Sanitary quality in seeds from species of Caatinga biome and control methods for fungi

Abstract

Sanitary and quality of seeds of three native species (Aroeira, Jucá, and Mofumbo) from the Caatinga biome, Brazil, and different control methods of microorganisms in seeds were evaluated. We used 500 seeds of each species, 100 for each treatment: control, superficial disinfestation, Enzimatic II, Captana and Mancozebe. Seeds were distributed in Petri dishes containing potato-dextrose-agar culture medium and stored in incubator type Biochemical Oxygen Demand at 28 ± 2 °C for five days. For germination tests, 100 seeds were arranged in germitest paper previously sterilized and moistened with sterilized distilled water, and incubated for 19 (mofumbo), 20 (jucá), and 25 (aroeira) days in incubators at 28 ± 2 °C. Aspergillus flavus, A. niger, A. ochraceus, Lasiodiplodia sp., Penicillium sp. and Rhizopus sp. are associated with the studied seeds. Captana reduced the incidence of Aspergillus and Penicillium in the seeds, being the most efficient in controlling the microfauna associated with these seeds. Enzimatic II also proved to be an efficient control for Aspergillus spp. There was no interference of the tested products on seed germination.

Headnote

ABSTRACT

Sanitary and quality of seeds of three native species (Aroeira, Jucá, and Mofumbo) from the Caatinga biome, Brazil, and different control methods of microorganisms in seeds were evaluated. We used 500 seeds of each species, 100 for each treatment: control, superficial disinfestation, Enzimatic II, Captana and Mancozebe. Seeds were distributed in Petri dishes containing potato-dextrose-agar culture medium and stored in incubator type Biochemical Oxygen Demand at 28 ± 2 °C for five days. For germination tests, 100 seeds were arranged in germitest paper previously sterilized and moistened with sterilized distilled water, and incubated for 19 (mofumbo), 20 (jucá), and 25 (aroeira) days in incubators at 28 ± 2 °C. Aspergillus flavus, A. niger, A. ochraceus, Lasiodiplodia sp., Penicillium sp. and Rhizopus sp. are associated with the studied seeds. Captana reduced the incidence of Aspergillus and Penicillium in the seeds, being the most efficient in controlling the microfauna associated with these seeds. Enzimatic II also proved to be an efficient control for Aspergillus spp. There was no interference of the tested products on seed germination.

Keywords: Combretum leprosum; Libidibia ferrea; Myracrodruon urundeuva; Seed storage

INTRODUCTION

The Caatinga biome integrates an important portion of the ecosystem in the Brazilian Northeast semiarid region. Comprehending a total are of 734,000 km2, this biome is present in all of nine states of the Northeast region, and a small part of the state of Minas Gerais. The rich and diverse biodiversity of Caatinga has been suffering from the impacts caused by deforestation and the exploitation of its natural resources. The intensive use of land with agricultural practices contributed to a loss of the productive areas due to erosion and nutrient withdrawal; the land use for pastures with exotic species, and the use of plants for firewood reduced the native vegetation and decreased the diversity of the forest (Moura et al., 2010).

Some of Caatinga's native species present great economic potential and may be used for wood supply and as a food source. Aroeira (Myracrodruon urundeuva Allemäo) is known for its wood and medicinal properties (Gomes et al., 2013). Jucá (Libidibia ferrea (Mart. ex Tul.) L.P.Queiroz) is widely used for landscaping of urban sites. Its leaves may serve for animal feeding and the wood is used in construction and joinery works. In addition to that, its extract has presented healing and antiseptic properties (Negri et al., 2009; Mota et al., 2012). Mofumbo (Combretum leprosum Mart.) is widely used in programs of reforestation of degraded lands (Pacheco et al., 2014) and in folk medicine as anti-inflammatory, expectorant, and in hemorrhages and flu treatments (Horinouchi et al., 2013). The longevity and large biomass production of these native trees are important characteristics regarding the reduction of environmental impacts in degraded areas (Gomes et al., 2013).

A recent Brazilian law (No. 12,615) protecting native lands from exploratory practices is fueling the increasing demand of seeds of forest native species in the country, mostly by programs aiming to recover and restore degraded areas (Brancalion et al., 2016). However, there are not many methodologies to formalize the activities for commercialization and quality control of seeds from such species, especially due to a lack of knowledge of some biological aspects of most of them (Wielewicki et al., 2006).

