Introduction

An artificial seed coating is a layer that covers the entire seed surface. It is usually formed by inert materials to provide adequate handling, promote particular microenvi­ronments and protect seeds against pathogens and insect damage (Giménez-Sampaio et al., 1992; Ziani et al., 2010; Zeng et al., 2012). Usually, seed coatings function as car­riers for pesticides which pro­tect the seeds and the emer-ging seedlings (Vavrina and McGovern, 1990; Kaufman, 1991). The coating agent should not be toxic or harm­ful for the plant or the envi­ronment. Chitosan is a natu-ral polymer that has shown good results when applied as seed coating for some crops (Benhamou et al., 1994; Bhaskara Reddy et al., 1999; Boonlertnirun et al., 2008; El Hadrami et al., 2010). This polymer is obtained from the deacetylation of chitin, the predominant component of arthropod exoskeletons and of cell walls of several fungi. Because it is biodegradable and non-toxic, and has anti­microbial properties, chitosan is seen as a versatile material for several agricultural appli­cations (Badawy and Rabea, 2011). It has been shown that chitosan elicits defense mech­anisms in plants through in­duction of glucanases, chiti­nases, phenolic compounds, terpenoids, PR proteins, prote­ase inhibitors, and compounds associated with oxidative burst, lignification and callose deposition (Bautista-Baños et al., 2006; Franco and Iriti, 2007; Mandal and Mitra, 2007; Hadwiger, 2013; Mejia- Teniente et al., 2013).

Chitosan has been used to coat corn, tomato, rice and wheat seeds and has been as­sociated with several effects that include better physiologi­cal quality, increased vigor, higher germination rates and induction of plant defenses (Benhamou et al., 1994; Bhaskara Reddy et al., 1999; Boonlertnirun et al., 2008; Ziani et al., 2010; Zeng et al., 2012). This underlines the value of chitosan used as an additive for seed coating. The use of agrochemicals in com­bination with chitosan has been evaluated and has yield­ed good results (Kashyap et al., 2015; Symonds et al., 2016). However, compatibility of this polymer with benefi­cial microorganisms that are frequently incorporated in some seed coats remains to be explored in depth.

Incorporation of beneficial agents into seed coatings has yielded good results in terms of plant protection, production or endophytic fungus coloniza­tion. The most common bene­ficial agents used for seed coating include Pseudomonas spp., Gliocladium spp., Glomus spp., Trichodermaspp. and Beauveria bassiana, which are associated with mineral solubi­lization, plant defense promo­tion, insect infection and an­tagonism on phytopathogens. Usually, these beneficial agents are mixed with adhesive com­ponents of polymeric nature, including xanthan gum, meth­ylcellulose, latex derivatives and synthetic adhesives to pro­duce seed coatings (Mao et al., 1997; Tefera and Vidal, 2009; Brownbridge et al., 2012; Colla et al., 2015). Never theless, combinations of chitosan and beneficial microorganisms in seed coatings are less fre­quent. Recently, the tolerance of some beneficial microor­ganisms (yeast cells and Trichoderma spores) to chi­tosan and some potential ap­plications have been demon­strated (Saifuddin and Raziah, 2007; Spasova et al., 2011)

Use of chitosan in seed coat­ings can potentially promote early plant defense responses, but in addition, the incorpo-ration of agrochemicals and microorganisms could increase seed and seedling protection. However, chitosan varies be­cause of the variability of its production (Lertsutthiwong et al., 2002; Abdou et al., 2008) and the nature of the agrochem­ical or biological agent to be incorporated, and each formula­tion should be evaluated. Therefore, the aim of this study was to evaluate application of various formulations of chitosan in mixtures with a fungicide or with conidia of beneficial fungi to determine their feasibility as a crop seed coating.

Materials and Methods

Preparation of seeds

Bean (Phaseolus vulgaris L.) ‘negro Jamapa’ seeds were ac­quired from a local supplier in Ciudad Victoria, Tamaulipas, Mexico, and damaged seeds were discarded. For fungicide retention tests and for testing coatings with conidia, seeds were disinfected with commer­cial 10% NaClO solution for 5min and rinsed three times for 5min with sterile distilled water. The seeds were then placed on sterile paper and air dried inside a laminar f low hood for 24h before coating and subsequent testing.

