- Email: [email protected]
Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity
ARTICLE IN PRESS
JID: SAJB
[m5G;November 1, 2019;16:30]
South African Journal of Botany 000 (2019) 1 6
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity L.L.S. Silvaa, I.M.S. Araujoa,b, P.M.O. Costac, A.P.S. Oliveiraa, A.P. Oliveirad, A.D.C. Santosd, ~oa, L.M. Dutrad, J.R.G.S. Almeidad, L.C.B.B. Coelhoa, C.M. Souza-Mottac, T.H. Napolea a, P.M.G. Paiva * Departamento de Bioquímica, Centro de Bioci^encias, Universidade Federal de Pernambuco, Recife, PE 50670-420, Brazil ria, Embrapa Agroindustria Empresa Brasileira de Pesquisa Agropecua Tropical, Fortaleza-CE, 60511-110, Brazil Departamento de Micologia, Centro de Bioci^encias, Universidade Federal de Pernambuco, Recife, PE 50670-420, Brazil d ~o Francisco, 56304-205 Petrolina, Pernambuco, Brazil Nucleo de Estudos e Pesquisas de Plantas Medicinais (NEPLAME), Universidade Federal do Vale do Sa a
b c
A R T I C L E
I N F O
Article History: Received 27 March 2019 Revised 17 September 2019 Accepted 12 October 2019 Available online xxx Keywords: Moringa oleifera Myracrodruon urundeuva Seed protection Termite infestation Phytopathogenic fungi
A B S T R A C T
Moringa oleifera seeds contain several biotechnologically-relevant molecules but are susceptible to termite attack. On the other hand, Myracrodruon urundeuva is strongly resistant to deterioration and it was previously reported the termiticidal activity of preparations from its heartwood against Nasutitermes corniger. This work evaluated whether methanolic (ME) and saline (SE) extracts from M. urundeuva heartwood would be able to protect M. oleifera seeds against N. corniger and improve seed sanity. Nuclear magnetic resonance (NMR) spectroscopy analysis was used to identify the chemical profile of the extracts. Seeds were sprayed for 5 days with ME (SME), SE (SSE), or positive control 1% (v/v) sodium hypochlorite (SSH) and then infested by workers and soldiers. Controls were also performed using seeds not treated with the extracts and infested (ST control) or not (S control). After death of all termites, the seeds were analyzed for vigor (by measurements of electric conductivity in the exudates) and sanity. The representative 1H NMR spectra showed phenolic compounds among the main components of the extracts. Mortality rate of workers and soldiers after 7 days was 100% in SME, SSE, and SSH while it was 50% in ST control. Perforations made by termites were observed in seeds from ST control and SME treatment but not in seeds treated with SE. Analysis of vigor revealed that seeds from control ST or those treated with ME or sodium hypochlorite were damaged by termites while SE promoted protection against N. corniger. Fusarium spp., Penicillium spp. and Trichoderma harziamum were identified in treatments and controls, revealing that the extracts did not improve seed sanity. In conclusion, SE from M. urundeuva heartwood conferred protection against N. corniger attack when sprayed on M. oleifera seeds and this effect can be related with lectin presence. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Seed germination and vigor depend on intrinsic factors, such as quality of the messenger RNAs stored during embryo maturation, proteostasis and DNA integrity, as well as on environmental factors and the conditions employed for harvesting, drying, and processing (Creech, 2012; Rajjou et al., 2012). During storage, seeds may also be affected by the attack of microorganisms (mainly fungi) and insects (such as termites, beetles, maggots, ants and wireworms) resulting in impairment of nutritional value, weight and germination power (Danielson et al., 1991). *Corresponding author. E-mail address: [email protected] (P.M.G. Paiva).
It is increasing the interest on the use of alternative techniques for controlling seed pests due to the high toxicity of antifungals and insecticides currently used. In the last years, studies focusing on natural products (e.g. plant extracts, powders, essential oils, lectins, enzyme inhibitors) for treating stored crops have been intensified aiming the protection of seeds and grains, with lower risks of intoxication to farmers and consumers (Camaroti, Oliveira, Paiva, Pontual, and Napole~ao, 2017; Memon et al., 2019; Pretty and Bharucha, 2015). Moringa oleifera (Moringaceae) is a pantropical tree whose seeds are source of compounds with a broad biotechnological potential, ~o such as in water treatment and production of cosmetics (Napolea et al., 2019). Its seeds contain coagulant molecules that are able to reduce turbidity, bacteria and cyanobacteria contamination, and helminth egg number in water and wastewaters (Ferreira et al., 2011;
https://doi.org/10.1016/j.sajb.2019.10.013 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
JID: SAJB 2
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Nishi et al., 2012; Santos et al., 2009; Sengupta et al., 2012). Studies showed that lectins from these seeds have larvicidal, ovicidal and oviposition-stimulant activity on Aedes aegypti (Coelho et al., 2009; Santos et al., 2012;Santos et al., 2014, 2019), antibacterial activity (Moura et al., 2015, 2017), nematicidal activity (Medeiros et al., 2018) and cytotoxic effects on cancer cells (Luz et al., 2017). In addition, the M. oleifera seeds have been studied as source of oil for biodiesel production (Rashid, Anwar, Moser, and Knothe, 2008). Although M. oleifera tree is considered to be resistant to most of pests, it is susceptible to the attack of fungi, ants and mainly termites € rndly, Reyes-Sa nchez, Salmero n-Miranda, and (Mendieta-Araica, Spo Halling, 2013). The protection of seeds and seedlings against termite attack is strongly recommended (Sauveur and Broin, 2010). Study showed that the lectins cMoL and WSMoL present in M. oleifera seeds were able to kill Nasutitermes corniger workers and soldiers only at high concentrations and thus these molecules probably fail in conferring resistance to the seeds against termite attack (Paiva et al., 2011). The N. corniger is a soil-feeding and wood-damaging termite species, being the most widely distributed of the genus, and considered a successful invasive pest in urban areas, causing considerable economic impact (Santos, Carrijo, Cancello, and Castro, 2017). Myracrodruon urundeuva (Anacardiaceae) is a timber tree known for its use in Brazilian building industry and medicinal properties. Its heartwood is strongly resistant to termite and phytopathogenic fungal attack and the biochemical basis of this resistance has been investigated. A chitin-binding lectin (MuHL) with termiticidal activity on N. corniger workers and soldiers was purified from saline extract of M. urundeuva heartwood; MuHL and the extract did not show repel et al., 2008, 2009a). On the other hand, methanolic lent activity (Sa extract from heartwood did not kill workers and soldiers but showed repellent effect on N. corniger (Sa et al., 2009a). Both saline and methanolic extracts, as well as MuHL, showed antifungal activity against et al., 2009a, 2009b). Fusarium species (Sa In this work we evaluated the efficacy of the treatments of M. oleifera seeds with saline (SE) and methanolic (ME) extracts from M. urundeuva in order to confer protection against damages caused by N. corniger on seeds and to improve seed sanity in regard to fungal presence.
