Treatment of bean seeds with plant extracts for controlling Zabrotes subfasciatus and its effects on physical and physiological quality during storage

1 Paraíba Estate University, R.Baraúnas, 351 Universitário, Campina Grande PB, 58429-500, Paraíba, Brazil. 2 Federal University of Campina Grande, Unidade Acadêmica da UFCG, Aprígio Veloso, 882 – Caixa Postal 10064 – 58109-970 – Campina Grande, Paraíba, Brazil. 3 Federal University of São Francisco Valley, Department of Agricultural Engineering, Av. Antonio Carlos Magalhães, 510, 48902-300, Juazeiro BA – Brazil. 4 Federal University of Pelotas, Seed Science and Technology, Campus Universitário s/n, 96010-001, Pelotas – RS – Brazil.

largest producer is Myanmar, followed by India (Fao, 2013).In the scenario of the northeastern agricultural production, given the infrequent rainfall, beans, because of their short cycle and tolerance to water stress, holds special relevance in the food supply and the composition of family income.Due to the observation, that there is lower occurrence of losses and hand labor during seasonal periods, as in the case of cultivation in a second season crop, because of the use of wet or irrigated lowlands (Frota and Pereira, 2000).
Beans can lose their physical and physiological quality after harvest, especially if incorrectly stored.This can happen due to fungal contamination, insect infestation and/or metabolic processes that reduce germination and vigor, causing, among other defects; seed coat darkening (especially the Carioca group); due to oxidation of phenols in the presence of oxygen (Lazzari, 2005).Therefore, the adoption of conservation practices is critical to the maintenance of seed quality during storage, to prevent loss in germination and vigor, as well as pest attack.
The presence of insects causes qualitative and quantitative losses.The insects consume the substrates and the respiration of the insects creates hot spots.These hot spots cause increase in temperature and inter granular humidity which create an environment for fungal growth (Lazzari, 1997;Rupolho et al., 2006).The losses of grains and seeds, resulting from contaminated storage in Brazil, are 10% (Viebrantz et al., 2016).
The grains and seeds can rot and become useless for consumption due to the appearance, smell and taste.According to Lorini et al. (2002), reducing water content of the beans up to 13% by drying, helps reduce losses due to fungal attack.Although without chemical control, this does not prevent the presence of insect pests during storage.In this context, extracts of plants can be used as an alternative to control insects and may also be used together with other pest control practices.The advantages are their rapid biodegradation which reduces the environmental contamination risk; and they are easy to obtain.
Given the unavailability of technical information on the control of Zabrotes subfasciatus L. using natural insecticide produced by plants, this work aims to evaluate the effect of leaves and barks extracts (Aspidosperma pyrifolium Mart.and Anadenanthera colubrina) and myrtle (Licania rigida benth) in the control of Z. subfasciatus associated to seed bean.It also aims to evaluate the seed treated during storage in pet bottles for a period of 180 days.

MATERIALS AND METHODS
The experiment was conducted in Agricultural Products Storage and Processing Laboratory) of the Academic Unit of Agricultural Engineering (UAEA) at the Federal University of Campina Grande (UFCG) Paraíba.Bean seeds were obtained from producers of Belo Jardim, PE, Brazil and seeds were obtained in the 2010/2011 crop season.
The initial quality of the seeds was evaluated by the purity analysis and the determination of moisture content, germination, vigor and infestation percentage of beans by Z. subfasciatus.
The moisture content was conducted in accordance with the Rules for Testing Seeds (Brazil, 2009).Moisture content was determined with two replications of 10 g of seeds and by using the oven method at 105 for 24 h.After treatment, the seeds were packed in pet packaging and stored in laboratory conditions without temperature and relative humidity control for a period of 180.They were evaluated every 45 days for moisture content, germination and first count of germination infestation, and weight loss.
The procedures of germination were carried out according to the Rules of Seed Analysis (BRASIL, 2009), but 200 seeds were used per treatment instead of 400.In this case, four replicates of 50 seeds were used per treatment (leaf and bark extracts and doses of 0, 1, 2, and 3 ml).The seeds were placed on germitest paper and the paper rolls were kept in a growth chamber at 25°C.The first count (vigor test) was performed together with the germination test by counting the normal seedlings found on the fifth day after sowing.Final germination was evaluated at 9 days (Brasil, 2009).
The infestation of Z. subfasciatus present in the stored beans was evaluated by observing the seeds per 100 g.The damaged seeds were then separated from the intact ones and were calculated over the total number of samples.The calculation was performed using the equation suggested by Almeida and Villamil (2000).
The percentage of beans' weight loss under storage conditions was conducted using 100 g of intact seeds and 100 of damaged seeds, and then calculated by the equation suggested by Almeida and Villamil (2000).