Forest species have many seed-associated pathogens, among which the fungi group stands as the most important causal agent (Muniz et al., 2007). Studies on seed health are important tools to understand the present microorganisms and the sanitary quality of these seeds. These factors may interfere directly in germination, bringing losses through deterioration, abnormalities, and lesions in plantlets (Netto and Faiad, 1995), besides the reduction in storage time (Asdal et al., 2019).

For these aforementioned factors, the objective of our study was to evaluate the health and quality of seeds of three different native species (aroeira, jucá, and mofumbo) of the Caatinga, and control methods for fungi that were incident.

MATERIALS AND METHODS

The experiment was conducted in the Laboratory of Microbiology and Plant Pathology at the Universidade Federal Rural do Semi-Árido - UFERSA, Mossoró, state of Rio Grande do Norte (RN), Brazil. Sample seeds of aroeira and mofumbo were collected in the city of Lagoa Nova-RN, and jucá seeds in Assu-RN (Table 1).

Sanitary quality and control of microorganisms

For sanitary evaluation and control of microorganisms in the seeds, the quantification and identification of fungi in the seeds were performed. Five hundred seeds of each species were used (100 per treatment): control treatment (no disinfestation); superficial disinfestation, Enzimatic II (Alltech do Brasil Agroindustrial Ltda, Maringá, Brazil), Captana (Adama Makhteshim LTD., Beer-Sheva, Israel), and Mancozebe (Indofil Industries Limited, Thane, India). The product Enzimatic II, currently under testing process (registration process), was conceived by Alltech Crop Science™.

Superficial disinfestation of seeds was done with ethanol 70% (for 30 secs) and sodium hypochlorite 2.5% (60s), with posterior rising in sterile distilled water. Seed treatment with Enzimatic II, Captana and Mancozebe were performed as presented in Table 2. Treated seeds were placed in Petri dishes containing potato-dextrose-agar (PDA) culture medium (supplemented with tetracycline 0.05g/L) (Boughalleb et al., 2006). Plates were stored in Biochemical Oxygen Demand (BOD) incubators for five days at 28 ± 2 °C (Fig. 1).

After the incubation period, fungi developing in the seed were identified by morphological characterization, using microscope and identification keys, and quantified. The incidence of each fungus (IF) was calculated with the following equation (a) (Boughalleb et al., 2006).

(ProQuest: ... denotes formula omitted.)

Germination tests

Germination tests were conducted to assess the effects of the products used in the control trial on the germination of aroeira, jucá, and mofumbo seeds. Dormancy of aroeira and jucá seeds were overcome by immersion in neutral detergent and scarification, respectively (Brasil, 2013). The treatment of sampled seeds followed the same methodology explained in the control session. One hundred seeds were used per treatment, with 4 replication with 25 seeds each, placed in germitest paper, previously sterilized and moistened with sterile distilled water (2.5x of water by weight of dry paper), and stored for 19 (mofumbo), 20 (jucá), and 25 (aroeira) days in BOD incubator at 28 ± 2 °C. Germination seeds were first counted after 7 (mofumbo), 9 (jucá), and 14 (aroeira) days, and at the end of the incubation period the percentage of germinated seeds was calculated (Alves et al., 2009; Brasil, 2013; Pacheco et al., 2014).

Statistical analysis

The completely randomized experimental design was used for the sanitary, control, and germination tests. Due to the non-normal distribution of the fungal incidence data, nonparametric Kruskal-Wallis test was carried out and means were compared using a multiple comparison test at 5% probability. Results from the seed germination assay were expressed as arithmetic means and subjected to analysis of variance and comparison of means using Tukey's test at 5% of probability (Ferreira et al., 2019; Matsoukis et al., 2015). All statistical analyses were performed in the R statistical software (R Development Core Team, 2013).

