Microorganism resistance to the existing products is yet another difficulty that agriculturalists have to deal with. In this context, the search for new agricultural products that can fight phytopathogens has become increasingly important. Plants have played an important role in this process, because they can serve as a source of new compounds for drug discovery. Plants belonging to the genus Pinus produce an oleoresin that protects the plant against herbivores and pathogens. With a view to developing products that can combat fungal pathogens without harming the environment, this work aimed to evaluate the antifungal activity of the oleoresins and fractions of Pinus elliottii Engelm and Pinus tropicalis against phytopathogens. The methodology based on NCCLS M38-A standards aided antifungal activity assessment. The microdilution method helped to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC). The oleoresins of P. elliottii and P. tropicalis afforded the most significant results—they displayed fungicidal activity against all the tested species. MIC values were promising, especially the MIC of the oleoresin of P. elliottii against S. rolfsii (1.95 μg ·mL -1). The MIC values of the oleoresins of P. elliottii and P. tropicalis ranged from 1.95 to 1000 μg · mL -1 and from 31.25 to 250 μg ·mL -1, respectively. Fraction PT2 of P. tropicalis furnished the best results among all the assayed fractions: MIC values lay between 125 and 500 μg ·mL -11. In conclusion, the oleoresin of P. tropicalis is a promising source of new antifungal agents for application in the treatment of phytopathogenic infections.
Phytopathogenic diseases affect the amount and quality of agricultural produce. For this reason, controlling pathogenic fungi has become a matter of great concern for agriculturalists worldwide [
All around the world, consumers have sought to purchase products of organic origin; i.e., products that do not contain chemical residues and that grow in conditions that harm the environment to a minimum. In this context, plant-derived products are an attractive alternative. In particular, Brazil is rich in plants that may contribute to the development of new antifungal agents. Besides that, the genus Pinus has been planted in Brazil for over thirty years, especially in the southern region, where these plants have thrived in favorable climate conditions [
Traditionally, the oleoresin derived from coniferous Pinus can 1) function as an antiseptics or analgesic, 2) act by relieving cough and inflammation, 3) aid treatment of skin burns and wound diseases, and 4) fight pulmonary tuberculosis [
Studies have shown that various classes of diterpenes display potential antifungal activity against plant pathogens such as Colletotrichum gloesporioides [
Pinus tropicalis is a species of the genus Pinus that is rich in diterpenoids. It is believed to possess fungicidal activity, particularly against phytopathogenic fungi. Literature reports have stated that the oleoresin of Pinus tropicalis has other important biological activities. Leyva et al. [
Pinus elliottii Engelm is also known as common pine or American pine [
On the basis of evidence that plants belonging to the genus Pinus exert antifungal activity, and given the fact that the search for new agricultural products to combat these microorganisms has increased, this study aimed to evaluate the in vitro antifungal activity of the oleoresins and fractions of Pinus elliottii and Pinus tropicalis against eight phytopathogens.
Certified oleoresin of Pinus elliottii Engelm (PE; 100.0 mg) and Pinus tropicalis (PT; 120.0 mg) was kindly provided by ARESB (Associação dos Resinadores do Brasil). Bioguided-assay fractionation with P. tropicalis (PT) was performed, due to the fact that this oleoresin showed to be very effective against a panel of fungal. This plant material was subjected to vacuum chromatography over silica gel 60H (500 g; Merck, art. 7736) using n-hexane and increasing amounts of ethyl acetate as eluant (1500 mL each fraction). After solvent evaporation, this procedure afforded five fractions (PT1 - PT6); which were also assayed against fungal.
The phytopathogenic fungi assayed in this study, namely Macrophomina phaseolina, Lasiodiplodia theobromae, Colletotrichum gloeosporioides, Pestalotiopsis sp., Sclerotium rolfisii, Fusarium solani, Fusarium oxysporum, and Phytophthora infestans, were donated by Brazilian Corporation of Agricultural Research.
The fungal isolates were maintained in Laboratory of Research in Applied Microbiology of the University of Franca. The samples were kept in sterile water, at room temperature. The fungi employed here were isolated from infected plants, as depicted in
The Minimum Inhibitory Concentration (MIC) values were determined by the microdilution broth method in 96-well microplates, in triplicate. The samples were dissolved in dimethylsulfoxide―DMSO (Sigma-Aldrich) at 1.0 mg∙mL−1, followed by dilution in RPMI (Difco) with MOPS pH 7.2; concentrations ranging from 0.98 to 2000.0 μg∙mL−1 were achieved. The final DMSO content was 5%.
The inoculum was prepared on the basis of the standards recommended by the NCCLS M-38A [
Amphotericin B was the positive control at concentrations ranging from 0.031 to 16 µg∙mL−1. A reference strain of Aspergillus fumigatus (ATCC 204305) was included as reference fungi. The following controls were employed: negative (RPMI broth only), positive (RPMI plus inoculum, without addition of antifungal agent), and diluent (DMSO and inoculum).
