INTRODUCTION

The production of black beans (Phaseolus vulgaris L.) in Brazil is intended for domestic consumption, with family farming being responsible for a large part of this production. One of the fundamental agricultural inputs for productivity is good quality seeds, which guarantee sustainability and food security, both for this type of agriculture and for domestic consumption (^{Sousa and Wander, 2014}; ^{Fernandes et al., 2019}).

Seed longevity and viability are classified into categories, such as orthodox, recalcitrant and intermediate, depending on their germination performance against desiccation tolerance and storage under different environmental conditions (^{Walters et al., 2013}; ^{Marcos-Filho, 2015}). Conservation aims to prolong durability, maintain quality and reduce losses after harvest, providing a longer shelf life and marketing of products of agroeconomic interest, such as seeds. The storage period is one of the most important stages in the agricultural sector, ensuring the maintenance of balance from the distributor market to the final consumer.

Storage, as an agricultural management practice, aims to ensure the integrity and viability of seeds for their qualitative attributes, genetic, physical, physiological and sanitary, between storage periods. However, there is an interaction among seeds and moisture content, due to hygroscopic processes, the type of packaging, and the conservation environment, all of which can have both positive and negative effects, particularly on the physiological quality of seeds.

In general, desiccation and lowering the temperature are techniques used only for orthodox seeds. Other factors for the conservation of seed quality are the presence and action of phytopathogens and insects, relative humidity and air temperature, oxygen availability, and storage period (^{Carvalho and Nakagawa, 2012}).

The inadequacy of storage conditions favors deterioration and, consequently, a reduction in seed quality, evidenced during the germination and initial development of the seedlings. The speed at which this deterioration occurs can be mitigated by adjusting the initial quality of the lot, the maturity stage, the longevity, the moisture content, the physical conditions of the seeds, and the phytosanitary treatment (^{Gomes et al., 2016}; ^{Menegaes et al., 2021a}; ^2021b).

Several authors, ^{Santos et al. (2005)}, ^{Maia et al. (2011)}, ^{Silva et al. (2014)} and ^{Zucareli et al. (2015)}, emphasize the importance of the correct storage conditions for black bean seeds, since their qualities, especially physiological ones, are negatively affected by uncontrolled temperatures and humidity conditions, by accelerating deterioration, impacting the establishment of plants in the field. The objective of this study was to assess the physiological and sanitary viability of black bean seeds after cold storage at different initial seed moisture contents and for different storage time periods.

MATERIALS AND METHODS

The experiment was carried out from March 2020 to March 2021, at the Seed Research and Teaching Laboratory of the Department of Plant Science of the Federal University of Santa Maria (UFSM), Brazil, located in Santa Maria, RS (29º43' S; 53º43' W and altitude of 95 m). The climate in the region is humid subtropical (Cfa), according to the Köppen-Geiger classification, with average annual precipitation of 1,769 mm, average annual temperature close to 19.2ºC and air humidity around 78.4% (^{Alvares et al., 2013}).

Black bean plants belonging to the IPR88 Uirapuru cultivar were grown in the municipality of Santa Maria, Brazil, and harvested in March 2020. The seeds were stored in a cold chamber (15ºC and 40% RH) in Kraft paper bags (brown type of 1.0 kg). This cultivar was chosen due to its excellent adaptation in the region, the possibility of harvesting in a mechanized cultivation medium and the potential for harvesting in a culture medium.

The design used was completely randomized, in a 3×5 factorial scheme (seed moisture levels and storage periods), with seed moisture levels of 10, 13 and 16%, following the methodology by the Rules for Seed Analysis manual (^{Brazil, 2009a}), and storage periods of 0, 3, 6, 9 and 12 months in cold chambers, with four replications, each experimental unit composed of 50 seeds. The first assessment of physiological and sanitary qualities was 48 h after seed harvest, considering this as zero storage period (0).

