Extracts of Caesalpinia ferrea an d Trichoderma sp . on the control of Colletotrichum sp . transmission in Sideroxylon obtusifolium seeds

Recent research reports the importance of preserving plants in Brazilian semiarid regions, in this context, the scientific literature has reported different pharmacological studies from plant extracts with an antifungal potential, coming from forest species that can contribute as a control and management strategy in the transmission of phytopathogens. This study aimed to evaluate the effect of biotech treatments in controlling the transmission of Colletotrichum sp. in seeds of S. obtusifolium. In this study, 100 seeds were subjected to the following preventive treatments: fungicide Captan®, extract of Caesalpinia ferrea Mart. Ex. Tul., and biological control with Trichoderma spp. The biological control with Trichoderma spp. and the alternative control using C. ferrea extract provided a greater protection to seeds and seedlings of S. obtusifolium facing the transmissibility of Colletotrichum sp.The treatment based on plant extract is more efficient for this purpose only in large seeds and does not interfere on the germination percentage and speed. Therefore it is necessary to perform other studies with Trichoderma spp. and C. ferrea extract to test different doses of these products.


INTRODUCTION
Sideroxylon obtusifolium (Roem & Schult.)Penn. is a specie of the caatinga biome that because of intense use, it is at risk of local extinction in the Northeast of Brazil (Silva & Dantas, 2013).This species is popularly known as quixaba, sapotiaba, sacutiaba, coronilha, coca, miri, rompe-ribão, but it is not a cultivated fruit (Silva et al., 2012a), however, it is widely utilized in the medicine due to its phytotherapeutic properties, which indicate the presence of some compounds of pharmacological interest (Gomes et al., 2010).This way, the establishment of forest recovery programs through the production of seedlings from seeds quality and genetic variability is essential (Beltrão et al., 2008).In this context, the sanity and physiological quality of seeds becomes important once the production of seedlings from them will reflect on their ability to develop healthy plants (Mondego et al., 2014).
The germination capacity it affected by the presence of fungi, as they serve up the seeds as a vehicle for transport and as shelter for survival, and thus involved in the continuity of the life cycle of these pathogens from one to another generation of the host plant.Thus, the research associated with transmission the seed and seedling contributes to defining certain control strategies involving the management of disease, since it thus defines if the inoculum of causative agent reached new areas through seed transmission or other random and pathway of foci of fungal infection (Poletto et al., 2014).
However, there is still a lack of research regarding the condition of forest seeds and the efficiency of chemical and alternative products, for the implementation of sanitary methods in the production of tree nurseries (Mertz et al., 2009).In this context, the scientific literature has reported different pharmacological studies (Marreiro et al., 2014), from plant extracts with antifungal potenticiality, coming from forest species, among them Caesalpinia ferrea Mart.Ex.Tul. which is an endemic species of north and northeast in Brazil (Ferreira & Soares, 2015).Therefore, become necessary research to find new bioactive molecules with relevance in the control of pathogens because it was believed that it is difficult to develop resistance to these compounds present in plant extracts with components that differ as fromtheir mode of action and apparently from their chemical constitution (Ferreira et al., 2013).
Moreover, taking into consideration the great potential of using S. obtusifolium in reforestation, ecology and pharmaceutical industry (Oliveira et al., 2012), the present study aimed to evaluate the effect of biotechnological treatments in controlling transmission of Colletotrichum sp. in seeds of Sideroxylon obtusifolium (Roem & Schul.)Penn.
After harvesting, the fruits were placed in polyethylene bags and taken to the Laboratory of Seed Analysis (LSA) and Plant Pathology of the phytotechnic and environmental Sciences Department, at the Agricultural Science Center of the Federal University of Paraiba, where the experiments were conducted.The fruits were underwent to natural fermentation for 72 hours, after this period they were rinsed in running water to obtain the seeds which were dried on paper towels at indoor temperature at the laboratory environment (25 ± 2 °C) for 72 hours (Silva et al., 2012a).

Length and Seed moisture content
Initially, the seed samples in each lot (mother trees trees) were measured using a digital caplier (0.001 mm) to determine length.The biometric data associated with the length of the seeds from the different mother trees (M 1 -9.99 cm, M 2 -9.94 cm, M 3 -7.69cm, M 4 -7.51 cm, M 5 -7.45 cm and M 6 -6.54 cm) served as a basis for obtaining the initial quality of the samples.The water content was obtained by using the oven method at 105 °C for 24 h, with four replications of 25 seeds per mother tree in each seed lot and was carried out according to the Rules for Seed Testing (Brasil, 2009).