RESULTS AND DISCUSSION

According to data from the phytosanitary evaluation, six fungi are associated with the studied seeds (Aspergillus flavus, A. niger, A. ochraceus, Lasiodiplodia sp., Penicillium sp. e Rhigopus sp.). The genera Apergillus was the most frequent in seeds of aroeira, jucá, and mofumbo, where the A. niger was the most frequent species in nondisinfected seeds, with averages of 92%, 45%, and 27%, respectively (Fig. 2). The A. niger was also present in all of the three species when seeds were treated for superficial disinfestation with sodium hypochlorite (in 96% of aroeira seeds, 60% of mofumbo, and 51% of jucá). Jucá and mofumbo seeds disinfected with sodium hypochlorite also presented A. flavus (12 % and 10%, respectively). Aspergillus is one of the main representants of the "storage fungi", which invade seeds during the postharvest and storage processes causing seed decay and deterioration (Bala, 2017; Vechiato, 2010), reducing the seed germination and vigor. Rhigopus sp., another typical fungus of storage (Bhattacharya and Raha, 2002), occurred in nondisinfected seeds of mofumbo (48%) and aroeira (20%). In seeds of jucá disinfected with sodium hypochlorite, we also observed the presence of Lasiodioplodia sp., a fungus commonly found in seeds from native forest species in Brazil (Botelho et al., 2008). To a lesser extent, the nondisinfected seeds also presented A. ochraceus (in aroeira, 2%), A. flavus (3% of jucá and 2% of mofumbo), and Penicillium sp. (7% of mofumbo) (Fig. 2).

Overall, the seed treatment with Captana, Mancozebe, and Enzimatic II presented satisfactory results in reducing the incidence of the main fungi in seeds of aroeira, jucá, and mofumbo (Table 3), showing significant differences (p < 0.05) when compared to the control and superficial disinfestation.

The treatment with Captana reduced in almost 100% the incidence of Aspergillus in all three species (Fig. 3). Silva et al. (2011) also reported the efficiency of Captana in controlling Aspergillus in seeds of five different seeds from the Mata Atlántica (Anadenanthera macrocarpa, Dalbergia nigra, Tababuia chrysotricha, T. heptaphylla, and Senna siamea). The Captana also inhibited Penicillium sp. and reduced the incidence of Rhigopus sp. in seeds of mofumbo and jucá. Classified as a nonspecific fungicide (Parisi and Santos, 2011), the Captana presents a wide range of action, acting against many fungi from the Oomycota division (known as not true fungi) until the true fungi (Ascomycota and Basidiomycota) (Blaney and Kotanen, 2001). Other studies also report the efficiency of Captana on Aspergillus spp. (Souza et al., 2003; Gallo et al., 2013) and other genera of seed fungi (Medeiros et al., 2012).

Mancozebe inhibited the development of fungi in seeds of mofumbo, but it was not efficient in controlling Apergillus spp. in seeds of aroeira and Easiodiplodia sp. in jucá (Fig. 3). However, other studies have reported the efficiency of Mancozebe in the control of Apergillus spp. in forest species (Gallo et al., 2013) and cultivated crops (Saleem et al., 2012). Several studies in the literature report that different active ingredients behaved in different ways regarding their efficiency in controlling fungi in seeds, what may be related to factors such as the seed, form of application of the product, microorganism diversity in the seed, among others (Coutinho et al., 1999).

The Enzimatic II presented promising results, reducing the overall incidence of fungi (Table 3) and, especially, the incidence of Apergillus spp. in all three species (Fig. 3). The Eurotium genera was not observed in the control group nor in seeds disinfected with sodium hypochlorite but was present in aroeira seeds treated with Enzimatic II (Fig. 3), what may be due to the action of the product on some fungal species that inhibited the development of such species by competition (Coutinho et al., 1999) or other antagonism mechanisms. Eurotium sp. has already been reported as an endophytic fungus in seeds (Mota et al., 2017).

Thus, with the lack of chemical products registered for treatment of forest seed (Parisi et al., 2019) and the scarcity of information on the use of alternative productions in these species, our results bring a promising option to assist on this processing stage, reducing the incidence of microorganisms in seeds and helping to moderate the use of harmful chemicals to the environment and human health.

The use of different methods of control did not interfere in the seed germination of any of the assessed species, presenting no statistical difference by the Tukey test (p < 0.05), showing that there was no phytotoxic effect of the products on the seeds (Table 4).

CONCLUSIONS

The fungi associated with seeds of aroeira, mofumbo, and jucá collected in areas of Caatinga forest, in the state of Rio Grande do Norte, are Aspergillus flavus, A. niger, A. ochraceus, Easiodiplodia sp., Penicillium sp., and Rhigopus sp. Captana reduced the incidence of Aspergillus and Penicillium in the assessed seeds, showing the greatest efficiency in controlling the microfauna associated with these seeds. Enzimatic II showed efficiency in controlling Aspergillus spp. in seeds of all tree native species. Treatment of aroeira, mofumbo, and jucá seeds with Captana, Mancozebe or Enzimatic II did not cause any damage to the seeds nor affected the germination.

