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 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 inhibited seedling growth and failed to sterilise seeds to the extent required for laboratory analyses. Fungal on the seed surface were most effectively eliminated by 0.5% 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 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).

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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, , mercury chloride) or organic compounds (ethanol, formalin and , in particular , penicillin and ). 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)

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– Dithane M-45 0.1 or 0.2% (Indofil Industries Ltd., India) – nystatin 0.45 or 0.90% (, 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, ; 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 for one hour in

123 ANNA BOCHENEK, IRENA GIEŁWANOWSKA, MONIKA CZERMIŃSKA AND KLAUDIA BOJAROWSKA

(A) 100

80

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40

20 Final germination (%) germination Final

0 ABCDEFGHI JKLM

(B) 50 a cd de a d c de cd ef de f cde f 40

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Infected seeds (%) Infected 10

0 ABCDEFGHI JKLM Figure 1. The effect of fungicides and antifungal drugs on the final germination percentage (A) and proportion of infected seeds (B) of barnyard millet. In (A) there were no significant differences; in (B) columns with the same lowercase letter are not significantly different (P > 0.05). Letters on X-axis: (A) control (deionised water); (B) Captan 0.1%; (C) Captan 0.2%; (D) Funaben-T 0.1%; (E) Funaben-T 0.2%; (F) Dithane M-45 0.1%; (G) Dithane M-45 0.2%; (H) nystatin 0.45%; (I) nystatin 0.9%; (J) natamycin 0.25%; (K) natamycin 0.5%; (L) natamycin 0.25% one hour; (M) natamycin 0.5% one hour.

0.25% aqueous suspension of natamycin did not differ significantly from that of seeds incubated in water. The vigour index was lowest in seeds incubated with natamycin and higher concentrations of Dithane M-45 and Funaben-T throughout the entire experiment (figure 2B).

Experiment 2 A microscopic analysis of radicles did not reveal necrotic changes in the apices of roots growing in deionised water (control). Small (typical) amounts of fluorescent callose were detected in the walls of root hair cells and root tissues (figure 3 A-D). In comparison with the control sample, the callose content of the walls of all meristem cells was higher in the apices of seedling roots growing in 0.25% suspension of natamycin. Fluorescence of callose deposits was also visible in hair root tips (figure 3 E-H). Necrosis was observed

124 ANTIFUNGAL EFFECTS ON BARNYARD MILLET SEEDS

(A) 200 a abcde abcde abcde abcde bcde e abc abcde cde de ab abcd

160

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(mm) 80

40

0 ABCDEFGHI JKLM

(B) 20000 a abcd abcd abcd bcd abcd d abc abcd cd d ab abcd

16000

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Vigour index (mm%)4000 length of seedlings Mean

0 ABCDEFGHI JKLM Figure 2. The effect of fungicides and antifungal drugs on the mean length of seedlings (A) and the vigour index (B) of barnyard millet seeds. Capital letters on the X-axis are explained in figure 1. Columns with the same lowercase letter are not significantly different (P > 0.05). Vertical bars indicate standard deviation. in the apices of seedling roots growing in 0.50% suspension of natamycin throughout the entire experiment (figure 3 I-L) and in the apices of roots exposed to a higher concentration of natamycin for one hour (figure 3 Q-T). The observed changes in the apices of barnyard millet roots were typical of necrosis, they affected entire cell groups, and were more or less extensive (figure 3 E-H, I-L, Q-T). Natamycin applied at a higher concentration clearly reduced cell resistance. Cell walls in the necrotised areas were deformed and contained far less structural material (including the polysaccharide callose). It seems that the observed necrotic changes were a typical response to abiotic stress (exposure to a chemical agent) (figure 3 E-H, I-L, Q-T). Only the apices of seedling roots treated with 0.25% natamycin for one hour were free of necrotic changes and changes in callose content. Similarly to the control sample grown in deionised water (figure 3 A-D), small amounts of fluorescent callose were detected in the longitudinal and transverse walls of root cells and in the walls of root hair cells (figure 3 M-P).