Aliquots of the MIC wells were transferred to Sabouraud Dextrose Agar―SDA (Difco) plates without the drug. The plates were incubated at 28˚C for seven days, to provide the concentration that was fungicidal ―Minimum Fungicidal Concentration (MFC)―defined as the lowest concentration of the compound that did not generate visible microbial growth in the medium.
The present work evaluated the antifungal activity of P. tropicalis and P. elliottii against phytopathogenic fungi. According to Holetz et al. [
The antifungal activity of the oleoresin of P. elliottii varied from 1.95 to 1000 µg∙mL−1. This oleoresin was the most active against S. rolfisii (MIC = 1.95 µg∙mL−1) as well as F. solani and M. phaseolina (MIC of 62.5 µg∙mL−1
Fungi | Oleoresin | P. tropicalis fractions | Oleoresin | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P. tropicalis | PT1 | PT2 | PT3 | PT4 | PT5 | PT6 | P. elliottii | ||||||||||||
MIC | MFC | MIC | MFC | MIC | MFC | MIC | MFC | MIC | MFC | MIC | MFC | MIC | MFC | MIC | MFC | ||||
Pestalotiopsis sp. | 250 | 250 | 1000 | 1000 | 125 | 250 | 500 | 500 | 500 | 500 | 1000 | 1000 | 1000 | 1000 | 1000 | 1000 | |||
C. gloesporioides | 31.25 | 31.25 | 250 | 250 | 250 | 250 | 250 | 250 | 250 | 250 | 250 | 500 | 500 | 500 | 125 | 125 | |||
S. rolfsii | 250 | 250 | 1000 | 1000 | 500 | 500 | 500 | 500 | 500 | 500 | 250 | 250 | 125 | 125 | 1.95 | 1.95 | |||
F. oxysporum | 125 | 125 | 250 | 500 | 125 | 125 | 500 | 500 | 500 | 1000 | 250 | 250 | 500 | 1000 | 500 | 500 | |||
P. infestans | 250 | 250 | 500 | 1000 | 500 | 1000 | 1000 | 1000 | 1000 | 1000 | 1000 | 2000 | 1000 | 2000 | 1000 | 1000 | |||
L. theobromae | 250 | 250 | 250 | 250 | 125 | 125 | 125 | 125 | 250 | 250 | 500 | 500 | 500 | 500 | 1000 | 1000 | |||
F. solani | 62.5 | 62.5 | 1000 | 1000 | 500 | 1000 | 1000 | 1000 | 1000 | 2000 | 250 | 250 | 250 | 500 | 62.5 | 62.5 | |||
M. phaseolina | 125 | 125 | 500 | 1000 | 250 | 250 | 500 | 500 | 500 | 500 | 1000 | 1000 | 1000 | 1000 | 62.5 | 62.5 | |||
in both cases). This oleoresin displayed moderate activity against C. gloesporioides and F. oxysporum; it exerted a fungicidal effect on all the other tested fungi. To ensure the accuracy of the technique, amphotericin B was tested against A. fumigatus (ATCC 204305), to give a MIC value of 1.0 µg∙mL−1, which lay within the expected standard established by NCCLS M38-A [
To date, there have been no reports on the antifungal activity of P. tropicalis and P. elliottii, which has precluded comparisons with the work of other authors. Therefore, here we present some data obtained by other authors for the same pathogens; however, the compounds assayed in such works and the assessment techniques differed from those employed in the present study.
Most literature papers have evaluated the antifungal activity by incorporating the tested extracts in the culture medium, followed by inoculation with the tested fungus. The authors then monitored fungus growth.
Lins et al. [
David et al. [
Domingues et al. [
According to Rozwalka et al. [
Costa et al. [
Seixas et al. [
Analysis of the MIC and MFC results obtained in the present work evidenced that the fractions derived from the oleoresin of P. tropicalis afforded better MIC values than the corresponding oleoresin. Therefore, the antifungal properties of the oleoresin of this plant are possibly related to the synergism of the compounds present in it.
In conclusion, fraction PT3 of the P. tropicalis and the oleoresins of P. elliottii and P. tropicalis exerted a fungicidal effect on all the tested phytopathogens. Hence, the oleoresins of these species constitute a potential source of new antifungal agents to treat infections caused by phytopathogens, mainly C. gloesporioides the main causative agent of anthracnose in tropical fruits such as papaya, mango, guava and passion fruit, and F. solani, which causes root rot in soybeans. The antifungal activity of the oleoresins probably originates from the synergism of the compounds that constitute the oleoresin.
We thank CNPq (Brazilian National Research Council) which has awarded us a grant for this research.