ACKNOWLEDGMENTS

To the Centro de Pesquisa Leopoldo Américo Miguez de Mello (CENPES/PETROBRAS) for the technical and financial support provided for our study.

Authors' contributions

All authors contributed effectively to this study. NASCIMENTO conducted and evaluated the experiments, and wrote the paper; NOGUEIRA did the literature search and wrote the paper; ALVES and ARAÚJO conducted and evaluated the experiments; DOMBROSKI and MACHADO coordinated the research project and analyzed the data; AMBROSIO coordinated the research project and the students, designed the study, and gave valuable contributions to the paper.

Sidebar

Received: 11 September 2019; Accepted: 12 December 2019

Footnote

"Corresponding author:

Luan Vítor Nascimento, Crop Protection, Department of Plant Pathology and Plant Protection. Georg-August-Universität Göttingen, 37077, Göttingen, Germany. E-mail: [email protected]

References

REFERENCES

Alves, E. U., R. D. L. Bruno, A. P. de Oliveira and A. U. Alves. 2009. Sulfuric acid to overcome the seed dormancy of pau ferro (Caesalpinea ferrea Mart. ex Tu. var. leiostachya Benth.). Rev. Caatinga. 22(1): 37-47.

Asdal, A., G. Brodal, S. Ø. Solberg, F. Yndgaard, R. von Bothmer and E. Meen. 2019. Seed Longevity and Survival of Seed Borne Diseases after 30 Year's Conservation in Permafrost: Report from the 100 Year Storage experiment. Accessed from: http://www.norden.diva-portal.org/smash/get/diva2:1370439/ FULLTEXT01.pdf. [Last accessed on 2020 Jan 10].

Bala, B. K. 2017. Drying and Storage of Cereal Grains. 2nd ed. Wiley Blackwell, United Kingdom.

Bhattacharya, K. and S. Raha. 2002. Deteriorative changes of maize, groundnut and soybean seeds by fungi in storage. Mycopathologia. 155(3): 135-141.

Blaney, C. S. and P. M. Kotanen. 2001. Effects of fungal pathogens on seeds of native and exotic plants: a test using congeneric pairs. J. Appl. Ecol. 38(5): 1104-1113.

Botelho, L. S., M. H. D. Morais and J. O. M. Menten. 2008. Fungi associated to the seeds of ipe-amarelo (Tabebuia serratifolia) and ipe-roxo (Tabebuia impeti gin osa): incidence, germination effect and seedlings transmission. Summa Phytopathol. 34(4): 343-348.

Boughalleb, N., N. Tarchoun and W. Dallagi. 2006. Effect of fungicides on in vitro infestation level of radish, carrot and pepper seeds. Plant Pathol. J. 5(3): 388-392.

Brancalion, P. H., L. C. Garcia, R. Loyola, R. R. Rodrigues, V. D. Pillar and T M. Lewinsohn. 2016. A critical analysis of the native vegetation protection law of Brazil (2012): updates and ongoing initiatives. Nat. Conservaçâo. 14(1): 1-16.

Brasil. 2013. Instruçöes Para Análise de Sementes de Espécies Florestais. Available from: http://www.agricultura.gov.br/ assuntos/laboratorios/arquivos-publicacoes-laboratorio/ florestal_documento_pdf-ilovepdf-compressed.pdf. [Last accessed on 2019 Jul 29].

Coutinho, W. M., E. Araújo and P. H. L. Magalhāes. 1999. Efeitos de extratos de plantas anacardiáceas e dos Fungicidas químicos benomyl captan sobre a micoflora e qualidade fisiológica de sementes de Feijoeiro (Phaseolus vulgaris l.). Cie. Agrotecnol. 23: 560-568.

Ferreira, S., G. M. O. Piovanni, C. R. Malacrida and V. R. Nicoletti. 2019. Influence of emulsification methods and spray drying parameters on the microencapsulation of turmeric oleoresin. Emir. J. Food Agric. 31(7): 491-500.

Gallo, R., R. M. R. Neto, L. Eburneo and H. R. Nascimento. 2013. Efficiency of fungicides on peroba-mica seeds (Aspidosperma desmanthum) and their effects on germination. Rev. Tróp. 7(2): 111-121.

Gomes, M. P., D. M. Duarte, M. M. L. Carneiro, L. C. Barreto, M. Carvalho, A. M. Soares, L. R. G. Guilherme and Q. S. Garcia. 2013. Zinc tolerance modulation in Myracrodruon urundeuva plants. Plant Physiol. Biochem. 67: 1-6.