PP = (I -D/I) × 100
where PP is the weight loss (%), I is the seeds loss integrity (g), and D is the weight damaged seeds (g).
The experiment of the extracts of leaves were separated, weighed and dried in an oven with forced air circulation for 72 h.Then, the plant materials were ground separately and placed in amber glass container containing 8 L of ethanol at 70% for maceration under occasional shaking.The extracted liquid passed through a first filtration filter paper under reduced pressure and a second filtration by gravity to hopper closed with cotton.Therefore, the filtrate was concentrated on evaporator under reduced pressure at a temperature of ± 80°C, for separating ethanol.The obtained solution was placed in water bath at a constant temperature of 65°C to evaporate the water and obtain solid material (Costa et al., 2011).
The hydro alcoholic extracts were used in doses that are sufficient to kill the insects, within seven days of life.The extracts were applied by the pipetting method directly on the mass of beans.Homogenizing was done by manual agitation and one plot was used for control.Then, the seeds were spread on polyethylene trays for a period of 24 h at room temperature in order to increase extract absorption for the seed mass.
After this time, seeds were distributed in plastic pet containers, with capacity of 500 g which were infested with 30 adult insects of Z. subfasciatus each.Insect were collected from bean seeds which are stored in plastic bottles.They were multiplied after selection, separation and identification using a microscopic.Insects were inoculated into a mass of beans, previously purged, inside a glass container of 300 ml capacity, sealed at the top with organdy cloth to allow natural ventilation.
The same procedure was used for untreated seeds.They were then stored in laboratory without temperature conditions and relative humidity control, for a period of 180 days.After this period, the percentage of infested seeds, weight loss, germination and the moisture content were performed.The control did not receive The experiment was conducted in a completely randomized design, in which the experiments were arranged in a factorial design with four replications and the means were compared by Tukey test at 1 and 5% probability.Effects of quantitative factors were analyzed using regression analysis: linear and quadratic equations for all variables and their interactions were examined.The data were evaluated with the software ASSISTAT version 7.6 (Silva and Azevedo, 2009).