2. Materials and methods
2.2. Methanolic and saline extracts from M. urundeuva heartwood The M. urundeuva heartwood was powdered (40 mesh). Methanolic extract (ME) was obtained by soaking 3 g of powdered heartwood in 10 mL of methanol; the mixture was homogenized at 25 °C for 3 h under magnetic stirring and afterwards it was filtered on filter et al., 2009a). paper and dried on a rotary evaporator to yield ME (Sa Saline extract (SE) was obtained by homogenization of the powdered heartwood (10 g) in 0.15 M NaCl (100 mL) for 16 h at 4 °C under magnetic stirring. After filtration, the homogenate was filtered through gauze and filter paper, centrifuged (3000 g, 15 min, 4 °C), and the et al., 2009a). supernatant corresponded to SE (Sa 2.3. Nuclear magnetic resonance (NMR) spectroscopy analysis For NMR analysis, 25 mg of each extract was solubilized in 500 mL of deuterated solvent. 1D and 2D NMR data were acquired at 298 K in D2O on a Bruker AVANCE III 400 NMR spectrometer (Bruker AXS, Inc., Madison, WI, USA) operating at 9.4 T, observing 1H and 13C nuclei at 400 and 100 MHz, respectively. The NMR spectrometer was equipped with a 5-mm direct detection probe (BBO) with z-gradient. Besides, one-bond heteronuclear (1H-13C) and homonuclear (1H-1H) correlations were assessed by heteronuclear single quantum coherence (HSQC) and correlation (COSY) spectroscopies. 1H and 13C NMR chemical shifts were given in ppm referenced to TMSP-d4 signal at 0.00 ppm, and the coupling constants (J) in Hz. The experiments were performed on a single replicate and the obtained spectra were processed in Topspin software version 3.2.0 (Bruker AXS). 2.4. Total phenol content Total phenol content of ME and SE was determined by the Folin-Ciocalteu method based on the reduction of phosphomolybdic-phosphotungstic reagent by phenols in alkaline solution (Morais, Nascimento, Queiroz, Pilo-Veloso, and Drumond, 1999). Folin-Ciocalteu reagent (1:10 solution in distilled water; 2.5 mL) and sodium carbonate (75 g/L solution in distilled water; 2 mL) were added to SE or ME (0.5 mg/mL solution in distilled water; 0.5 mL). The mixtures were kept at 50 °C for 5 min. After cooling for 30 min, the absorbance was measured at 760 nm. The phenol content was determined using a standard calibration curve of tannic acid (9.6 48 mg/mL in distilled water; 0.5 mL).
2.1. Insects and plant materials 2.5. Hemagglutinating activity and protein content Nests of N. corniger were collected from a fragment of Atlantic Forest at the campus of the Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil. The colonies were removed from the trunk of a tree with a machete, transferred into black plastic bags to the laboratory, and maintained at 28 °C and 75% relative humidity. After that the termites used in the assays were collected, the nest was returned to the collection place. M. oleifera seeds were obtained from mature pods collected in the campus of the Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil. The seeds were washed with tap water followed by distilled water, pre-selected according to physic integrity (excluding malformed, punctured, cracked and/or curled seeds) and dried for 24 h at 28 °C. A sample of M. urundeuva heartwood from a tree of 6 m ~o, of height and 20 cm of diameter was collected in Caxias, Maranha Brazil. A voucher specimen is archived under number 054 at the Herbarium Aluisio Bittencourt, Centro de Estudos Superiores de Caxias ~o, Brazil. (CESC), Universidade Estadual do Maranha Collection of termites, M. oleifera seeds and M. urundeuva heart~o da wood was authorized by the Instituto Chico Mendes de Conserva¸c a Biodiversidade from the Brazilian Ministry of the Environment (authorization 38690). The accesses were recorded (A4915DF) in the ~o do Patrimo ^nio Genetico e do Conheciin the Sistema Nacional de Gesta mento Tradicional Associado (SisGen).
Hemagglutinating activity (HA) assay was carried out in microtiter plates (Kartell S.P.A., Italy) according to Paiva and Coelho (1992) using suspension (2.5% v/v) of rabbit erythrocytes previously treated with glutaraldehyde (Bing, Weyand, and Stavistsky, 1967). The sample (50 mL) was serially two-fold diluted in 0.15 M NaCl in a row of a 96well microplate and then 50 mL of the erythrocyte suspension were added to each well. Erythrocytes incubated only in 0.15 M NaCl were used as control. The HA was defined as the reciprocal of the highest dilution of the sample promoting full agglutination of erythrocytes. ME and SE were evaluated for protein concentration according to Lowry, Rosebrough, Farr, and Randal (1951) using a standard curve of bovine serum albumin (31.25 500 mg/mL). The Ethics Committee on Animal Use of the Universidade Federal de Pernambuco approved the erythrocytes collection method (process 23076.033782/2015-70). 2.6. Treatment of M. oleifera seeds with the extracts and infestation with N. corniger The seeds previously selected as described in Section 2.1 were divided using the manual method according to the instructions of the Brazilian Ministry of Agriculture, Animal Husbandry and Food Supply (2009). In each plastic vessel, 10 g of seeds were placed and sprayed (2 mL) once
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
ARTICLE IN PRESS
JID: SAJB
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
3
solution containing the exudate was evaluated using conductivimeter ~o Ltda., Sa ~o Paulo, Brazil). Four (Marte Balan¸c as e Aparelhos de Precisa independent experiments were performed in triplicate.
time daily (for 5 days) with SE or ME (SSE and SME treatments, respectively). Next, 16 workers and 4 soldiers of N. corniger were added to each vessel, which was covered by nonwoven fabric allowing the equilibrium between the water content of seeds and the relative humidity and temperature of the storing environment. The bioassays were maintained during 22 days at 28 °C and the number of live insects was recorded 7 and 22 days after the assay beginning. At the end of the experiment, the seeds were weighed and evaluated for the damages caused by infestation level as described in the next sections. Two negative control assays were performed: S control, containing only seeds without treatment and not infested by termites; and ST control, containing seeds without treatment but infested by termites. Assays were also performed spraying (2 mL) 1% sodium hypochlorite on seeds (SSH treatment; positive control). Four independent experiments were performed in triplicate.
Five seeds from controls and each treatment were equidistantly distributed on a filter paper sheet (10 cm £ 10 cm) moistened with sterile distilled water (1 mL) in a petri plate. The plates were incubated in a bio-oxygen demand (BOD) chamber at 20 °C for 4 days. After this period, the seeds were individually examined on stereomicroscope. The detected fungi were isolated and identification was performed according to morphologic characteristics of the mycelia. Four independent experiments were performed in triplicate.
2.7. Evaluation of damages caused by N. corniger
2.9. Statistical analysis
Seeds from controls and treatments with SE and ME were analyzed for presence of perforations and cracks made by termites using a stereomicroscope (Nikon SMZ745T; Nikon Corporation, Tokyo, Japan) and photographed using a digital camera (Moticam 5, 5.0 MP; Motic Instruments Inc., Richmond, Canada). Seed vigor was evaluated by measuring the electric conductivity in seed exudates according to Loeffler, Tekrony, and Egli (1988). This assay evaluated the release of electrolytes from seeds in the course of imbibition of them in distilled water. Fortyfive seeds from each treatment were placed in plastic vessel containing 100 mL of distilled water. The seeds remained immerse during 24 h at 25 °C. Next, the seeds were withdrawn and electric conductivity in the
The data were analyzed by the Student’s t-test (significance level of p < 0.05) using the software MicrocalTM Origin 6.0.
2.8. Evaluation of seed sanity
3. Results and discussion NMR spectroscopy has been used to assess a wide array of metabolites and to identify the maximum number of compounds in a complex mixture (Emwas et al., 2019). The 1H NMR spectra of SE and ME showed signals in aromatic and aliphatic regions. However, the aromatic signals attributed to phenolic compounds at d 5.60 7.01 were more expressive in SE compared to ME (Figs. 1A and 1B). In addition,
Fig. 1. Representative 1H NMR 400 MHz spectra (region between 0 10 ppm) of saline (A) and methanolic (B) extracts from Myracrodruon urundeuva heartwood. Extracts were analyzed in solution.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
ARTICLE IN PRESS
JID: SAJB 4
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Table 1 Survival of Nasutitermes corniger infesting Moringa oleifera seeds treated or not with methanolic (ME) or saline (SE) extracts from Myracrodruon urundeuva heartwood. Treatment
Live insects (%) Days after infestation beginning
ST control SME SSE SSH
Workers Soldiers Workers Soldiers Workers Soldiers Workers Soldiers
0 100 100 100 100 100 100 100 100
7 50 § 4.61 50 § 18.4 0.0 0.0 0.0 0.0 0.0 0.0
22 0.0 0.0
ST control: seeds not treated but infested by termites. SME and SSE: seeds treated with ME and SE, respectively, and infested by termites. SSH: seeds treated with 1% sodium hypochlorite.