Sanitary test
To identify the microflora in seeds it was used the filter paper method (Blotter Test), with 200 seeds of each mother tree divided into eight replicates of 25 seeds subjected to surface disinfection by immersion in sodium hypochlorite at 2% for five minutes and then placed in petri dishes with sterile filter paper moistened with sterile distilled water.After a seven day incubation period at indoor temperature (25 ± 2 °C), fungal structures were analyzed with the aid of stereoscopic and optical microscope.Confirmation of fungi genus level was performed using an identification key (Barnett & Hunter, 1972).
Although for the Colletotrichum sp. has found moderate incidence, according to Vechiato & Parisi (2013), nothing can be stated in regard to the damage that can be caused, given there is no search results on transmission rates that can quantify the damage to forest seeds.Thus we proceeded to obtain the isolates of Colletotrichum sp.following the methodology described by Medeiros et al. (2013), where the seeds were incubated in petri dishes containing PDA medium (1000 ml of distilled water, 200 g of potato, 20 g dextrose and 17 g agar) sterilized.
After seven days of incubation period at indoor temperature (25 ± 2 °C and a 12 h photoperiod), the fungal structures were analyzed with the aid of a stereoscopic and optical microscope.The fungus (Colletotrichum sp.) was isolate and replicated in petri dishes containing sterilized PDA medium, following the method described previously by Medeiros et al. (2013) and then the genera of the pathogen was confirmed using identification keys (Barnett & Hunter, 1972).
The C. ferrea leaves, a species of the Fabaceae Caesalpiniaceae family, known as juca, jucaina and pau-ferro (Brazilian ironwood), was collected at the Agricultural Science Center of the Federal University of Paraiba, Areia, PB, geographically located at the coordinates 6°57′42″ S, 35°41′43″ W, and used to obtain the crude alcoholic extract (CAE).They were collected and placed in Kraft paper bags and then taken to the LSA where they were weighed on a precision scale (0,001 g) and put into portions of 500 g each.
Shortly after, the leaves were sterilized for five minutes using a solution of sodium hypochlorite (2%) and then they were oven dried at a temperature of 40 °C for a period of 72 hours and completely grindded using an electric grinder until no pieces of intact leaves were visible (Stange et al., 2009).The CAE was obtained as described by Silva et al. (2012b) with slight modifications, where 60 g of the ground leaves were infused in 700 mL of absolute alcohol (99.5%) and left at indoor temperature for 24 h.After extraction, the extract was filtered at a temperature of 50 °C and taken to the vacuum evaporator (Biothec ® Model BT 350/4) to remove the solvent, the residue was then stored in previously sterilized and dried amber bottles with lids.

Seed treatment and inoculation
The concentration of the fungal suspension was determined using a Neubauer chamber with about 2 x 10 5 conidia/mL of Colletotrichum sp., in which the seeds from each mother tree were inoculated by immersing them in the same fungal suspension for a 12 h period with four replications of 25 seeds.The seeds were kept at indoor temperature (25 ± 2 °C) and dried on paper towels for 30 minutes, they were then disinfected with sodium hypochlorite (2%) for two minutes and 70% ethanol for 30 seconds, afterwards they were rinsed twice in sterile distilled water (SDW) and dried as previously described (Ferraz & Calvi, 2010).The treatments were composed by (T 1 ) untreated and no inoculated seeds, (T 2 ) seeds inoculated with Colletotrichum sp., (T 3 ) inoculated seeds treated with Captan ® fungicide, (T 4 ) inoculated seeds treated with C. ferrea extract and (T 5 ) inoculated seeds treated with Trichoderma spp.
The chemical treatment was carried out manually, by applyinng the fungicide Captan ® at a concentration of 240 g per 100 kg of seeds, subsequently, the seeds were placed in a polyethylene plastic bag and shaken until homogeneous.The biological and alternative treatments were conducted by immersing the seeds for a 24h period in 20 mL of the bio-fungicide Trichodel ® , the dosage used was based on the manufacturer recommendations, 1 x 10 9 viable cells per milliliter of Trichoderma spp.and the C. ferrea extract was diluted at a ratio of 0.156 mg per 200 mL, this dosage was based on preliminary tests using SDW.