RESULTS AND DISCUSSION
The seeds were initially stored with 13.29% moisture content and 96% germination was observed.The results of the evaluation of the initial quality of the seeds prior to storage with respect to moisture content was in the range suggested for storage by Delouche et al. (1976), Popinigis (1985) and Carvalho and Nakagawa (2000).
According to the results shown in Table 1, it appears that the infestation reduced from 9.49 (control) to 6.82, 6.48 and 2.88% with a dose of 3 ml of the leaves extracts of A. pyrifolium Mart., A. colubrina and L. rigida, respectively and similar behavior was observed in extracts from barks of the same species.It can be noted that the 3 ml dose was the best for controlling insect infestation and extracts from L. rigida leaf and bark of A. pyrifolium Mart.showed the best results in controlling the infestation of Z. subfasciatus present in seed mass of stored beans.It was also observed that extracts of L. rigida (2.88) and A. pyrifolium Mart.barks (3.96) in 3 ml dose, showed no statistic difference in controlling insect infestation.
The low percentages of insect infestation in controlled bean seeds, reveal the efficiency of the extracts used in the control of insect infestations.This efficiency is probably due to its secondary metabolite action, especially in Anadenanthera colubrine extracts (bark or leaf); which it does by leaving a thin protective waterproof film.This thin lubricant layer serves as a barrier to insect.According to Silva et al. (2013), repellent effects are observed in insect sensory system when they are exposed to undesirable substances.Costa (2011) studying Z. subfasciatus infestation in a mass of beans, treated with extracts of jackfruit (Artocarpus heterophyllus L.) and Chenopodium ambrosioides L. verified reduction of infestation by 90% using doses of 6 to 8 ml, during 120 days of storage.Ali et al. ( 2011) observed that the amount of damage to grains caused by weevils increased with storage time and this devalued the product sale.Garcia et al. (2000) noted that the treatment of beans with ground black pepper with doses 4 and 6 g per kg -1 had absolute control on Z. subfasciatus during the storage period.It is noted that, it is feasible to use alternative plant extracts in order to control insect infestations in seeds and grains during storage.
For interaction with extracts procedure as shown in Table 2, it is noted that when inoculated with A. colubrina and myrtle leaves extracts or A. pyrifolium Mart.and myrtle shells, no statistic differences were observed.The best results were observed in extracts from A. pyrifolium Mart.leaves and A. colubrina barks.However, uninoculated beans showed better control of Z. subfasciatus with A. pyrifolium Mart.leaves and myrtle bark extracts which did not differ statistically; this result was followed by L. rigida leaves (Silva et al., 2013).
When the bean seeds were inoculated with A. colubrina and myrtle leaves extracts, no statistical differences were observed.
The low percentage of infestation demonstrates the efficient action of the extracts in the study.However, we noticed an emollient action on stored seeds treated with myrtle extracts, this action promoted a strong bond between the seeds.These residues are probably due to the chemical compounds (saponins) present in the myrtle extract, indicating that these seeds should not be stored for more than 180 days under these conditions, when treated with myrtle extracts.Garcia et al. (2003) studying the bioactivity of A. colubrina and myrtle extracts, observed results which show that the extract had active substances against insects (mortality).Barbosa et al. (2000) observed resistance associated with species of bean arcelin Z. subfasciatus protein, implying that some substances derived from the secondary metabolism of plants can affect the biology of insects.Analysis of the data contained in Table 3, exhibits equal statistics of doses in controlling Z. subfasciatus present in beans paste stored at room condition for 180 days in pet packaging.However, for procedures within each dose (column), the dose of 3 ml showed the best control of Z. subfasciatus, where the infestation after 180 days of storage was 5.73% (average).These results are due, probably, to the quantity of the dose as secondary plant constituents present in the extracts; this dose operates with higher efficiency due to better distribution upon its application to the seeds.
According to Costa (2011), when the extracts A. heterophyllus and C. ambrosioides were used to control the infestation of Z. subfasciatus, in P. vulgaris, for a storage period of 120 days, equal statistical result was observed in both the inoculated and non-inoculated procedure with doses of 6 and 8 ml.
As shown in Table 4, for interaction with the procedure time, there is increased infestation of Z. subfasciatus in seed stored in the mass, with the passage of storage time, gradually, in both procedures with superiority in the inoculated seeded process.It appears also that after three months (90 days) of storage, the percentage of infestation was 6.66% for the mass of seeds infested with Z. subfasciatus.It can be seen that during storage the extracts were loosing their bioactivity due to the effects of their volatile constituents, which might explain the increased infestation with time.The insecticide effect of plant extracts was also analyzed by Pessoa (2004) for corn seed storage for popcorn with positive results in controlling infestation of Sitophilus zeamais.Santos et al. (1998) and Costa (2011) also controlled infestation of Z. subfasciatus in bean seed with plant extracts.Silva Junior (2011) working with hydroalcoholic extracts of sugar apple and black pepper on the infestation of corn with S. zeamais, inoculated the seed mass; after 180 days in storage, he observed lower infestation for the highest levels of these extracts, which partly agree with this research.