metabolic alterations in the aliphatic region were observed, mainly due the singlets at d 2.35, 3.19 and 3.50, relative to methyl hydrogens present only in ME. Modifications in region at 0.90 1.30 characteristic of methyl groups were showed in both extracts. Finally, the singlet at d 1.77 attributed to acetate was revealed, but more intense in SE (Fig. 1A). These signals can be associated with the presence of carbohydrates, organic acids and amino acids in these extracts. The extraction procedure using methanol yielded 76 mg of phenolic compounds. The presence of proteins and HA were not detected in ME. On the other hand, SE showed low phenol content (10 mg), high protein concentration (20 mg/mL) and presence of lectin (HA of 32,768). The results obtained here are in according to those reported by S a et al. (2009a) and confirms ME as a lectin-free preparation richer in phenolic compounds and SE as a protein preparation with lectin (MuHL) activity and poorer than ME in phenols. Since it has been reported that these constituents (both lectin and phenolic compounds) can be involved in the defense mechanisms of the heart et al., 2008, 2009a), we wood against attack by termites and fungi (Sa investigated the potential of ME and SE to combat biodeterioration of M. oleifera seeds. Table 1 shows the survival rates of termites from SSE, SME, ST control, and SSH treatments. In SSE, SME, and SSH treatments, all termites were dead 7 days after infestation while in ST control the mortality rate of workers and soldiers was 50§0.0% after 7 days. These results et al., 2009a) was preindicate that termiticidal activity of SE (Sa served in the experimental conditions used in the infestation assay. These authors showed that the heartwood lectin (MuHL) act as active principle of SE and the hemagglutinating activity detected in the
extract used here assures the lectin presence. On the other hand, ME was reported to be non-termiticidal to N. corniger but a repellent et al., 2009a). agent (Sa The analysis of M. oleifera seeds under stereomicroscope reveal the presence of perforations made by termites in seeds treated with ME (Fig. 2B) and seeds from ST control. On the other hand, no perforations or cracks were observed in seeds treated with SE (Fig. 2A), which showed the same aspect of seeds in S control, without alterations in size, color and weight. Electric conductivity measurements were performed aiming to determine if the termites damaged seed vigor and integrity. High exudate conductivity is related with disorganization level of cell membrane systems and thus impairment of the physiological potential (vigor) of the seed (Krzyzanowski, Vieira, and Fran¸c a Neto, 1999). Table 2 shows that the mean electric conductivity in the exudate released from seeds of S control (no infestation) was significantly (p < 0.05) lower than the values found in exudate from seeds from ST control (presence of termites), revealing that termites damaged the seed integrity. There was no significant difference (p > 0.05) between the conductivity in the exudate released from ST control seeds and those treated with ME, indicating that this extract did not prevent the seed attack by termites although it was previ et al., 2009a). It was ously reported that it exerts repellent activity (Sa probable that the repellent effect of methanolic extract on the termites led to the death of all of them after 7 days due to blocking of feeding. However, this effect may have been a bit slowly so that termites could also damage the seeds for a time period before they feel repelled. Also, conductivity in the exudate from seeds of SSH treatment was not significantly (p < 0.05) different of that determined in control exudate and thus the broadly used disinfectant sodium hypochlorite did not prevent termite attack. Distinctly, the conductivity in exudate released from seeds previously treated with SE was similar (p < 0.05) to that of exudate from seeds of S control (Table 2) revealing that the saline extract, containing lectin, not only promoted the death of termites but preserved the integrity of seed cell membranes. This protection was probably conferred by a rapid termiticidal action of the extract. The seeds were also evaluated for fungi presence in order to evaluate if the M. urundeuva heartwood extracts would also interfere on fungal development in addition to cause termite mortality. Fungal growth was detected in seeds from S and ST controls as well as in seeds from SME and SSE assays. Thus, the extracts failed in promote improvement in the sanity of seeds. The main fungi identified in the seeds from each treatment were from Fusarium and Penicillium genera. Trichoderma harzianum was detected in seeds from controls and SME treatment. This species is mainly present in soil and able to
Fig. 2. Aspects of Moringa oleifera seeds treated with saline (A) and methanolic (B) extracts from Myracrodruon urundeuva heartwood after infestation with Nasutitermes corniger. Magnification: 5£.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
JID: SAJB
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6 Table 2 Electric conductivity in exudates from Moringa oleifera seeds treated or not with saline (SE) and methanolic (ME) extracts from Myracrodruon urundeuva heartwood. Treatment
Conductivity (ms/cm/g)
S control ST control SME SSE SSH
0.43 § 0.16 a 0.91 § 0.21 b 1.17 § 0.43 c 0.27 § 0.14 a 2.17 § 0.01 c
S control: seeds not treated and not infested by termites. ST control: seeds not treated but infested by termites. SME and SSE: seeds treated with ME and SE, respectively, and infested by termites. SSH: seeds treated with 1% sodium hypochlorite. Different letters indicate significant (p<0.05) differences.
degrade cell walls of pathogenic fungi and thus is used as a fungicide (Elad, Zimand, Zaqs, Zuriel, and Chet, 1993). The presence of this species in M. oleifera seeds may be positive for them in order to prevent a great infection and the absence of antifungal effect of ME can be interesting by this point of view. On the other hand, this fungus was not identified in seeds from SSE assay and thus antifungal activity of SE (S a et al., 2009a) may have affected this species. 4. Conclusion Saline extract from M. urundeuva heartwood, containing lectin, phenols, carbohydrate, organic acids and amino acids conferred protection against N. corniger attack when sprayed on M. oleifera seeds. On the other hand, the methanolic extract, with similar composition but lacking lectin, did not protect the seeds. The saline and methanolic heartwood extracts did not improve seed sanity in regard to presence of Fusarium spp. and Penicillium spp. The results indicate that the lectin present in the saline extract is the key component in the prevention of moringa seeds against attack by termites. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors express their gratitude to the Conselho Nacional de gico (CNPq) for financial support Desenvolvimento Científico e Tecnolo (472546/2012-0; 407192/2018-2) research grants and fellowship (JRGSA, LCBBC, CMSM, THN, PMGP). We are also grateful to the ~o de Amparo a Ci^encia e Tecnologia do Estado de Pernambuco Funda¸c a (FACEPE; APQ-0661-2.08/15) and the Brazilian Ministry of Science, Technology and Innovation (MCTI; 01200.003711/2011-11) for financial support. LLSS would like to thank CNPq for Scientific Initiation scholarship (PIBIC-CNPq-UFPE). IMS Araujo would like to thank the ~o de Aperfei¸c oamento de Pessoal de Nível Superior (CAPES) Coordena¸c a for graduate scholarship. Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.10.013. References Bing, D.H., Weyand, J.G.M., Stavistsky, A.B., 1967. Hemagglutination with aldehydefixed erythrocytes for assay of antigens and antibodies. Proceedings of the Society for Experimental Biology and Medicine 124, 1166–1170. Brazilian Ministry of Agriculture, Animal Husbandry and Food Supply, 2009. Regras lise de sementes. Brazilian Ministry of Agriculture, Animal Husbandry and para ana Food Supply, Brasília, Brazil.