125 ANNA BOCHENEK, IRENA GIEŁWANOWSKA, MONIKA CZERMIŃSKA AND KLAUDIA BOJAROWSKA

(A) (B) (C) (D)

(E) (F) (G) (H)

(I) (J) (K) (L)

(M) (N) (O) (P)

(Q) (R) (S) (T)

(A-T) 100 μm

Figure 3. Morphological and physiological changes in the root apices of germinating barnyard millet seeds following exposure to natamycin. (A-D) Apex of a root growing in deionised water – control. Visible fluorescence of small (typical) amounts of callose in the walls of root hair cells and root tissues. (E-H) Apex of a root growing in natamycin suspension (0.25%). The content of callose in the walls of all meristem cells is higher than in the control sample (G, arrowheads). Fluorescence of callose deposits is also visible in hair root tips (H, arrows). (I-L) Deformed apex of a root growing in 0.50% natamycin suspension, with a clear necrotic area (I, J, K, arrows). Necrotic protoplasts (K, arrows) and deformed cell walls with minimal amounts of fluorescent callose (K, arrowheads) are visible in meristem cells. In the undamaged part, root hairs have normal structure and small amounts of callose (L, arrows). (M-P) Radicle emerging from a germinating seed treated with natamycin suspension (0.25%) for one hour and transferred to water. Small amounts of fluorescent callose are visible in the longitudinal and transverse walls of root cells and in the walls of root hair cells. (Q-T) Radicle emerging from a germinating seed treated with natamycin suspension (0.50%) for one hour with necrotic meristem cells. Necrotic cells form a large and dense area in the root apex (Q, R, arrows). In the necrotised region of the root, cells have clearly deformed walls (S, arrows) with smaller amounts of fluorescent callose. Healthy and deformed root hairs in the undamaged part of the root (T, arrows) with small amounts of callose in cell walls.

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Discussion

There is a general scarcity of published studies investigating the effectiveness of antifungal treatments for seed disinfection for laboratory testing, in particular the diaspores of wild plants including weeds. Due to the low effectiveness of popular inorganic and organic compounds and agricultural fungicides in disinfecting seeds for biochemical and physiological analyses (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), Michalczyk and Pawlicka (1980) compared the disinfecting effects of nystatin and sodium hypochlorite on rye kernels. Nystatin did not inhibit seed germination, and it was more effective in eliminating fungi from the seed surface than sodium hypochlorite. Nystatin, a mycostatic drug that is widely used in medicine, did not modify the activity of selected hydrolytic enzymes in kernels (Michalczyk and Pawlicka, 1984). Bochenek and co-workers (Pawlicka and Bochenek, 1993; Bochenek et al., 1994) compared the disinfecting properties of several drugs, including nystatin and natamycin, and agricultural fungicides in a study of rye and wheat seeds. Fungicides (Captan, Funaben-T and Dithane M-45) effectively disinfected cereal kernels, but they also inhibited respiratory processes and seedling growth. Natamycin eliminated fungi from kernel surfaces, it did not inhibit respiration, but it retarded the growth of seedlings (Pawlicka and Bochenek, 1993). Microscopic analyses revealed that root growth was inhibited by a rigid callose layer covering the tip and sides of the root, which was particularly thick in seedlings incubated with fungicides (Bochenek et al., 1994). In view of the above preliminary findings, the authors made attempts to disinfect barnyard millet seeds with drugs that are widely used in medicine, nystatin and natamycin. Barnyard millet seeds, similarly to the seeds of all wild plant species, were highly infected with fungi. In the appropriate concentrations and incubation times, natamycin and nystatin effectively eliminated fungi from the surface of seeds. To date, medical mycostatics have been rarely used in agriculture, probably due to the relatively high prices of these substances (Dalhoff, 2017). However, only small amounts of disinfecting agents are required in laboratory studies, therefore, the relevant costs are not the most important consideration. Disinfecting agents applied in medicine and laboratory seed analyses have to have much higher antifungal efficacy than conventional agricultural disinfectants. Natamycin is also used in the food industry (Delves-Broughton et al., 2006). Humans and animals are often infected with fungal pathogens that belong to the same taxonomic groups or even the same species and genera as plant pathogens. Fungi of the genus Fusarium are such pathogens (Allen et al., 2004; Agrios et al., 2005; Ryan, 2014). Therefore, medical mycostatics could be expected to effectively eliminate fungal infections in seeds. The above hypothesis was confirmed by previous experiments performed on cereal kernels (Michalczyk and Pawlicka, 1980; 1984; Pawlicka and Bochenek, 1993; Bochenek et al., 1994) and in the present study of barnyard millet seeds. Similar results were obtained by the authors in a study investigating the seeds of other species such as Galium aparine L. (data unpublished). The tested disinfectants inhibited the growth of cereal seedlings by forming a rigid callose layer with a thickness of up to 30 μm on the tip and sides of the root and by