Chitosan preparation

For coatings, two types of chitosan were used. Chitosan from shrimp was prepared from commercial chitin (Sigma- Aldrich, St. Louis, MO, USA) by deacetylation in 70% NaOH solution at 120ºC for 1h and left to rest for 12h at room temperature before extensive washing with distilled water. Chitosan was dried at 60ºC for 12h and then dissolved to a concentration of 2% in 5% acetic acid by constant stirring. The solution pH was adjusted to 6 with 2M NaOH, and dia­lyzed for salt removal using distilled water for 3 days with three daily water changes. Dialysis was done with a stan­dard RC membrane Spectra/ Por® 6 and pre-wetted dialysis tube with 25kDa molecular weight cutoff (SpectrumLabs, USA). After this, the solution was adjusted to pH 6 using 1% HCl. This solution was used to formulate treatments, adjusting it to the required concentration in each experiment. The degree of deacetylation was determined by the potentiometer titration method (Yuan et al., 2011).

Insect chitosan was obtained from adult corpses of Ptero-phylla beltrani (Bolivar & Bolivar), which were processed with adaptations to the proce­dure of Torres-Castillo et al. (2015). Ground insects were first washed with 250mM NaOH and then with 500mM NaOH at 90ºC, followed by five rinses with distilled water. After this, the resulting material was subjected to deacetylation as indicated above. This chi­tosan solution was also subject­ed to dialysis and the degree of deacetylation was determined by the potentiometer titration method (Yuan et al., 2011).

Seed germination with commercial chitosan and mixtures of chitosan with fungicide

Six treatments were prepared under aseptic conditions. DW: distilled water (control); FS: 0.5% fungicide solution Ziram® (FMC Agroquímica of Mexico, Zapopan, Jalisco, Mexico) in distilled water; C0.25: 0.25% chitosan in dis­tilled water; C1: 1% chitosan in distilled water; C0.25F: 0.25% chitosan solution mixed with 0.5% Ziram® fungicide; and C1F: 1% chitosan solution mixed with 0.5% Ziram® fun­gicide. The fungicide was dis­solved in water, to be later in­corporated into the mixture of chitosan, and stirred periodi­cally to prevent aggregate for­mation or sedimentation. Previously disinfected and dried seeds were individually immersed in their respective solution for 2-5s and then dried for 48h at room temperature (28-30 C). In each treatment, a sterile glass beaker with 200ml of solution was used to im­merse the seeds. To evaluate germination, 100 seeds per treatment were tested in Petri dishes with Whatman No. 1 filter paper moistened with 5ml of sterile distilled water. Seeds were considered germinated when roots at least 5mm long were present; the number of germinated seeds was recorded daily and germination percent­age was calculated after 72h. The experiment was performed in triplicate.

Fungicide retention test

Because chitosan has been suggested as an agent for re­tention and gradual release of various compounds, the ability of shrimp chitosan to retain Ziram® fungicide on the sur­face of bean seeds was as­sessed. A total of 200 seeds per treatment were coated by individual immersion in the corresponding solution for 2-5s and dried for 24h at room tem­perature (28-30ºC). A group of 50 seeds per treatment was reserved until use, the remain­ing 150 per treatment were washed in sterile distilled wa­ter. The fungicide retention test was related to permanence of the fungicide effect on seeds from treatments FS, C0.25F and C1F after washing with sterile water. Groups of 150 seeds per treatment were placed in containers with 500ml of sterile distilled water, which was changed every 2h over an 8h period. After 1h, 50 washed seeds per treatment were removed; after 2h, anoth­er 50 seeds per treatment were removed; and finally, after 8h, the last 50 seeds per treatment were removed. All seeds were dried for 1h at room tempera­ture (28-30ºC) inside a laminar flow hood. Each 50-seed treat­ment had different exposure times to rinses. To determine permanence of the fungicide, all rinsed and dried seeds were placed in Petri dishes with po­tato dextrose agar (PDA, Bioxon) culture medium form­ing two groups of 25 seeds (experiment in duplicate). Then, each seed was inoculat­ed individually with 10μl of a solution of Fusarium oxyspo­rum conidia at a concentration of 1×105 conidia/ml. Conidia were harvested by washing the surface of a 5 days old F. ox­ysporum culture with 15ml of 0.05% Tween 80 (Sigma- Aldrich) sterile solution. Conidia concentration was ad­justed to 1×105. Petri dishes with inoculated seeds were in­cubated 72h at room tempera­ture (28-30ºC). Presence of my­celial growth on seeds was re­lated to conidia germination, and seeds with mycelial growth from treatments FS, C0.25F and C1F were considered to have lost fungicidal effect.