~o, T.H., 2017. PhyCamaroti, J.R.S.L., Oliveira, A.P.S., Paiva, P.M.G., Pontual, E.V., Napolea toinsecticides for controlling pests and mosquito vectors of diseases. In: Green, V. (Ed.), Biocontrol Agents: Types, Applications and Research Insights. Nova Science Publishers Inc, New York, pp. 147–188. ~o, T.H., Gomes, F.S., Ferreira, R.S., Zingali, R.B., et al., Coelho, J.S., Santos, N.D.L., Napolea 2009. Effect of Moringa oleifera lectin on development and mortality of Aedes aegypti larvae. Chemosphere 77, 934–938. Creech, C.F. (.2012). Effects of planting date, harvest date, and environmental conditions on germination of forage kochia accessions. All Graduate Theses and Dissertations, Paper 1264. Available in: http://digitalcommons.usu.edu/etd/1264. Danielson, S., Wright, R., Hein, G., & Peters, L. (1991). G91-1023 Insects that attack seeds and seedlings of field crops. Historical Materials from University of Nebraska-Lincoln Extension 1099. Elad, Y., Zimand, G., Zaqs, Y., Zuriel, S., Chet, I., 1993. Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions. Plant Pathology 42, 324–332. Emwas, A.-.H., Roy, R., McKay, R.T., Tenori, L., Saccenti, E., Gowda, G.A.N., et al., 2019. NMR spectroscopy for metabolomics research. Metabolites 9, 123. ~o, T.H., Santos, A.F.S., Sa, R.A., Carneiro-da-Cunha, M.G., Morais, Ferreira, R.S., Napolea M.M.C., et al., 2011 Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera. Letters in Applied Microbiology53, 186 192. Krzyzanowski, F.C., Vieira, R.D., Fran¸c a Neto, J.B., 1999. Vigor de sementes: conceitos e testes. ABRATES, Londrina, pp. 1.1–1.21. Loeffler, T.M., Tekrony, D.M.&, Egli, D.B., 1988. The bulk conductivity test as an indicator of soy bean seed quality. Journal of Seed Technology 12, 37–53. Lowry, D.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Luz, L.A., Rossato, F.A., Costa, R.A.P., Napole~ao, T.H., Paiva, P.M.G., Coelho, L.C.B.B., 2017. Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells. Toxicology in Vitro 44, 94–99. ~o, T.H., Paiva, P.M.G., Coelho, L.C.B.B., Bezerra, A.C.D.S., Medeiros, M.L.S., Moura, M.C., Napolea et al., 2018. Nematicidal activity of a water soluble lectin from seeds of Moringa oleifera. International Journal of Biological Macromolecules 108, 782–789. Memon, Q.U.A., Wagan, S.A., Chunyu, D., Shuangxi, X., Jingdong, L., Damalas, C.A., 2019. Health problems from pesticide exposure and personal protective measures among women cotton workers in southern Pakistan. Science of the Total Environment 685, 659–666. €rndly, E., Reyes-Sanchez, N., Salmero n-Miranda, F., Halling, M., Mendieta-Araica, B., Spo 2013. Biomass production and chemical composition of Moringa oleifera under different planting densities and levels of nitrogen fertilization. Agroforestry Systems 87, 81–92. Morais, S.A.L., Nascimento, E.A., Queiroz, C.R.A.A., Pilo-Veloso, D., Drumond, M.G., 1999. Studies on polyphenols and lignin of Astronium urundeuva wood. Journal of Brazilian Chemical Society 10, 447–452. ~o, T.H., Coriolano, M.C., Paiva, P.M.G., Figueiredo, R.C.B.Q., Moura, M.C., Napolea Coelho, L.C.B.B., 2015. Water-soluble Moringa oleifera lectin interferes with growth, survival and cell permeability of corrosive and pathogenic bacteria. Journal of Applied Microbiology 119, 666–676. ~o, T.H., Primon-Barros, M., Xavier, A.S., Carneiro, N.P., Moura, M.C., Trentin, D.S., Napolea et al., 2017. Multi-effect of the water-soluble Moringa oleifera lectin against Serratia marcescens and Bacillus sp.: antibacterial, antibiofilm and anti-adhesive properties. Journal of Applied Microbiology 123, 861–874. Napole~ao, T.H., Albuquerque, L.P., Santos, N.D.L., Nova, I.C.V., Lima, T.A., Paiva, P.M.G., Pontual, E.V., 2019. Insect midgut structures and molecules as targets of plantderived protease inhibitors and lectins. Pest Management Science 75, 1212–1222. https://doi.org/10.1002/ps.5233. Nishi, L., Vieira, A.M.S., Vieira, M.F., Bongiovani, M., Camacho, F.P., Bergamasco, R., 2012. Hybrid process of coagulation/flocculation with moringa oleifera followed by ultrafiltration to remove Microcystis sp. cells from water supply. Procedia Engineering 42, 865–872. Paiva, P.M.G., Coelho, L.C.B.B., 1992. Purification and partial characterization of two lectin isoforms from Cratylia mollis mart. (camaratu bean). Applied Biochemistry and Biotechnology 36, 113–118. Paiva, P.M.G., Santana, G.M.S., Souza, I.F.A.C., Albuquerque, L.P., Agra-Neto, A.C., Albuquerque, A.C., et al., 2011. Effect of lectins from Opuntia ficus indica cladodes and Moringa oleifera seeds on survival of Nasutitermes corniger. International Biodeterioration and Biodegradation 65, 982–989. Pretty, J., Bharucha, Z.P., 2015. Integrated pest management for sustainable intensification of agriculture in Asia and Africa. Insects 6, 152–182. Rajjou, L., Duval, M., Gallardo, K., Catusse, J., Bally, J., Job, C., et al., 2012. Seed germination and vigor. Annual Review of Plant Biology 63, 507–533. Rashid, U., Anwar, F., Moser, B.R., Knothe, G., 2008. Moringa oleifera oil: a possible source of biodiesel. Bioresource Technology 99, 8175–8179. , R.A., Argolo, A.C.C., Napolea ~o, T.H., Gomes, F.S., Santos, N.D.L., Melo, C.M.L., et al., Sa 2009a. Antioxidant, Fusarium growth inhibition and Nasutitermes corniger repellent activities of secondary metabolites from Myracrodruon urundeuva heartwood. International Biodeterioration and Biodegradation 63, 470–477. , R.A., Gomes, F.S., Napolea ~o, T.H., Santos, N.D.L., Melo, C.M.L., Gusm~ao, N.B., et al., 2009b. Sa Antibacterial and antifungal activities of Myracrodruon urundeuva heartwood. Wood Science and Technology 43, 85–95. , R.A., Napole~ao, T.H., Santos, N.D.L., Gomes, F.S., Albuquerque, A.C., Xavier, H.S., et al., Sa 2008. Induction of mortality on Nasutitermes corniger (Isoptera, termitidae) by Myracrodruon urundeuva heartwood lectin. International Biodeterioration and Biodegradation 62, 460–464. Santos, A.F., Carrijo, T.F., Cancello, E.M., Castro, A.C.M., 2017. Phylogeography of Nasutitermes corniger (Isoptera: termitidae) in the neotropical region. BMC Evolutionary Biology 17, 230.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
5
JID: SAJB 6
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Santos, A.F.S., Luz, L.A., Argolo, A.C.C., Teixeira, J.A., Paiva, P.M.G., Coelho, L.C.B.B., 2009. Isolation of a seed coagulant Moringa oleifera lectin. Process Biochemistry 44, 504–508. Santos, N.D.L., Moura, K.S., Napole~ao, T.H., Santos, G.K.N., Coelho, L.C.B.B., Navarro, D.M.A.F., et al., 2012. Oviposition-stimulant and ovicidal activities of Moringa oleifera lectin on Aedes aegypti. PLoS ONE 7, e44840. ~o, T.H., Benevides, C.A., Albuquerque, L.P., Pontual, E.V., Santos, N.D.L., Napolea Oliveira, A.P.S., et al., 2019. Effect of gamma irradiation of Moringa oleifera seed lectin on its larvicidal, ovicidal, and oviposition-stimulant activities against Aedes aegypti. South African Journal of Botany. https://doi.org/10.1016/j.sajb.2018.05.001.