From the results obtained in Table 5, it appears that the behavior of infestation by Z. subfasciatus is progressive and increases with time regardless of the dose.It was observed that the best controls were those with higher doses of 1 and 3 ml, respectively (line), with a dose of 1 ml, the infestation of Z. subfasciatus was controlled more efficiently within the time (column) when compared to the dose of 3 ml.It appears that the lower volume of extract was the most effective in controlling infestation, probably by the interaction of its chemical constituents in the form of application and speed of exposure of extracts on the seeds.
Resende et al. ( 2008) studied bean storage, observed after 84 days, a significant increase of 91.67% in grain population of insect pests stored in a mass of seeds treated with plant extracts.
In this study, the maximum infestation of Z. subfasciatus after 180 days of storage was 13.73% with a 5 ml dose compared to 10.91% with a dose of 1 ml.Therefore, these two doses are confirmed as being the most efficient for the control of weevil in beans seeds.Harborne (1980) stated that the leaves of angiosperms accumulate flavonoids which serve as insect repellant.A similar result was found in A. colubrina and A. pyrifolium Mart.which are present in the extracts of the leaves and flavonoids of the barks.
The A. colubrina leave extract was also more effective in the control of Z. subfasciatus at the rate of 2.91, 4.88 and 9.16% in 45, 90, and 135 days, respectively.The bark extracts from myrtle was also found to be more efficient, it showed a result of 3.78 and 5.40% infestation in 45 and 90 days of storage, respectively.This level of effectiveness is followed by A. pyrifolium Mart.extract.
As shown in Table 6, there is infestation of Z. subfasciatus in seed mass, during storage and the extracts of A. colubrina leaves showed higher results in infestation control at all times.This result is followed the by A. colubrina bark.
The percentage of infestation of leaf extracts was statistically equal for all times, however, there was no statistical difference for all leaves and bark extracts analyzed during the 180 days of storage.There was a lower infestation occurrence in A. pyrifolium Mart.bark with 10.31%, followed by A. colubrina bark with 11.56%.Neves et al. (2000) used botanical products to control Z. subfasciatus in bean seeds.Assessments were performed at 30, 60 and 90 days after infestation and  Navickiene et al. (2007) after testing organic extracts from the seeds, leaves and stalks of Piper tuberculatum, ascertained that these extracts showed potential insecticidal activity, showing a process of rapid poisoning against Anticarsia gemmatalis (Lepidoptera: Noctuidae) causing 80% mortality when doses greater than 800 insect -1 cg were administered.According to Torres and Marcos Filho (2001), the susceptibility of plants to insects extracted from plant allelochemicals, depends on the organ and plant species, form of extraction and insect species.According to Chagas et al. (2003), a range of different compounds can be isolated depending on the solvent used to obtain the extract.
In the analysis of germination percentage of stored bean seeds (Table 7) and their interaction with extract products, it appears in the inoculated procedure that the highest percentage of germination was given to the shell A. pyrifolium Mart.extract (92.00%) followed by A. pyrifolium Mart.leaf extract (91.00%), then the L. rigida bark (80.00%) and the lowest for the L. rigida leaf extract (78.00%).
In the non-inoculated procedure, all extracts showed the same efficiency in maintaining the viability of seeds, this is revealed by the average germination rate which was 91%.The efficiency showed by all extracts was with the exception of the A. pyrifolium Mart.extracts sheet and A. pyrifolium Mart.shell, which though showed the same germination in the non-inoculated procedure, the seeds remained higher than that of the inoculated procedure.This shows that the presence of Z. subfasciatus caused damage to the beans seeds.
According to Olanda et al. (2011), when the leaf extract of Casearia sylvestris Sw. was used to treat bean seeds in a concentration of 0.78%, the germination percentage of 12.5 was observed but from 25 to 50%, complete death of the seeds was observed.Thus, a high concentration of Echinodorus macrophyllus (Kunth) Micheli bark extract showed phytotoxicity on the bean seeds.However, when applied in lower concentrations, the extract showed a beneficial effect on germination; thus, demonstrating the potential of E. macrophyllus (Kunth) Micheli in bean seed treatment.
The addition of millet extracts reduced the percentage of germination rate in bean seeds (Farias et al., 2009).Karunakaran et al. (2001) viewed fungal growth in wheat seeds stored with 19% moisture content at temperatures between 20 and 35°C, only after the germination of the seeds have reduced the percentage below 90%.Matioli et al. (1978) found that in corn seeds infested by Sitophilus oryzae, there was increased insect population and reduced weight and seed germination.Garcia et al. (2000) found effects of ground black pepper extract at a dose of 4 g/1 kg of seeds, as the most efficient treatment in the control of Z. subfasciatus in bean seeds.