Germination of seeds coated with shrimp or insect chitosan and mixtures of each with conidia

Since chitosan is associated with the natural development of entomopathogenic and an­tagonistic fungi, we included B. bassiana and T. harzianum conidia in the formulations of shrimp and P. beltrani chitosan to coat bean seeds. Conidia were harvested by washing the surface of a 10 days old solid fungal culture with a sterile solution of 0.05% Tween 80 (Sigma-Aldrich) and concentra­tion was adjusted to 2.5×105. Treatments for the experiment with B. bassiana included DW: distilled water control; C0.25: shrimp chitosan 0.25%; C1: shrimp chitosan 1%; C0.25Bb: shrimp chitosan 0.25% with B. bassiana conidia; C1Bb: shrimp chitosan 1% with B. bassiana conidia; CP0.25: P. beltrani chitosan 0.25%; CP1: P. beltrani chitosan 1%; CP0.25Bb: P. beltrani chitosan 0.25% with B. bassiana conid­ia; and CP1Bb: P. beltrani chi­tosan 1% with B. bassiana co­nidia. For the experiment with T. harzianum conidia, the treat­ments were: DW: distilled wa­ter control; C0.25: shrimp chi­tosan 0.25%; C1: shrimp chi­tosan 1%; C0.25Th: shrimp chitosan 0.25% with T. harzia­num conidia; C1Th: shrimp chitosan 1% with T. harzianum conidia; CP0.25: P. beltrani chitosan 0.25%; CP1: P. beltra­ni chitosan 1%, CP0.25Th: P. beltrani chitosan 0.25% with T. harzianum conidia; and CP1Th: P. beltrani chitosan 1% with T. harzianum conidia. Seeds pre­viously disinfected and dried were subjected to individual immersion in the respective solution during 2-5s and then deposited onto clean waxed paper and dried for 48h at room temperature (28-30ºC). To evaluate germination, 100 seeds were used for each treat­ment in Petri dishes with Whatman No. 1 filter paper. Seeds were moistened with 5ml of sterile distilled water; moisture was kept by adding 1.5ml of sterile distilled water every other day. Germination was recorded daily and germi­nation percentage for each treatment was calculated after 72h. The experiment was per­formed in triplicate.

Statistical analysis

The data were analyzed us­ing a completely randomized design by analysis of variance using the Statistical Analysis System version 6.2 (SAS Institute, Inc., Cary, North Carolina). Means were com­pared by Tukey (p<0.05).

Results and Discussion

Effect of coatings on germination

Coatings from the two sources of chitosan formed a continuous translucent film with a dusty appearance when dry. FS, C0.25F and C1F pro­duced seeds with dusty whitish coatings; seeds treated with B. bassiana and T. harzianum had dusty translucent coatings. Viability of conidia mixed with chitosan was related to myceli­al growth on some seeds; abundant mycelial growth for B. bassiana but scarce for T. harzianum was observed.

Despite the wide diversity of applied treatments and the 70% germination in some treat­ments, no significant differenc­es in seed germination percent­age were observed (p<0.05). This confirmed that chitosan can be useful as an adherent agent (Figure 1) since it did not affect the germination pro­cess. Seeds coated with 0.25% to 1% chitosan mixtures and their respective mixtures with fungicides showed no signifi­cant difference in germination rate (Figure 1a). Furthermore, germination of seeds coated with the two types of chitosan and at different concentrations was not statistically different from the control. In addition, none of the two types of chi­tosan mixed with conidia of fungal species negatively af­fected seed germination (Figure 1b and c). These re­sults show that application of these chitosan coatings and derivatives did not affect the hydration process or germina­tion percentages of bean seeds. Regarding the effects of chi­tosan on germination, different reports indicate an inductive effect of germination of some species, such as Sorghum or Egyptian anise, but can inhibit germination of lettuce seeds, while others report that it may or may not affect germination rates compared to control treat­ments. These reports indicate that the effects are variable and will depend on the nature of the chitosan, its molecular size, crop characteristics and growth conditions (Lizárraga-Paulín et al., 2011; Goñi et al., 2013; Mahdavi and Rahimi, 2013; Hameed et al., 2013, 2014). In our study, the degree of deacetylation was 68.5% for shrimp chitosan and 72.4% for chitosan from P. beltrani; and the molecular weight was >25kDa, relative to the dialysis membrane used. Retention of fungicide on the surface of seeds treated with chitosan

Application of chitosan for gradual release of drugs and agrochemicals has been re-ported (Teixeira et al., 1990; Bansal et al., 2011). For this reason, a fungicidal agent was included as part of the chitosan coating formulation.