~o, K.S., Napolea ~o, T.H., Trindade, P.B., Pinto, M.R., Coelho, L.C.B.B., Santos, N.D.L., Paixa et al., 2014. Evaluation of Moringa oleifera seed lectin in traps for the capture of Aedes aegypti eggs and adults under semi-field conditions. Parasitology Research 113, 1837–1842. Sauveur, A.S., Broin, M., 2010. Growing and processing Moringa leaves. Moringa Association of Ghana (MAG), Accra, Ghana. ttir, G.R., Sengupta, M.E., Keraita, B., Olsen, A., Boateng, O.K., Thamsborg, S.M., Palsdo et al., 2012. Use of Moringa oleifera seed extracts to reduce helminth egg numbers and turbidity in irrigation water. Water Research 46, 3646–3656.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
JID: SAJB
[m5G;November 1, 2019;16:30]
South African Journal of Botany 000 (2019) 1 6
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity L.L.S. Silvaa, I.M.S. Araujoa,b, P.M.O. Costac, A.P.S. Oliveiraa, A.P. Oliveirad, A.D.C. Santosd, ~oa, L.M. Dutrad, J.R.G.S. Almeidad, L.C.B.B. Coelhoa, C.M. Souza-Mottac, T.H. Napolea a, P.M.G. Paiva * Departamento de Bioquímica, Centro de Bioci^encias, Universidade Federal de Pernambuco, Recife, PE 50670-420, Brazil ria, Embrapa Agroindustria Empresa Brasileira de Pesquisa Agropecua Tropical, Fortaleza-CE, 60511-110, Brazil Departamento de Micologia, Centro de Bioci^encias, Universidade Federal de Pernambuco, Recife, PE 50670-420, Brazil d ~o Francisco, 56304-205 Petrolina, Pernambuco, Brazil Nucleo de Estudos e Pesquisas de Plantas Medicinais (NEPLAME), Universidade Federal do Vale do Sa a
b c
A R T I C L E
I N F O
Article History: Received 27 March 2019 Revised 17 September 2019 Accepted 12 October 2019 Available online xxx Keywords: Moringa oleifera Myracrodruon urundeuva Seed protection Termite infestation Phytopathogenic fungi
A B S T R A C T
Moringa oleifera seeds contain several biotechnologically-relevant molecules but are susceptible to termite attack. On the other hand, Myracrodruon urundeuva is strongly resistant to deterioration and it was previously reported the termiticidal activity of preparations from its heartwood against Nasutitermes corniger. This work evaluated whether methanolic (ME) and saline (SE) extracts from M. urundeuva heartwood would be able to protect M. oleifera seeds against N. corniger and improve seed sanity. Nuclear magnetic resonance (NMR) spectroscopy analysis was used to identify the chemical profile of the extracts. Seeds were sprayed for 5 days with ME (SME), SE (SSE), or positive control 1% (v/v) sodium hypochlorite (SSH) and then infested by workers and soldiers. Controls were also performed using seeds not treated with the extracts and infested (ST control) or not (S control). After death of all termites, the seeds were analyzed for vigor (by measurements of electric conductivity in the exudates) and sanity. The representative 1H NMR spectra showed phenolic compounds among the main components of the extracts. Mortality rate of workers and soldiers after 7 days was 100% in SME, SSE, and SSH while it was 50% in ST control. Perforations made by termites were observed in seeds from ST control and SME treatment but not in seeds treated with SE. Analysis of vigor revealed that seeds from control ST or those treated with ME or sodium hypochlorite were damaged by termites while SE promoted protection against N. corniger. Fusarium spp., Penicillium spp. and Trichoderma harziamum were identified in treatments and controls, revealing that the extracts did not improve seed sanity. In conclusion, SE from M. urundeuva heartwood conferred protection against N. corniger attack when sprayed on M. oleifera seeds and this effect can be related with lectin presence. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Seed germination and vigor depend on intrinsic factors, such as quality of the messenger RNAs stored during embryo maturation, proteostasis and DNA integrity, as well as on environmental factors and the conditions employed for harvesting, drying, and processing (Creech, 2012; Rajjou et al., 2012). During storage, seeds may also be affected by the attack of microorganisms (mainly fungi) and insects (such as termites, beetles, maggots, ants and wireworms) resulting in impairment of nutritional value, weight and germination power (Danielson et al., 1991). *Corresponding author. E-mail address: [email protected] (P.M.G. Paiva).
It is increasing the interest on the use of alternative techniques for controlling seed pests due to the high toxicity of antifungals and insecticides currently used. In the last years, studies focusing on natural products (e.g. plant extracts, powders, essential oils, lectins, enzyme inhibitors) for treating stored crops have been intensified aiming the protection of seeds and grains, with lower risks of intoxication to farmers and consumers (Camaroti, Oliveira, Paiva, Pontual, and Napole~ao, 2017; Memon et al., 2019; Pretty and Bharucha, 2015). Moringa oleifera (Moringaceae) is a pantropical tree whose seeds are source of compounds with a broad biotechnological potential, ~o such as in water treatment and production of cosmetics (Napolea et al., 2019). Its seeds contain coagulant molecules that are able to reduce turbidity, bacteria and cyanobacteria contamination, and helminth egg number in water and wastewaters (Ferreira et al., 2011;
https://doi.org/10.1016/j.sajb.2019.10.013 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
JID: SAJB 2
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Nishi et al., 2012; Santos et al., 2009; Sengupta et al., 2012). Studies showed that lectins from these seeds have larvicidal, ovicidal and oviposition-stimulant activity on Aedes aegypti (Coelho et al., 2009; Santos et al., 2012;Santos et al., 2014, 2019), antibacterial activity (Moura et al., 2015, 2017), nematicidal activity (Medeiros et al., 2018) and cytotoxic effects on cancer cells (Luz et al., 2017). In addition, the M. oleifera seeds have been studied as source of oil for biodiesel production (Rashid, Anwar, Moser, and Knothe, 2008). Although M. oleifera tree is considered to be resistant to most of pests, it is susceptible to the attack of fungi, ants and mainly termites € rndly, Reyes-Sa nchez, Salmero n-Miranda, and (Mendieta-Araica, Spo Halling, 2013). The protection of seeds and seedlings against termite attack is strongly recommended (Sauveur and Broin, 2010). Study showed that the lectins cMoL and WSMoL present in M. oleifera seeds were able to kill Nasutitermes corniger workers and soldiers only at high concentrations and thus these molecules probably fail in conferring resistance to the seeds against termite attack (Paiva et al., 2011). The N. corniger is a soil-feeding and wood-damaging termite species, being the most widely distributed of the genus, and considered a successful invasive pest in urban areas, causing considerable economic impact (Santos, Carrijo, Cancello, and Castro, 2017). Myracrodruon urundeuva (Anacardiaceae) is a timber tree known for its use in Brazilian building industry and medicinal properties. Its heartwood is strongly resistant to termite and phytopathogenic fungal attack and the biochemical basis of this resistance has been investigated. A chitin-binding lectin (MuHL) with termiticidal activity on N. corniger workers and soldiers was purified from saline extract of M. urundeuva heartwood; MuHL and the extract did not show repel et al., 2008, 2009a). On the other hand, methanolic lent activity (Sa extract from heartwood did not kill workers and soldiers but showed repellent effect on N. corniger (Sa et al., 2009a). Both saline and methanolic extracts, as well as MuHL, showed antifungal activity against et al., 2009a, 2009b). Fusarium species (Sa In this work we evaluated the efficacy of the treatments of M. oleifera seeds with saline (SE) and methanolic (ME) extracts from M. urundeuva in order to confer protection against damages caused by N. corniger on seeds and to improve seed sanity in regard to fungal presence.
2. Materials and methods
2.2. Methanolic and saline extracts from M. urundeuva heartwood The M. urundeuva heartwood was powdered (40 mesh). Methanolic extract (ME) was obtained by soaking 3 g of powdered heartwood in 10 mL of methanol; the mixture was homogenized at 25 °C for 3 h under magnetic stirring and afterwards it was filtered on filter et al., 2009a). paper and dried on a rotary evaporator to yield ME (Sa Saline extract (SE) was obtained by homogenization of the powdered heartwood (10 g) in 0.15 M NaCl (100 mL) for 16 h at 4 °C under magnetic stirring. After filtration, the homogenate was filtered through gauze and filter paper, centrifuged (3000 g, 15 min, 4 °C), and the et al., 2009a). supernatant corresponded to SE (Sa 2.3. Nuclear magnetic resonance (NMR) spectroscopy analysis For NMR analysis, 25 mg of each extract was solubilized in 500 mL of deuterated solvent. 1D and 2D NMR data were acquired at 298 K in D2O on a Bruker AVANCE III 400 NMR spectrometer (Bruker AXS, Inc., Madison, WI, USA) operating at 9.4 T, observing 1H and 13C nuclei at 400 and 100 MHz, respectively. The NMR spectrometer was equipped with a 5-mm direct detection probe (BBO) with z-gradient. Besides, one-bond heteronuclear (1H-13C) and homonuclear (1H-1H) correlations were assessed by heteronuclear single quantum coherence (HSQC) and correlation (COSY) spectroscopies. 1H and 13C NMR chemical shifts were given in ppm referenced to TMSP-d4 signal at 0.00 ppm, and the coupling constants (J) in Hz. The experiments were performed on a single replicate and the obtained spectra were processed in Topspin software version 3.2.0 (Bruker AXS). 2.4. Total phenol content Total phenol content of ME and SE was determined by the Folin-Ciocalteu method based on the reduction of phosphomolybdic-phosphotungstic reagent by phenols in alkaline solution (Morais, Nascimento, Queiroz, Pilo-Veloso, and Drumond, 1999). Folin-Ciocalteu reagent (1:10 solution in distilled water; 2.5 mL) and sodium carbonate (75 g/L solution in distilled water; 2 mL) were added to SE or ME (0.5 mg/mL solution in distilled water; 0.5 mL). The mixtures were kept at 50 °C for 5 min. After cooling for 30 min, the absorbance was measured at 760 nm. The phenol content was determined using a standard calibration curve of tannic acid (9.6 48 mg/mL in distilled water; 0.5 mL).