It can be seen that seed germination was not influenced by A. pyrifolium Mart leaf and A. colubrina bark extracts.The percentages of germination of bean seeds were statistically similar at doses of 1, 3 and 5 ml.There was statistical similarity in the germination of bean seeds with doses 1, 3 and 5 ml.In general, germination was more affected by L. rigida leaf and bark extracts, which was statistically similar at doses of 3 and 5 ml, but within each dose (column) the 3 ml dose proved to be the overall best in the treatment of the seed in order to obtain seed quality.Whereas, the following A. pyrifolium Mart.with A. colubrina leaf and A. pyrifolium Mart.with A. colubrina bark resulted in a higher percentage of seed germination (92.00%) (Table 8).It is noted that in the procedures, equal statistic results were observed, except for the dose of 1 ml in the inoculated procedure which has the highest percentage of germination.It is also observed that there is difference in 3 and 5 ml doses with higher germination percentage for the non-inoculated procedure (Table 9).
In summary, it can be stated that germination was not affected in seeds treated with doses of these extracts.It is also observed through graphic representation that there is high coefficient and quality.This validates the use of equations in the conditions under which the study did; when you want to estimate intermediate points of the experiment.Brito (2010) found that Mimosa tenuiflora (Wild) extract negatively influenced all the variables studied mainly in corn germination, supporting the thesis that corn can be used as a model in bioassays in tests on allelopathy (Macias et al., 2000).Silva Junior (2011) reported the interaction of inoculated and uninoculated procedures in the treatment of corn seeds within a period of 180 days, showing a different result on germination.There was a record of 72% germination at the end of the storage period for inoculated seeds.This shows the efficiency of the extracts.
It appears that time was decisive in the germination results of stored bean seeds; those that were observed between 45 and 90 days showed similar results for the extracts.That is, significant effects of extracts on seed germination were only observed after 90 days of storage (column).However, after 180 days of storage, the A. colubrina leaf extract which showed 64.00% result and myrtle bark which showed 65.00% showed the worst germination result.This is followed by A. colubrina leaf extract (79.00%) (Table 10).L. rigida bark extracts (85.00%) and A. colubrina leaf (86.00%) were the least efficient, with the former being statistically, in the maintenance of seed germination after 135 days of storage.However, it was observed that the tough A. pyrifolium Mart.extract (88.00%) kept seed germination to the end of the storage period.It was also observed that the A. pyrifolium Mart.leaf extract (92.00%) and L. rigida leaf (90.00%) showed similar results at 135 days of storage.This result is due to the presence of natural substances with potential insecticide, present in the hydro alcoholic extracts used.This potential is revealed in the phytochemical study, where steroids and tannins showed bioactive effects on L. rigida and A. colubrina extracts.Medeiros et al. (2007) evaluated dry and green leaves of neem on the quality of cowpea seeds and they concluded that the extracts affected seed germination; the treatments which received neem seed powder differed significantly from the control.Silva (2007) observed reduced germination in corn seeds treated with extracts of M. tenuiflora (Wild Poiret).Haven given effect to the woody part, it was observed that germination of seeds of other grasses was not affected when treated with leaf extract of M. tenuiflora (Wild) Poiret).Silva and Aquila (2006) verified the effect of Erythroxyhum argentinum extracts, Luehea divaricata, Mysine guianensis and Ocotea peberula on the initial germination of lettuce.They observed that the treatment showed significant difference from the control group despite that those species presented allelopathic potential.Lima et al. (2007) observed that aqueous extracts of the aerial part of Crotalaria juncea, Canavalia ensiformes (L.) DC. and Sesamum indicum reduced the final germination of Bidens pilosa in a concentration of 20%.
It was also observed that there was reduction in seed germination with advance deterioration during storage time for both the inoculated and non-inoculated, statistically, equal to the procedures within 45 times and 90 days.
But in the final two times (135 and 180 days), the inoculated procedure (87.00 and 67.00%) was lower than the uninoculated procedure (90.00 and 86.00%), respectively (Table 11).Almeida et al. (2008) found that Croton sonderianus of aqueous extracts also promoted a reduction in the percentage of germination of Cassia tora.Germination decreased within storage period of 45 and 90 days (column), there were no significant statistical differences, but seed germination showed different behavior at 135 and 180 days.In addition, at 180 days, there was lower seed germination for the dose of 5 ml 69.00% (Table 12).
This result shows that the doses tested for this test are effective in controlling Z. subfasciatus and did not inhibit the germination of seed bean.
Promising results in the control of Z. subfasciatus were verified by Queiroga (2010) with vegetable oils in the treatment of stored beans.The insects were controlled and after 150 days of storage, there was an observation of 57.00% seed germination.Almeida (2003) studied the effects of plant extracts in the control of Callosobruchus maculatus and its effects on the bean Vigna unguiculata.From the research, it was concluded that the vigor and germination of seeds treated with Conospermum caeruleum extracts and Piper nigrum were positive, while this decreased over storage time.While the best extract in preserving the seed was P. nigrum.

