 1B). All of the investigated chemical treatments retarded seedling growth, but 0.25% natamycin suspesion exerted the weakest and least significant inhibitory effects on seedling growth after one hour of seed incubation. Seeds incubated with Dithane M-45 at a higher concentration produced the shortest seedlings (figure 2A). The vigour index of seeds incubated