2.1. Insects and plant materials 2.5. Hemagglutinating activity and protein content Nests of N. corniger were collected from a fragment of Atlantic Forest at the campus of the Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil. The colonies were removed from the trunk of a tree with a machete, transferred into black plastic bags to the laboratory, and maintained at 28 °C and 75% relative humidity. After that the termites used in the assays were collected, the nest was returned to the collection place. M. oleifera seeds were obtained from mature pods collected in the campus of the Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil. The seeds were washed with tap water followed by distilled water, pre-selected according to physic integrity (excluding malformed, punctured, cracked and/or curled seeds) and dried for 24 h at 28 °C. A sample of M. urundeuva heartwood from a tree of 6 m ~o, of height and 20 cm of diameter was collected in Caxias, Maranha Brazil. A voucher specimen is archived under number 054 at the Herbarium Aluisio Bittencourt, Centro de Estudos Superiores de Caxias ~o, Brazil. (CESC), Universidade Estadual do Maranha Collection of termites, M. oleifera seeds and M. urundeuva heart~o da wood was authorized by the Instituto Chico Mendes de Conserva¸c a Biodiversidade from the Brazilian Ministry of the Environment (authorization 38690). The accesses were recorded (A4915DF) in the ~o do Patrimo ^nio Genetico e do Conheciin the Sistema Nacional de Gesta mento Tradicional Associado (SisGen).
Hemagglutinating activity (HA) assay was carried out in microtiter plates (Kartell S.P.A., Italy) according to Paiva and Coelho (1992) using suspension (2.5% v/v) of rabbit erythrocytes previously treated with glutaraldehyde (Bing, Weyand, and Stavistsky, 1967). The sample (50 mL) was serially two-fold diluted in 0.15 M NaCl in a row of a 96well microplate and then 50 mL of the erythrocyte suspension were added to each well. Erythrocytes incubated only in 0.15 M NaCl were used as control. The HA was defined as the reciprocal of the highest dilution of the sample promoting full agglutination of erythrocytes. ME and SE were evaluated for protein concentration according to Lowry, Rosebrough, Farr, and Randal (1951) using a standard curve of bovine serum albumin (31.25 500 mg/mL). The Ethics Committee on Animal Use of the Universidade Federal de Pernambuco approved the erythrocytes collection method (process 23076.033782/2015-70). 2.6. Treatment of M. oleifera seeds with the extracts and infestation with N. corniger The seeds previously selected as described in Section 2.1 were divided using the manual method according to the instructions of the Brazilian Ministry of Agriculture, Animal Husbandry and Food Supply (2009). In each plastic vessel, 10 g of seeds were placed and sprayed (2 mL) once
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
ARTICLE IN PRESS
JID: SAJB
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
3
solution containing the exudate was evaluated using conductivimeter ~o Ltda., Sa ~o Paulo, Brazil). Four (Marte Balan¸c as e Aparelhos de Precisa independent experiments were performed in triplicate.
time daily (for 5 days) with SE or ME (SSE and SME treatments, respectively). Next, 16 workers and 4 soldiers of N. corniger were added to each vessel, which was covered by nonwoven fabric allowing the equilibrium between the water content of seeds and the relative humidity and temperature of the storing environment. The bioassays were maintained during 22 days at 28 °C and the number of live insects was recorded 7 and 22 days after the assay beginning. At the end of the experiment, the seeds were weighed and evaluated for the damages caused by infestation level as described in the next sections. Two negative control assays were performed: S control, containing only seeds without treatment and not infested by termites; and ST control, containing seeds without treatment but infested by termites. Assays were also performed spraying (2 mL) 1% sodium hypochlorite on seeds (SSH treatment; positive control). Four independent experiments were performed in triplicate.
Five seeds from controls and each treatment were equidistantly distributed on a filter paper sheet (10 cm £ 10 cm) moistened with sterile distilled water (1 mL) in a petri plate. The plates were incubated in a bio-oxygen demand (BOD) chamber at 20 °C for 4 days. After this period, the seeds were individually examined on stereomicroscope. The detected fungi were isolated and identification was performed according to morphologic characteristics of the mycelia. Four independent experiments were performed in triplicate.
2.7. Evaluation of damages caused by N. corniger
2.9. Statistical analysis
Seeds from controls and treatments with SE and ME were analyzed for presence of perforations and cracks made by termites using a stereomicroscope (Nikon SMZ745T; Nikon Corporation, Tokyo, Japan) and photographed using a digital camera (Moticam 5, 5.0 MP; Motic Instruments Inc., Richmond, Canada). Seed vigor was evaluated by measuring the electric conductivity in seed exudates according to Loeffler, Tekrony, and Egli (1988). This assay evaluated the release of electrolytes from seeds in the course of imbibition of them in distilled water. Fortyfive seeds from each treatment were placed in plastic vessel containing 100 mL of distilled water. The seeds remained immerse during 24 h at 25 °C. Next, the seeds were withdrawn and electric conductivity in the
The data were analyzed by the Student’s t-test (significance level of p < 0.05) using the software MicrocalTM Origin 6.0.
2.8. Evaluation of seed sanity
3. Results and discussion NMR spectroscopy has been used to assess a wide array of metabolites and to identify the maximum number of compounds in a complex mixture (Emwas et al., 2019). The 1H NMR spectra of SE and ME showed signals in aromatic and aliphatic regions. However, the aromatic signals attributed to phenolic compounds at d 5.60 7.01 were more expressive in SE compared to ME (Figs. 1A and 1B). In addition,
Fig. 1. Representative 1H NMR 400 MHz spectra (region between 0 10 ppm) of saline (A) and methanolic (B) extracts from Myracrodruon urundeuva heartwood. Extracts were analyzed in solution.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
ARTICLE IN PRESS
JID: SAJB 4
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Table 1 Survival of Nasutitermes corniger infesting Moringa oleifera seeds treated or not with methanolic (ME) or saline (SE) extracts from Myracrodruon urundeuva heartwood. Treatment
Live insects (%) Days after infestation beginning
ST control SME SSE SSH
Workers Soldiers Workers Soldiers Workers Soldiers Workers Soldiers
0 100 100 100 100 100 100 100 100
7 50 § 4.61 50 § 18.4 0.0 0.0 0.0 0.0 0.0 0.0
22 0.0 0.0
ST control: seeds not treated but infested by termites. SME and SSE: seeds treated with ME and SE, respectively, and infested by termites. SSH: seeds treated with 1% sodium hypochlorite.