Conclusions
Plant extracts showed insecticidal activity against Z. subfasciatus, killing them and/or inhibiting their development.In the treatment of bean seeds, the dose of 3 ml was the best to control the incidence of Z. subfasciatus, with better results from the use of L. rigida leaf extracts and A. pyrifolium Mart.bark.The procedures adopted in seed treatment with the extracts of plant species were efficient in maintaining the viability and did not affect germination during 180 days of storage.Germination was affected by insufficient storage time (less than 180 days), and the doses 1 and 3 ml were the most efficient for the maintenance of seed germination.

Table 1 .
Control of infestation (adult emergency) of Zabrotes subfasciatus using hydroalcoholic extracts of leaf and bark of A. pyrifolium mart, A. colubrina and L. rigida in inoculated and non-inoculated bean seeds after 180 days of storage in pet packaging.

Table 2 .
Efficiency (mortality) of hydro alcoholic extracts of leaf and bark A. pyrifolium Mart., A. colubrina and L. rigida applied in bean seeds inoculated and non-inoculated with Z. subfasciatus in 180 days of storage in pet type packaging.

Table 3 .
Mean values (%) of the infestation procedure of bean seeds, inoculated and non-inoculated with Zabrotes subfasciatus and treated with plants extracts in pet packaging, for a period of 180 days.

Table 5 .
Mean value (%) of Zabrotes subfasciatus infestations in inoculated and non-inoculated been seeds, treated with different doses of plant extracts, stored in pet containers, in a period of 180 days.

Table 6 .
Germination percentage (%) of bean seeds treated with hydroalcoholic extracts of leaves and barks of Aspidosperma pyrifolium Mart., A. colubrina and L. rigida, inoculated and not inoculated with Zabrotes subfasciatus for 180 days of storage packaging pet.

Table 7 .
Average germination values (%) of interaction in bean seeds treated with doses of leaves hydro alcoholic extracts ,bark of Aspidosperma pyrifolium Mart., A. colubrina and L. rigida, inoculated and not inoculated with Zabrotes subfasciatus for 180 days in storage with packaging pet.had the best results with a mixture of neem oil with oil Piper hispidinervum.The result was a 100% reduction in the numbers of eggs and damaged grains, at 90 days. they

Table 8 .
Average values (%) of germination of the interaction procedure with dose in bean seeds treated with hydroalcoholic extracts of leaves and bark of Aspidosperma pyrifolium Mart., A. colubrina and L. rigida, inoculated and not inoculated with Zabrotes subfasciatus for 180 days of storage packaging pet.

Table 9 .
Average values of germination percentage (%) for interaction of extract with bean seeds treated with hydroalcoholic extracts of leaves and bark Aspidosperma pyrifolium Mart., Anadenanthera colubrina, and L. rigida leaves inoculated and not inoculated with Zabrotes subfasciatus in pet type storage packaging for 180 days.

Table 10 .
Percentage of germination (%) for the interaction procedure in bean seeds treated with hydroalcoholic extracts of leaves and bark of Aspidosperma pyrifolium Mart., Anadenanthera colubrina and L. rigida inoculated and not inoculated with Zabrotes subfasciatus in storage packaging type pet for 180 days.

Table 11 .
Percentage (%) of germination for dose interaction in bean seeds treated with hydroalcoholic extracts of leaves and bark of Aspidosperma pyrifolium Mart., Anadenanthera colubrina and L. rigida; inoculated and not inoculated with Zabrotes subfasciatus in pet packaging storage for 180 days.