Horinouchi, C. D., D. A. Mendes, S. Soley, E. F. Pietrovski, V. A. Facundo, A. R. S. Santos, D. A. Cabrini and M. F. Otuki. 2013. Combretum leprosum Mart. (Combretaceae): Potential as an antiproliferative and anti-inflammatory agent. J. Ethnopharmacol. 145(1): 311-319.

Matsoukis, A., D. Gasparatos and A. Chronopoulou-Sereli. 2015. Mepiquat chloride and shading effects on specific leaf area and K, P, CA, Fe and Mn content cf Lantana Camara L. Emir. J. Food Agric. 27(1): 121-125.

Medeiros, J. G. F., B. B. Silva, A. C. A. Neto and L. C. Nascimento. 2012. Fungi associated with seeds of flamboyant-mirim (Caesalpinia pulcherrima): Incidence, effect on germination, transmission and control. Pesqui. Florest. Bras. 32(71): 303308.

Mota, F. C. M., J. C. S. Ferreira and J. Imaña-Encinas. 2012. Analysis of growth Caesalpinia ferrea MART. On campus of the University of Brasilia, federal district. Rev. Verde Agroecol Desenvolv. Sustentável. 7(4): 195-200.

Mota, J. M., M. P. Melo, F. F. S. Silva, E. M. J. Sousa, E. S. Sousa, B. M. Barguil and J. E. A. Jr. Beserra. 2017. Fungal diversity in lima bean seeds. Braz. J. Biosyst. Eng. 11(1): 79-87.

Moura, A. S. S., B. A. Maciel, C. A. Souza, D. C. Moura, D. M. Borge-Nojosa, E. C. Gomes, E. S. Alves, E. M. Riegelhaupt and E. V. Sampaio. 2010. Uso Sustentável E Conservaçâo dos Recursos Florestais Da Caatinga. Serviço Florestal Brasileiro, Brasilia, p. 367.

Muniz, M. F. B., L. M. E. Silva and E. Blume. 2007. The effect of asepsis and substrate composition on tree seed and seedling quality. Rev. Bras. Sementes. 29(1): 140-146.

Negri, L. C. G., A. F. Rosa and P. C. Zonetti. 2009. Dormancy-break of arboreal species seeds. Rev. Agron. Meio Ambiente. 2(3): 487-500.

Netto, D. A. M. and M. G. R. Faiad. 1995. Viability and seed health of forest tree species. Rev. Bras. Sementes. 17(1): 75-80.

Pacheco, M. V., F. D. S. Araújo, C. D. S. Ferrari and R. D. L. A. Bruno. 2014. Germination of Combreto leprosum Mart. seeds. Rev. Caatinga. 27(1): 154-162.

Parisi, J. J. D. and A. F. Santos. 2011. Métodos convencionais de detecçâo de fungos em sementes. In: Santos, A. F., J. J. D. Parisi and J. O. Menten, editors. Patologia de Sementes Florestais. Embrapa Florestas, Colombo, p. 49-61.

Parisi, J. J. D., A. F. D. Santos, C. J. Barbedo and P. F. Medina. 2019. Pathology of forest tree seeds: Damage, detection and control, a review. Summa Phytopathol. 45(2): 129-133.

R Development Core Team. 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing.

Saleem, M. J., R. Bajwa, A. Hannan and T A. Qaiser. 2012. Maize seed storage mycoflora in Pakistan and its chemical control. Pak. J. Bot. 44(2): 807-812.

Silva, L. G. D., F. C. Cosmi, J. Junior, A. F. D. Souza and W. B. Moraes. 2011. Effect of chemical control on the sanity of forest species seeds. Cien. Florestal. 21(2): 473-478.

Souza, A. A. D., R. D. L. Bruno, E. Araújo and G. B. Bruno. 2003. Mycoflora and physiological quality of cotton seeds treated with chemical fungicides and aroeira extract. Rev. Bras. Sementes. 25(1): 56-64.

Vechiato, M. H. 2010. Importância da Qualidade Sanitária de Sementes de Florestais na Produçâo de Mudas. Available from: http://www.infobibos.com/Artigos/2010_3/SementesFlorestais/ index.htm. [Last accessed on 2019 Jul 04].

Wielewicki, A. P., C. Leonhardt, G. Schlindwein, A. C. S. Medeiros. 2006. Proposal for germination and water content standards for seeds of some southern Brazilian native tree species. Rev. Bras. Sementes. 28(3): 191-197.