metabolic alterations in the aliphatic region were observed, mainly due the singlets at d 2.35, 3.19 and 3.50, relative to methyl hydrogens present only in ME. Modifications in region at 0.90 1.30 characteristic of methyl groups were showed in both extracts. Finally, the singlet at d 1.77 attributed to acetate was revealed, but more intense in SE (Fig. 1A). These signals can be associated with the presence of carbohydrates, organic acids and amino acids in these extracts. The extraction procedure using methanol yielded 76 mg of phenolic compounds. The presence of proteins and HA were not detected in ME. On the other hand, SE showed low phenol content (10 mg), high protein concentration (20 mg/mL) and presence of lectin (HA of 32,768). The results obtained here are in according to those reported by S a et al. (2009a) and confirms ME as a lectin-free preparation richer in phenolic compounds and SE as a protein preparation with lectin (MuHL) activity and poorer than ME in phenols. Since it has been reported that these constituents (both lectin and phenolic compounds) can be involved in the defense mechanisms of the heart et al., 2008, 2009a), we wood against attack by termites and fungi (Sa investigated the potential of ME and SE to combat biodeterioration of M. oleifera seeds. Table 1 shows the survival rates of termites from SSE, SME, ST control, and SSH treatments. In SSE, SME, and SSH treatments, all termites were dead 7 days after infestation while in ST control the mortality rate of workers and soldiers was 50§0.0% after 7 days. These results et al., 2009a) was preindicate that termiticidal activity of SE (Sa served in the experimental conditions used in the infestation assay. These authors showed that the heartwood lectin (MuHL) act as active principle of SE and the hemagglutinating activity detected in the
extract used here assures the lectin presence. On the other hand, ME was reported to be non-termiticidal to N. corniger but a repellent et al., 2009a). agent (Sa The analysis of M. oleifera seeds under stereomicroscope reveal the presence of perforations made by termites in seeds treated with ME (Fig. 2B) and seeds from ST control. On the other hand, no perforations or cracks were observed in seeds treated with SE (Fig. 2A), which showed the same aspect of seeds in S control, without alterations in size, color and weight. Electric conductivity measurements were performed aiming to determine if the termites damaged seed vigor and integrity. High exudate conductivity is related with disorganization level of cell membrane systems and thus impairment of the physiological potential (vigor) of the seed (Krzyzanowski, Vieira, and Fran¸c a Neto, 1999). Table 2 shows that the mean electric conductivity in the exudate released from seeds of S control (no infestation) was significantly (p < 0.05) lower than the values found in exudate from seeds from ST control (presence of termites), revealing that termites damaged the seed integrity. There was no significant difference (p > 0.05) between the conductivity in the exudate released from ST control seeds and those treated with ME, indicating that this extract did not prevent the seed attack by termites although it was previ et al., 2009a). It was ously reported that it exerts repellent activity (Sa probable that the repellent effect of methanolic extract on the termites led to the death of all of them after 7 days due to blocking of feeding. However, this effect may have been a bit slowly so that termites could also damage the seeds for a time period before they feel repelled. Also, conductivity in the exudate from seeds of SSH treatment was not significantly (p < 0.05) different of that determined in control exudate and thus the broadly used disinfectant sodium hypochlorite did not prevent termite attack. Distinctly, the conductivity in exudate released from seeds previously treated with SE was similar (p < 0.05) to that of exudate from seeds of S control (Table 2) revealing that the saline extract, containing lectin, not only promoted the death of termites but preserved the integrity of seed cell membranes. This protection was probably conferred by a rapid termiticidal action of the extract. The seeds were also evaluated for fungi presence in order to evaluate if the M. urundeuva heartwood extracts would also interfere on fungal development in addition to cause termite mortality. Fungal growth was detected in seeds from S and ST controls as well as in seeds from SME and SSE assays. Thus, the extracts failed in promote improvement in the sanity of seeds. The main fungi identified in the seeds from each treatment were from Fusarium and Penicillium genera. Trichoderma harzianum was detected in seeds from controls and SME treatment. This species is mainly present in soil and able to
Fig. 2. Aspects of Moringa oleifera seeds treated with saline (A) and methanolic (B) extracts from Myracrodruon urundeuva heartwood after infestation with Nasutitermes corniger. Magnification: 5£.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
JID: SAJB
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6 Table 2 Electric conductivity in exudates from Moringa oleifera seeds treated or not with saline (SE) and methanolic (ME) extracts from Myracrodruon urundeuva heartwood. Treatment
Conductivity (ms/cm/g)
S control ST control SME SSE SSH
0.43 § 0.16 a 0.91 § 0.21 b 1.17 § 0.43 c 0.27 § 0.14 a 2.17 § 0.01 c
S control: seeds not treated and not infested by termites. ST control: seeds not treated but infested by termites. SME and SSE: seeds treated with ME and SE, respectively, and infested by termites. SSH: seeds treated with 1% sodium hypochlorite. Different letters indicate significant (p<0.05) differences.
degrade cell walls of pathogenic fungi and thus is used as a fungicide (Elad, Zimand, Zaqs, Zuriel, and Chet, 1993). The presence of this species in M. oleifera seeds may be positive for them in order to prevent a great infection and the absence of antifungal effect of ME can be interesting by this point of view. On the other hand, this fungus was not identified in seeds from SSE assay and thus antifungal activity of SE (S a et al., 2009a) may have affected this species. 4. Conclusion Saline extract from M. urundeuva heartwood, containing lectin, phenols, carbohydrate, organic acids and amino acids conferred protection against N. corniger attack when sprayed on M. oleifera seeds. On the other hand, the methanolic extract, with similar composition but lacking lectin, did not protect the seeds. The saline and methanolic heartwood extracts did not improve seed sanity in regard to presence of Fusarium spp. and Penicillium spp. The results indicate that the lectin present in the saline extract is the key component in the prevention of moringa seeds against attack by termites. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors express their gratitude to the Conselho Nacional de gico (CNPq) for financial support Desenvolvimento Científico e Tecnolo (472546/2012-0; 407192/2018-2) research grants and fellowship (JRGSA, LCBBC, CMSM, THN, PMGP). We are also grateful to the ~o de Amparo a Ci^encia e Tecnologia do Estado de Pernambuco Funda¸c a (FACEPE; APQ-0661-2.08/15) and the Brazilian Ministry of Science, Technology and Innovation (MCTI; 01200.003711/2011-11) for financial support. LLSS would like to thank CNPq for Scientific Initiation scholarship (PIBIC-CNPq-UFPE). IMS Araujo would like to thank the ~o de Aperfei¸c oamento de Pessoal de Nível Superior (CAPES) Coordena¸c a for graduate scholarship. Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.10.013. References Bing, D.H., Weyand, J.G.M., Stavistsky, A.B., 1967. Hemagglutination with aldehydefixed erythrocytes for assay of antigens and antibodies. Proceedings of the Society for Experimental Biology and Medicine 124, 1166–1170. Brazilian Ministry of Agriculture, Animal Husbandry and Food Supply, 2009. Regras lise de sementes. Brazilian Ministry of Agriculture, Animal Husbandry and para ana Food Supply, Brasília, Brazil.