Evaluation of physiological and sanitary quality
The evaluation of the physiological and sanitary quality (transmissibility) was performed mutually in a germination chamber at a constant temperature of 30 °C with a 12 h light/12 h dark period using fluorescent lights (4 x 20 W).Initially, the seeds were manually scarified with 80 grit sandpaper on the opposite side of the hilum region and sown in transparent plastic boxes (11.0 x 11.0 x 3.5 cm) which were sterilized with sodium hypochlorite (2%) containing vermiculite substrate (Silva et al., 2012a), they were then moistened with distilled water at 60% of its retention capacity (Brasil, 2009) with four repetitions of 25 seeds per treatment, the seeds were placed in substrate at a depth of two centimeters.
The evaluations were performed every other day from the 15th to the 30 th day, taking as a criterion the seedling emergence and the results are expressed in percentages.On the 30th day, the final transmissibility test was performed using the seeds submitted to the last four treatments (T 2 ) seeds inoculated with Colletotrichum sp., (T 3 ) inoculated seeds treated with Captan ® fungicide, (T 4 ) inoculated seeds treated with C. ferrea extract and (T 5 ) inoculated seeds treated with Trichoderma spp.previously described.The incidence of the disease in the inoculated S. obtusifolium seeds was determined as well as the rates of infection and transmission in shoots and primary roots associated to Colletotrichum sp..During this period, plants that showed Colletotrichum sp.infection symptoms in the cotyledons, roots, stems or leaves were considered sick plants.To confirm the etiology of the pathogen, symptomatic seeds, seedling fragments and roots were previously sterilized according to the method described by Walker et al. (2013) and Mondego et al. (2014) and were incubated in Petri dishes containing sterilized PDA medium (1000 mL of distilled water, 200 g of potato, 20 g of dextrose and 17 g of agar).
After a seven day incubation period at indoor temperature (25 ± 2 °C) and a 12 h photoperiod, the fungal structures were analyzed with the aid of a stereoscopic and optical microscope.The genera of the fungus were obtained using identification keys (Barnett & Hunter, 1972), the transmission of the pathogen to the emerged plant and to the dead non-emerged seedlings was considered positive when at least one seed, fragment of the symptomatic or asymptomatic plant presented mycelial growth.
At the end of the germination test, the percentage (Labouriau, 1983) and germination speed index (Maguire, 1962) was determined, the rate of transmission of the fungus to the seedlings was calculated using the formula adapted from Teixeira & Machado (2003): T(%) = I.T(%)x100/ I.D(%), where I.T = infection rate in seedlings with symptoms of the selected fungus; I.D = incidence of disease in artificially inoculated seeds.

Experimental design and statistical analysis
The experiment was conducted in an entirely randomized design, with treatments distributed in a 6 x 5 factorial arrangement for the physiological quality (six mother trees and five treatments), and a 6 x 4 factorial arrangement for the sanitary quality analysis (six mother trees and four treatments), both with four replications of 25 seeds.Data were subjected to the variance analysis test (ANOVA) using the SAS ® statistical software (Statistical Analysis System) and means were compared by Tukey test at 1% probability (SAS/STAT, 2011).