Subsequently, the qualities were assessed by the following tests:

Standard germination test (SGT): seeds were distributed on a roll of germination paper, moistened with distilled water at a rate of 2.5 times the dry paper mass. The rolls were kept in a BOD (Box for Organism Development), with a photoperiod of 24 h and a temperature of 25±2ºC (^{Brazil, 2009a}). Germination evaluations were performed at 5 and 9 days after sowing (DAS), and the results are expressed as a percentage of seedlings.

Length and dry mass of seedlings: the seeds were kept in the same conditions as the SGT, and at 5 DAS the total length of the seedling, including the stem and the root of ten normal seedlings of each repetition, was measured using a millimeter ruler. Subsequently, the total dry mass was determined by drying the material in a forced ventilation oven at 65±5ºC for 48 h and then recording the mass on a digital scale (^{Kryzanowski et al., 2020}).

Mass electrical conductivity: performed with four repetitions of 50 seeds, weighed and placed in disposable plastic cups, with a capacity of 200 mL, containing 75 mL of distilled water. The cups were placed in a BOD type germinator set at 25ºC, and readings were taken 24 h after imbibition using a table conductivity meter (^{Kryzanowski et al., 2020}).

Sanitary test: seeds were distributed in transparent plastic boxes for germination in a paper substrate (Blotter Test), moistened with distilled water corresponding to 2.5 times the mass of the dry paper. Seed germination was inhibited by freezing for 24 h at 6±1ºC, then the boxes were kept in BOD for 5 d with a photoperiod of 12 h light and 12 h dark at 20±2ºC (^{Brazil, 2009b}). They were evaluated under a magnifying glass (stereoscopic microscope) for the identification of phytopathogens at the genus level, and the results expressed as percentage of total infested seeds (SIT).

Data expressed as percentages were transformed into arcsine x100. Analysis of variance (ANOVA) of the data and comparison of means by the regression test (P<0.05) were performed with the aid of the SISVAR program (^{Ferreira, 2014}).

RESULTS AND DISCUSSION

Tests to verify the physiological quality of black bean seeds showed significant interaction (Fig. 1). The variables of first germination count (FGR) and germination (GER), at 5 and 9 DAS, respectively, gave similar results, with general averages of 95, 92, 77, 71 and 64% of FGR and GER for storage periods of 0, 3, 6, 9 and 12 months in cold chambers, in that order. ^{Fernandes et al. (2019)} found that black bean seeds showed similar FGR and GER, even when subjected to saline conditions.

Figure 1. (A) First germination count (FGR), (B) germination (GER), (C) mass electrical conductivity (EC), (D) thousand seed mass (TSM), (E) seedling length (SL) and (F) seedling dry mass (SDM) of black bean seeds (Phaseolus vulgaris L.) cultivar IPR88 Uirapuru subjected to different storage periods and seed moisture levels. 

According to Normative Directive no. 45/2013 of the Ministry of Agriculture, Livestock and Supply (MAPA) (^{Brazil, 2013}), black bean seeds must have at least 70% germination for the basic commercial category and for other categories at least 80% germination. Thus, data from this study indicate that the commercialization of black bean seeds is possible for up to nine months of storage at seed moisture levels of 10 and 13%, and for up to 6 months at 16% seed moisture content.

Seed deterioration was confirmed both by the reduction of germination percentage and by the mass electrical conductivity (EC), which worsened during storage, mainly in seeds at a higher degree of humidity. ^{Boiago et al. (2013)} verified by the EC test that storage of cowpea seeds (Vigna unguiculata (L.) Walp.) for more than 90 d resulted in increased deterioration with negative implications for germination percentage.

The reduction in germination percentage of these seeds may be attributed to the interaction of the genetic characteristics of the species with the storage environment. These results are similar to those verified by ^{Menegaes et al. (2021a}; ^2021b) for safflower seeds (Carthamus tinctorius L.) stored for 0, 4, 8 and 12 months in cold chambers. For the aforementioned authors, the EC test confirms that as the storage period of the seeds increases, their germination potential is reduced, inducing an increase in their deterioration, even under controlled conditions of relative humidity and temperature. According to ^{Marcos-Filho (2015)}, greater electrolyte leaching in the EC test reflects greater deterioration of seed quality, negatively affecting the emergence of seedlings in the field.