~o, T.H., 2017. PhyCamaroti, J.R.S.L., Oliveira, A.P.S., Paiva, P.M.G., Pontual, E.V., Napolea toinsecticides for controlling pests and mosquito vectors of diseases. In: Green, V. (Ed.), Biocontrol Agents: Types, Applications and Research Insights. Nova Science Publishers Inc, New York, pp. 147–188. ~o, T.H., Gomes, F.S., Ferreira, R.S., Zingali, R.B., et al., Coelho, J.S., Santos, N.D.L., Napolea 2009. Effect of Moringa oleifera lectin on development and mortality of Aedes aegypti larvae. Chemosphere 77, 934–938. Creech, C.F. (.2012). Effects of planting date, harvest date, and environmental conditions on germination of forage kochia accessions. All Graduate Theses and Dissertations, Paper 1264. Available in: http://digitalcommons.usu.edu/etd/1264. Danielson, S., Wright, R., Hein, G., & Peters, L. (1991). G91-1023 Insects that attack seeds and seedlings of field crops. Historical Materials from University of Nebraska-Lincoln Extension 1099. Elad, Y., Zimand, G., Zaqs, Y., Zuriel, S., Chet, I., 1993. Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions. Plant Pathology 42, 324–332. Emwas, A.-.H., Roy, R., McKay, R.T., Tenori, L., Saccenti, E., Gowda, G.A.N., et al., 2019. NMR spectroscopy for metabolomics research. Metabolites 9, 123. ~o, T.H., Santos, A.F.S., Sa, R.A., Carneiro-da-Cunha, M.G., Morais, Ferreira, R.S., Napolea M.M.C., et al., 2011 Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera. Letters in Applied Microbiology53, 186 192. Krzyzanowski, F.C., Vieira, R.D., Fran¸c a Neto, J.B., 1999. Vigor de sementes: conceitos e testes. ABRATES, Londrina, pp. 1.1–1.21. Loeffler, T.M., Tekrony, D.M.&, Egli, D.B., 1988. The bulk conductivity test as an indicator of soy bean seed quality. Journal of Seed Technology 12, 37–53. Lowry, D.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Luz, L.A., Rossato, F.A., Costa, R.A.P., Napole~ao, T.H., Paiva, P.M.G., Coelho, L.C.B.B., 2017. Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells. Toxicology in Vitro 44, 94–99. ~o, T.H., Paiva, P.M.G., Coelho, L.C.B.B., Bezerra, A.C.D.S., Medeiros, M.L.S., Moura, M.C., Napolea et al., 2018. Nematicidal activity of a water soluble lectin from seeds of Moringa oleifera. International Journal of Biological Macromolecules 108, 782–789. Memon, Q.U.A., Wagan, S.A., Chunyu, D., Shuangxi, X., Jingdong, L., Damalas, C.A., 2019. Health problems from pesticide exposure and personal protective measures among women cotton workers in southern Pakistan. Science of the Total Environment 685, 659–666. €rndly, E., Reyes-Sanchez, N., Salmero n-Miranda, F., Halling, M., Mendieta-Araica, B., Spo 2013. Biomass production and chemical composition of Moringa oleifera under different planting densities and levels of nitrogen fertilization. Agroforestry Systems 87, 81–92. Morais, S.A.L., Nascimento, E.A., Queiroz, C.R.A.A., Pilo-Veloso, D., Drumond, M.G., 1999. Studies on polyphenols and lignin of Astronium urundeuva wood. Journal of Brazilian Chemical Society 10, 447–452. ~o, T.H., Coriolano, M.C., Paiva, P.M.G., Figueiredo, R.C.B.Q., Moura, M.C., Napolea Coelho, L.C.B.B., 2015. Water-soluble Moringa oleifera lectin interferes with growth, survival and cell permeability of corrosive and pathogenic bacteria. Journal of Applied Microbiology 119, 666–676. ~o, T.H., Primon-Barros, M., Xavier, A.S., Carneiro, N.P., Moura, M.C., Trentin, D.S., Napolea et al., 2017. Multi-effect of the water-soluble Moringa oleifera lectin against Serratia marcescens and Bacillus sp.: antibacterial, antibiofilm and anti-adhesive properties. Journal of Applied Microbiology 123, 861–874. Napole~ao, T.H., Albuquerque, L.P., Santos, N.D.L., Nova, I.C.V., Lima, T.A., Paiva, P.M.G., Pontual, E.V., 2019. Insect midgut structures and molecules as targets of plantderived protease inhibitors and lectins. Pest Management Science 75, 1212–1222. https://doi.org/10.1002/ps.5233. Nishi, L., Vieira, A.M.S., Vieira, M.F., Bongiovani, M., Camacho, F.P., Bergamasco, R., 2012. Hybrid process of coagulation/flocculation with moringa oleifera followed by ultrafiltration to remove Microcystis sp. cells from water supply. Procedia Engineering 42, 865–872. Paiva, P.M.G., Coelho, L.C.B.B., 1992. Purification and partial characterization of two lectin isoforms from Cratylia mollis mart. (camaratu bean). Applied Biochemistry and Biotechnology 36, 113–118. Paiva, P.M.G., Santana, G.M.S., Souza, I.F.A.C., Albuquerque, L.P., Agra-Neto, A.C., Albuquerque, A.C., et al., 2011. Effect of lectins from Opuntia ficus indica cladodes and Moringa oleifera seeds on survival of Nasutitermes corniger. International Biodeterioration and Biodegradation 65, 982–989. Pretty, J., Bharucha, Z.P., 2015. Integrated pest management for sustainable intensification of agriculture in Asia and Africa. Insects 6, 152–182. Rajjou, L., Duval, M., Gallardo, K., Catusse, J., Bally, J., Job, C., et al., 2012. Seed germination and vigor. Annual Review of Plant Biology 63, 507–533. Rashid, U., Anwar, F., Moser, B.R., Knothe, G., 2008. Moringa oleifera oil: a possible source of biodiesel. Bioresource Technology 99, 8175–8179. , R.A., Argolo, A.C.C., Napolea ~o, T.H., Gomes, F.S., Santos, N.D.L., Melo, C.M.L., et al., Sa 2009a. Antioxidant, Fusarium growth inhibition and Nasutitermes corniger repellent activities of secondary metabolites from Myracrodruon urundeuva heartwood. International Biodeterioration and Biodegradation 63, 470–477. , R.A., Gomes, F.S., Napolea ~o, T.H., Santos, N.D.L., Melo, C.M.L., Gusm~ao, N.B., et al., 2009b. Sa Antibacterial and antifungal activities of Myracrodruon urundeuva heartwood. Wood Science and Technology 43, 85–95. , R.A., Napole~ao, T.H., Santos, N.D.L., Gomes, F.S., Albuquerque, A.C., Xavier, H.S., et al., Sa 2008. Induction of mortality on Nasutitermes corniger (Isoptera, termitidae) by Myracrodruon urundeuva heartwood lectin. International Biodeterioration and Biodegradation 62, 460–464. Santos, A.F., Carrijo, T.F., Cancello, E.M., Castro, A.C.M., 2017. Phylogeography of Nasutitermes corniger (Isoptera: termitidae) in the neotropical region. BMC Evolutionary Biology 17, 230.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013
5
JID: SAJB 6
ARTICLE IN PRESS
[m5G;November 1, 2019;16:30]
L.L.S. Silva et al. / South African Journal of Botany 00 (2019) 1 6
Santos, A.F.S., Luz, L.A., Argolo, A.C.C., Teixeira, J.A., Paiva, P.M.G., Coelho, L.C.B.B., 2009. Isolation of a seed coagulant Moringa oleifera lectin. Process Biochemistry 44, 504–508. Santos, N.D.L., Moura, K.S., Napole~ao, T.H., Santos, G.K.N., Coelho, L.C.B.B., Navarro, D.M.A.F., et al., 2012. Oviposition-stimulant and ovicidal activities of Moringa oleifera lectin on Aedes aegypti. PLoS ONE 7, e44840. ~o, T.H., Benevides, C.A., Albuquerque, L.P., Pontual, E.V., Santos, N.D.L., Napolea Oliveira, A.P.S., et al., 2019. Effect of gamma irradiation of Moringa oleifera seed lectin on its larvicidal, ovicidal, and oviposition-stimulant activities against Aedes aegypti. South African Journal of Botany. https://doi.org/10.1016/j.sajb.2018.05.001.
~o, K.S., Napolea ~o, T.H., Trindade, P.B., Pinto, M.R., Coelho, L.C.B.B., Santos, N.D.L., Paixa et al., 2014. Evaluation of Moringa oleifera seed lectin in traps for the capture of Aedes aegypti eggs and adults under semi-field conditions. Parasitology Research 113, 1837–1842. Sauveur, A.S., Broin, M., 2010. Growing and processing Moringa leaves. Moringa Association of Ghana (MAG), Accra, Ghana. ttir, G.R., Sengupta, M.E., Keraita, B., Olsen, A., Boateng, O.K., Thamsborg, S.M., Palsdo et al., 2012. Use of Moringa oleifera seed extracts to reduce helminth egg numbers and turbidity in irrigation water. Water Research 46, 3646–3656.
Please cite this article as: L.L.S. Silva et al., Evaluation of the use of Myracrodruon urundeuva heartwood extracts to protect Moringa oleifera seeds against Nasutitermes corniger attack and improve sanity, South African Journal of Botany (2019), https://doi.org/10.1016/j. sajb.2019.10.013