RESULTS AND DISCUSSION
The moisture content of the S. obtusifolium seeds were about 10%, and the higher percentages of germination (Table 1) occurred in the seeds that did not receive treatment and were not inoculated (Rate of germination (%) = M 1 -95, M 2 -90, M 3 -93, M 4 -92 and M 6 -88), associated with all mother trees except for mother tree 5 (85%).However, these did not differ statistically (P ≤ 0.01) from mother tree 1 (98%) that consisted of inoculated seeds and were treated with Captan ® (T 3 ) and also mother tree 2 (88%) that had inoculated seeds and were subjected to the alternative control using C. ferrea extract (T 4 ).It also appears that the worst germination and vigor performance was associated with the seeds from mother tree 6 (T 4 ), regardless of the parameter evaluated in relation to the germination speed index * Means followed by the same letter, lowercase and uppercase in the column on the line, do not differ significantly at 1% probability (P ≤ 0.01) by Tukey test.T 1 = untreated seeds and not inoculated, T 2 = only inoculated seeds with Colletotrichum sp., T 3 = inoculated seeds and fungicide treated, T 4 = inoculated seeds and subjected to alternative control with C. ferrea extract, T 5 = inoculated seeds and submitted the biological control with Trichoderma spp.NOTE: Length of the seeds of different mother trees (M 1 -9.99 cm, M 2 -9.94 cm, M 3 -7.69cm, M 4 -7.51 cm, M 5 -7.45 cm and M 6 -6.54 cm). of the S. obtusifolium seeds (Tables 1 and 2).
As for the germination speed index of all the mother trees (Table 2) the best results came from the untreated and uninoculated seeds shown in Table 1 (T 1 ) however, there were no statistical differences (P ≤ 0.01) between the inoculated seeds from mother tree 1 and 2, and the seeds subjected to chemical control (T 3 ) and seeds from mother tree 2 treated with an alternative control, C. ferrea extract (T 4 ).
Similarly, the fungicide treatment with Captan ® and vegetal extract provided significant eradication of Colletotrichum sp. in seeds of Platypodium elegans Vog.(Machado, 2000) and Ceiba speciosa St. Hill.(Lazarotto et al., 2010), obtaining the highest percentage and germination speed index.In seeds of other native species like Tabebuia serratifolia (Vahl.)Nich.(Botelho et al., 2008) and Tabebuia impetiginosa (Mart.ex DC.) Standl.(Mertz et al., 2009), the chemical treatment constituted an efficient method to control pathogens associated with those seeds.
As for the C. Ferrea extract, it was discovered that the secondary metabolism of those species is a natural source of various chemical substances with anti-fungal properties.Among them are flavonoids, saponines, tannins and proteolytic inhibitors (Ferreira et al., 2013).Thus, the anti-fungal potential of C. ferrea extract in controlling pathogens such as Colletotrichum guaranicola and Fusarium oxysporum were described by Bariani et al. (2012), in studies with husk extracts of C. ferrea in the evaluation of sporulation and mycelial growth of fungi in vitro, confirming the results obtained in this study.
In general, the larger seeds of S. obtusifolium had a better germinative performance most likely because they have a lower imbibition rate and were well nourished during their development resulting in well-formed embryos with a higher quantity of substance storage, which are the reasons why their tissues are more resistant to deleterious actions caused by microorganisms that slowly colonize and consume them.These attributes contribute to the reason that these seeds expressed higher physiological qualities in relation to the mother trees consisting of smaller seeds.Similar results were obtained by Oliveira et al. (2003), when the influences of fungi and seed size on germination and vigor of Rheedia gardneriana (Planch & Triana) were studied.
The germination percentage of inoculated and untreated seeds (T 2 ) is in accordance with the data obtained by Medeiros et al. (2015), who found that the fungal infection in C. ferrea seeds severely affected the physiological quality of the seeds, and in some cases completely inhibited germination.Medeiros et al. (2013) also reported that Pterogyne nitens Tul.seeds which are predisposed to the action of microorganisms, reduce the survival rate of pathogens and enhance the percentage and germination rate when treated.Thus, according to Cavalheiro et al. (2009), C. ferrea can be a new alternative in the search for active ingredients that are of interest to the biotechnology industry.With this in sight, more studies on the concentration and purification of compounds of the extract of this species is necessary.Hence, the use of products extracted from plants may be a viable alternative to control pathogens associated with seeds, with the added advantage of lessening the environmental impact of agrochemicals (Lazarotto et al., 2009).
Table 3 shows that the majority of the S. obtusifolium seed mother trees had higher values of symptomatic seedlings and the transmission rate of Colletotrichum sp. was higher in seeds that were only inoculated (T 2 ) and subjected to treatment with TABLE 2. Germination speed index (GSI) of Sideroxylon obtusifolium seeds, subjected to different treatments to control Colletotrichum sp.
* Means followed by the same letter, lowercase and uppercase in the column on the line, do not differ significantly at 1% probability (P ≤ 0.01) by Tukey test.T 1 = untreated seeds and not inoculated, T 2 = only inoculated seeds with Colletotrichum sp., T 3 = inoculated seeds and fungicide treated, T 4 = inoculated seeds and subjected to alternative control with C. ferrea extract, T 5 = inoculated seeds and submitted the biological control with Trichoderma spp.NOTE: Length of the seeds of different mother trees (M 1 -9.99 cm, M 2 -9.94 cm, M 3 -7.69cm, M 4 -7.51 cm, M 5 -7.45 cm and M 6 -6.54 cm).chemical fungicide (T 3 ) making them stand out from the others where in mother trees 1, 2 and with 36, 32 and 37% (T 2 ) and mother tree six with 30% (T 3 ) of the seeds contaminated also infected seedlings with transmission rates of 49, 55, 52 and 39% respectively.It is also noted that Colletotrichum sp.infected all of the seed lots except for those subjected to preventive treatments using C. ferrea extract (T 4 ) and Trichoderma spp.(T 5 ).Symptoms caused by Colletotrichum sp., such as necrotic lesions in cotyledons, young leaves, roots and stem seedlings followed by their falling over were observed in the evaluation of sanitary and physiological quality, through the transmission rate achieved during the seed germination process of S. obtusifolium.Similar results were obtained by Seneme et al., (2012) when evaluating the influence of Colletotrichum sp. on the sanitary quality of Peltophorum dubium (Sprengel) Taubert.seeds, as well as by Lazarotto et al. (2010) during the seedling production of Ceiba speciosa A. St.-Hil.which initially found wounds in the cotyledons and later in the shoots, starting with the wilt apex seedling, stem strangulation and death of the seedling.