Coating formulations with chitosan allowed functionality and retention of the fungicide on the seed surface. Rinse times of seeds were related with the absence or presence of F. oxysporum mycelia on the seed surface as an indica­tion of either prevalence or loss of fungicidal effect. Presence of mycelia at 72h in most seeds in DW, C0.25 and C1 was observed. In contrast, in the case of FS, C0.25F and C1F, the presence of mycelia was minimal for water immer­sion times of 0 and 1h. However, as immersion time increased, more mycelia-cov­ered seeds were observed. Seeds from FS showed gradual mycelial growth in accord with rinse time. When comparing the three formulations with fungicide, most of the seeds with the same rinse times had mycelia after 8h. Therefore, the longer the contact with the medium, the greater the release of the agrochemical when no adherent is present. In the case of C0.25F and C1F, the fungi­cidal effect remained on most of the seeds, even after immer­sion in water for 8h (Figure 2), confirming that chitosan coat­ings retained the fungicide and prevented fast release into the medium, as suggested by Roy et al. (2014). This theoretically would increase the protection time on the seed and in its surroundings and decrease the impact on soil microbiota by preventing diffusion into the soil, due to confined fungicide application.

Seed coating with chitosan and conidia

The feasibility of a biofunc­tional coating based on chitosan and Trichoderma conidia for plant protection was explored in vitro with the combination of chitosan and T. harzianum spores against sapstain fungi, and also on controlling Fusariumand Alternaria strains (Chittenden and Singh, 2009; Spasova et al., 2011). Therefore, the possibility of forming seed coatings that al­low germination and retention of conidia of beneficial fungi on the seed surface was evaluatedB. bassiana and T. harzia­num conidia were incorporated into coatings based on the two types of chitosan and were applied homogeneously. During the tests, no changes were ob­served in percentage of seed germination as shown above (Figure 1b and c). In the case of B. bassiana, mycelial growth in 45% of the treated seeds was recorded; while T. harzianum mycelia appeared on only 2% of the treated seeds. The fact that B. bassi­ana conidia could generate mycelia in the coating on a significant percentage of the seeds can be linked to the abil­ity of the fungus to take ad­vantage of the chitosan as a source of C and N (Palma- Guerrero et al., 2010). In the case of T. harzianum, evalua­tion is required to determine whether the conidia remained viable in the coating or were unable to germinate. For both fungi, it would be of interest to conduct extensive tests for han­dling times and conditions that trigger germination when in­corporated into the coating to increase success of in situ biocontrol.

Chitosan is considered an trigger agent of plant defenses, as well as having antimicrobial effect (Bautista-Baños et al., 2006). These properties have made chitosan one of the most widely recognized agents for defense induction in several crops (Thakur and Sohal, 2013). Chitosan concentrations used in this study did not af­fect germination of B. bassiana conidia, similar to results re­ported previously. Nor was F. oxysporum conidial germina­tion inhibited, a result opposite to other reports (Palma- Guerrero et al., 2008), but this may be related to the nature of chitosan used. However, an inhibitory effect on the germi­nation of conidia of T. harzia­num cannot be ruled out. Palma-Guerrero et al. (2008) observed inhibition of T. har­zianumand T. atroviridae ex­posed to 0.01 and 1mg·ml-1, which could be indicative of generalized sensitivity of some Trichoderma species. Although concentrations of up to 1% chitosan can be used to include active conidia of some benefi­cial fungi such as B. bassiana, the viability of seed coatings requires conidia tolerance, which will depend on the fun­gal species or strains. Therefore, evaluation of each case is recommended. Like the inclusion of a fungicide agent and conidia, it is possible to include compounds such as Bacillus thuringiensis toxins, antimicrobial peptides or bioac­tive proteins (protease inhibi­tors, cyclotides, chitinases), phytohormones, nitrogen-fixing bacteria or growth promoters, among other components that could protect the seeds or seedling establishment (Pérez- Quiñones et al., 2010; Fan et al., 2012).

The success of chitosan as a retaining agent for the develop­ment of seed coatings with agrochemicals and biological agents was confirmed. Germination of bean seeds was not affected using chitosan coatings. Conidia germination of B. bassianawas higher than that of T. harzianum, which indicates differential sensitivity to chitosan. Success of seed coatings depends on many fac­tors, including surface charac­teristics, architecture of seed surface, presence of trichomes, seed physiology, additive na­ture of chitosan, seed genetics and propagules or microorgan­ism responses. Therefore, it is important to emphasize the need for a holistic perspective in generating coatings and studying their effect on germi­nation stages subsequent to determining their impact on the establishment of the seed­lings and on the rhizosphere.

Acknowledgements

The authors acknowledge project PFI2014-60 (“Coating Seeds Using Chitosan for Protection from Saprophytic Fungi and Agricultural Pests”) of UA AT 2014 research pro­gram and the Institute for Applied Ecology of the Universidad Autónoma de Taumalipas for the facilities provided for this study.

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Notes