It was observed that the mass of a thousand seeds (TSM) showed an interaction between the storage period and seed moisture content (Fig. 1D), with a reduction of the masses during storage. ^{Scariot et al. (2017)} attributed the reduction of TSM of wheat seeds (Triticum aestivum L.) during the storage period to the maintenance of the hygroscopic balance and the permeability of the storage form, considering that there is an energy expenditure for this maintenance.

It was found that there was a similarity in mean seedling length (SL) and seedling dry mass (SDM) of black beans at all moisture content levels, with averages of 18, 17, 14, 13 and 13 cm, and 45, 44, 41, 37 and 36 mg/plant for storage periods of 0, 3, 6, 9 and 12 months in cold chambers, respectively. ^{Zucareli et al. (2015)} found that during storage periods of up to 18 months, seeds of the Carioca beans variety had a reduction in physical quality during the general form.

The storage period directly influenced the total number of infested seeds, regardless of the seed moisture levels (Fig. 2). From this evaluation, the presence of phytopathogens of the genera Aspergillus, Fusarium, Penicillium and Sclerotinia (Fig. 2B, 2C, 2D and 2E) was verified in the stored black bean seeds. ^{Gomes et al. (2016)} emphasize the importance of seed health quality for productivity, especially for fava bean (Phaseolus lunatus L.) seeds, which, when infested by phytopathogens, show reduced emergence, negatively affecting the establishment of plants in the field.

Figure 2. (A) Total infested seeds (TIS), (B) Aspergillus spp. (ASP), (C) Fusarium spp. (FUS), (D) Penicillium spp. (PEN) and (F) Sclerotinia spp. (SCL) of black bean (Phaseolus vulgaris L.) seeds, cultivar IPR88 Uirapuru, subjected to different storage periods. 

It was observed that the regression trends on the incidence of the Aspergillus,Fusarium, Penicillium and Sclerotinia phytopathogens, were similar at all humidity levels of black bean seeds over different storage periods. On the other hand, it was noted that Aspergillus fungi (Fig. 2B) showed an increasing regression trend as the storage period increased for all moisture levels. ^{Menegaes et al. (2021b)} found similar fungal behavior for safflower seeds stored for 0, 4, 8 and 12 months.

The opposite was verified for Penicillium and Sclerotinia (Fig. 2D and 2E), which showed a decreasing trend according to the storage period, with the lowest percentage of fungal incidence at 12 months of storage for all moisture levels. ^{Marino et al. (2008)} attribute the difference in incidence of pathogens on black bean seeds to the storage environment. Care must be taken from this harvest to avoid the association of pathogens with the seeds as much as possible, since the pathogen survives longer, maintaining its viability and characteristics, and can be easily disseminated in different environmental conditions, at levels considered to be contamination, as seen in Fig. 2C for Fusarium pathogens.

The results of the present study corroborate the findings of ^{Santos et al. (2005)}, ^{Maia et al. (2011)}, ^{Silva et al. (2014)} and ^{Zucareli et al.(2015)}, in which all authors verified the importance of storing bean seeds under controlled conditions in order to avoid accelerating their deterioration. It was observed that with the extension of storage periods in all seed moisture levels, there was a reduction in the physiological quality of black bean seeds and a considerable increase in the incidence of pathogens on the seeds. Since the storage conditions were not enough to help in the phytosanitary control of these seeds, further studies are required on this subject.

CONCLUSION

It is possible to store black bean seeds at different initial seed moisture contents and periods in a cold chamber. The physiological and sanitary qualities of black bean seeds are maintained for up to nine months at 10 or 13% seed moisture contents and up to 6 months at 16% seed moisture content.