Mother trees
Given the results observed, Colletotrichum sp.possibly colonized the S. obtusifolium embryonic tissues and then transmitted it to the seedlings.It was found that by treating the plants or seeds using plant extract the seeds were greatly protected, this is probably because this defense treatment was conducted by systemic action through their inhibition.According to Vechiato & Parisi (2013), this can be related to a possible intra-embryonic infection followed by a localized infection, since the symptoms appear during germination and the pathogen is driven by the cotyledons, causing symptoms in the shoots.The analysis of germination characteristics and sanity of forest seeds is an important factor, since there are few studies about this, especially with native species which are used in the process of forest restoration (Fantinel et al., 2013).
Several studies on forest species confirmed the seedling seed transmission of Colletotrichum sp.Lazarotto et al. (2010)  Different results from those detected in this study were reported by Oliveira et al. (2003), who compared control methods for Peltophorum dubium (Spreng.)Taub.seeds controlling Aspergillus niger, Colletotrichum sp. and Fusarium sp.where it was detected that the percentage of infected seeds did not affect germination, but this association may allow the survival of the fungus and its dissemination.In other hosts plant, such as Blepharocalyx salicifolius (H.B.K.) Berg.(Rego et al., 2012) and C. speciosa (Lazarotto et al., 2010), symptoms caused by Colletotrichum sp. to the seedlings have not been verified.According to the first author, even though fungal transmission from the seeds to the seedlings was not observed, it is known that fungi can cause damage both in storage, at germination and during subsequent growth stages.
However, similar results from those detected * Means followed by the same letter, lowercase and uppercase in the column on the line, do not differ significantly at 1% probability (P ≤ 0.01) by Tukey test.T 2 = only inoculated seeds with Colletotrichum sp., T 3 = inoculated seeds and fungicide treated, T 4 = inoculated seeds and subjected to alternative control with C. ferrea extract, T 5 = inoculated seeds and submitted the biological control with Trichoderma spp.NOTE: Length of the seeds of different mother trees (M 1 -9.99 cm, M 2 -9.94 cm, M 3 -7.69cm, M 4 -7.51 cm, M 5 -7.45 cm and M 6 -6.54 cm).

CONCLUSION
Biological control with Trichoderma spp.and the alternative test with C. ferrea extract provide greater protection to the seed and seedling of S. obtusifolium to the transmissibility of Colletotrichum sp., been the treatment based on the plant extract the most effective for this purpose, but only in larger seeds for not interfering in the germination percentage and speed.
studied the detection and fungal transmission on C. speciosa seeds and Vechiato & Parisi (2013) evaluated the influence of Colletotrichum sp., in the sanitary and physiological seed quality of Lithraea brasiliensis March., Myracrodruon urundeuva Fr.All., T. impetiginosa Mart.ex DC.Standl., Apeiba tibourbou Aubl., C. fissilis Vell.and Dalbergia nigra (Vell.)Allemão ex Benth.the destructive effects caused by this pathogen were noted and in some cases completely inhibited seed germination.

TABLE 3 .
Ferreira et al. (2013)tic seedlings (SS) and transmission rate (TR) of pathogens in seeds of Sideroxylon obtusifolium seeds, subjected to different treatments to control Colletotrichum sp. in this study were reported byPedro et al. (2012), according to these authors, the efficiency of chemical fungicides and some other formulas based on Trichoderma harzianum reduced the incidence of pathogens such as Colletotrichum sp.however,Ferreira et al. (2013)verified that the antifungal effect of C. ferrea extract on controlling C. lindemuthianum and C. truncatum, confirming the results obtained in the research with S. obtusifolium.