127 ANNA BOCHENEK, IRENA GIEŁWANOWSKA, MONIKA CZERMIŃSKA AND KLAUDIA BOJAROWSKA increasing the callose content of root hairs (Bochenek et al., 1994). In other studies of plants, callose was detected in healthy somatic and generative cells, in damaged cells or cells whose growth was inhibited (Rodkiewicz et al., 1989; Wareing and Philips, 1997; Shi et al., 2016; Musiał and Kościńska-Pająk, 2017). According to some authors, callose is a protective substance and a leak sealant in plants, and it is capable of filtering the uptake of substances at the molecular level. The above findings suggest that callose synthesis in roots is intensified to prevent the uptake of foreign and possibly toxic chemicals, such as fungicides and antifungal drugs (Rodkiewicz et al., 1989; Bochenek et al., 1994; Wareing and Philips, 1997). The callose layer inhibits root growth. Under exposure to antifungal compounds, the content of callose increased in the cell walls of barnyard millet roots, but its deposition was considerably lower than in cereal root cells (Bochenek et al., 1994). A microscopic analysis revealed that root growth was inhibited mainly by cell necrosis. Chemical compounds have easier access to the seeds and radicles of barnyard millet which are far smaller than cereal kernels, which could explain the observed differences in responses to the tested antifungal agents. Additional callose was accumulated in root cell walls, but before the callose layer was thick enough to filter out undesirable substances at the molecular level, toxic compounds induced necrosis in the protoplasts of meristem cells. As a result, necrotic changes in root apex cells were the most probable cause of inhibited growth and lower vigour of barnyard millet seedlings which were exposed to antifungal drugs and fungicides. The analysed antifungal drug, natamycin, shows considerable promise, and it could also be used to disinfect explants for in vitro cultures of plant tissues (Liao et al., 2013). Explants and seeds for laboratory analyses are generally disinfected with the same inorganic and organic chemical substances. These compounds are often ineffective, and they can alter metabolic processes in both seeds and plant tissues (Davey and Anthony, 2010; Teixeira da Silva et al., 2015; Duan et al., 2016). The results of our preliminary study (data not published) indicate that explants can be effectively disinfected with natamycin in combination with an antibacterial. Further research is required to confirm the results of the preliminary study and develop an effective sterilisation protocol.

Conclusion

Incubation of barnyard millet seeds in the continuous presence of fungicides and nystatin inhibited seedling vigour and failed to disinfect seeds to the extent required for laboratory analyses. A suspension containing 0.5% natamycin applied continuously or for one hour was most effective in eliminating fungal infections from the surface of the analysed seeds. However, at the above concentration, natamycin retarded the growth of seedlings and decreased the vigour index. Incubation of barnyard millet seeds with 0.25% natamycin suspension for one hour was the most effective disinfection method that was least detrimental to seed physiology. Under exposure to a higher concentration of natamycin, the growth of barnyard millet seedlings was compromised due to necrosis of root apex cells.

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Acknowledgements

Support for the research was received through grant 12.620.039-300 from the University of Warmia and Mazury. The authors wish to thank Dr. Ewa Pawlicka for the inspiration.

References

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