The negative impact of synthetic pesticides on the environment and human health has highlighted the use of natural products as a promising alternative for biorational plant pest management7. Fungal secondary metabolites are considered a valuable source of molecules with a wide variety of biological applications. Various reports have pointed out the potential of these compounds for the control of insects and nematodes. One of the first detailed works on the insecticidal properties of fungal metabolites was published by Cole and Rolinson9. More recently, Asaff et al.4 showed that dipicolinic and oxalic acids, isolated from Paecilomyces fumosoroseus, have strong insecticidal effects on nymphs of Bemisia species. Likewise, Gaich and Mulzer12 showed that the sesquiterpenoid, penifulvin A (Penicillium griseofulvum) has antifeedant effects on Spodoptera frugiperda. In addition, nematotoxic activity has been reported in a wide variety of fungal metabolites, such as cytochalasin B, enniatin B, moniliformin, verrucarin A20, trichodermin36,37, and butyric acid from Paecilomyces lilacinus 602933.

Our research group has undertaken a long-term project on bioprospection of mycodiversity and its biotechnological application. In this sense, the search for microfungi in the tropical regions of Mexico has yielded a fungal collection whose mycelial extracts have been evaluated on several pathogenic bacteria and fungi, as well as the plant parasitic nematode Meloidogyne incognita13,29. None of these native fungi have been assessed against phytophagous insects. Therefore, in the present work, with the aim of broadening the search for new and more eco-friendly agents to control plant pests, twelve saprophytic fungi (Table 1) were selected, based on their biological profile. The fungi were cultured and their organic extracts were evaluated on the root-knot nematode Meloidogyne javanica (Neal) Chitwood (Nematoda: Meloidogynidae), the poligophagous Lepidoptera Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), and aphids Myzus persicae Sulzer (Hemiptera: Aphididae) and Rhopalosiphum padi L. (Hemiptera: Aphididae). In addition, one of the most active extracts was partitioned and its active constituents were identified through bioassay-guided fractionation.

The results of settling inhibition, feeding reduction, and nematotoxic activities are shown in Table 2. None of the extracts inhibited the mobility of M. javanica (MI<50%) or the feeding of S. littoralis (FR<70%). In contrast, the EA extracts of G. masseei, Perelegamyces parviechinulatus and Verticillium sp. TH28, and the ME extracts of Clonostachys rosea and Corynespora cassiicola, Phialophora verrucosa, and Spadicoides sp. inhibited the settling of M. persicae and R. padi (SI>80%; Table 2). The extracts of C. rosea and G. masseei caused strong settling inhibition of both aphid species. The dose–response evaluations for these extracts are shown in Table 3. For M. persicae, the ME extract of C. rosea was the most active, with an EC50 value of 8μg/cm2; whereas for R. padi, the EA extract of G. masseei was the most active with an EC50 value of 19μg/cm2.

Molecular identification by using ITS1 and ITS4 primers resulted in a DNA band of ∼650bp. After edition, 561bp were used as query in BLAST, finding the highest homology with G. masseei (first hit accession AB540554.1). Query cover about 97% of known G. masseei ITS fragment, with an E value of 0.0 and 99% of identity.

The chemical composition of the EA extract of G. masseei was determined by a bioassay-guided fractionation. The hexane (ARGM-1A) and acetonitrile (ARGM-1B) fractions had strong effects on settling inhibition of M. persicae (>85%) and R. padi (SI>70%) (Table 3). Analysis of fraction ARGM-1A and its methyl derivative (ARGM-4) by GC–MS and by comparison with commercial standards (free and methyl esters) revealed the presence of four fatty acids as major components. Fatty acids included hexadecanoic acid (16:0), octadecanoic acid (18:0), 9-octadecenoic acid (18:1) and 9,12-octadecadienoic acid (18:2) (Table 4). The most abundant components were hexadecanoic acid (74%) and 9,12-octadecadienoic acid (16.8%). In addition, ARGM-1A fraction was methylated and the product, ARGM-4, also had an effect on the settling of both aphid species.

Purification of the second fraction ARGM-1B by silica gel columns yielded a complex mixture of compounds which included ergosterol. This compound was isolated and identified by comparison with a commercial sample (E6510-10G, Sigma–Aldrich). The subfractions were not active on aphid settling.

The compounds isolated in this work and commercial standards were evaluated on both aphid species (Table 5). Methyl hexadecanoate had a low effect on the settling of R. padi and was not tested on M. persicae. Methyl 9-octadecenoate strongly inhibited the settling of both aphid species (SI>70%); likewise, methyl 9,12-octadecadienoate only inhibited the settling of M. persicae (SI>70%). Ergosterol showed no activity. Dose–response evaluation of both active pure esters showed that methyl 9-octadecenoate was the most active compound, with an EC50 value of 16μg/cm2 for M. persicae and 35μg/cm2 for R. padi (Table 5).

In the present work, the evaluation of organic extracts of microfungi showed that the ME and EA extracts had different effects on the plant pest targets. The extracts had very low or no effect on S. littoralis or M. javanica. However, various extracts displayed a strong effect on the settling of aphids M. persicae and R. padi. To our knowledge, the fungal extracts evaluated in the present work had not been previously assessed on aphid settling. The results showed that 30% of the evaluated fungi produced metabolites that deterred aphids. The most active were the EA extract of G. masseei (EC50, 15–19μg/cm2) and the ME extract of C. rosea (EC50, 8–38μg/cm2).

The molecular analysis identified fungus G. masseei. Minor discrepancies between the query and retrieved sequences were observed, however identity was supported by the congruency of molecular identification with traditional taxonomy performed according to Kiyuna et al.19. Thus, the ITS fragment was enough to identify the fungus and discrepancies can be attributed to usual polymorphism among strains. The fungus G. masseei is a cosmopolitan species that has antimicrobial properties28; however, no studies on identification of secondary metabolites produced by this species have been reported. The primary fractionation (ARGM-1A and 1B) of the extract revealed that G. masseei produces metabolites that affect aphid settling. These metabolites were detected in the low and medium polarity fractions. In the low polarity fraction, fatty acids and their derivatives are the most abundant components. Although hexadecanoic acid (75%) was the major constituent, its effect on the settling of M. persicae was produced only by their derivatives methyl and ethyl esters (5.5–8.7μg/cm2), in agreement with previous reports32. Surprisingly, these compounds showed no effect on the settling of R. padi. Other components of the low polarity fraction were the methyl esters of 9,12-octadecadienoic acid (17.8%) and 9-octadecenoic acid (9.3%). In this case, previous studies have shown that the acid forms of these compounds have anti-aphid properties8. In the present study, methyl 9-octadecenoate was the most active compound with an EC50 of 16 and 35μg/cm2, for M. persicae and R. padi, respectively (Table 5). Methyl 9,12-octadecadienoate, in contrast, only inhibited the settling of M. persicae (EC50=20μg/cm2), but not of R. padi.

During the purification of the extract, we observed that the settling inhibition activity remained in low and medium polarity fractions for M. persicae, but decreased for R. padi, where SI values decreased from crude extract (91.9) to 70 and 76%, respectively. An opposite trend was observed for the natural methylated ARGM-4 fraction (SI=84%). We then verified the activity of the isolated methyl ester mixture and an artificial mixture of commercial standards of the major constituents present in the fraction ARGM-1A. The mixture was prepared with the methyl esters of the hexadecanoic, 9,12-octadecadienoic, and 9-octadecenoic acids (27:6:1). The methyl 9-octadecenoate and natural mixture of hexane fraction inhibited the settling of both aphids (SI>70%); however, 9,12-octadecadienoate and the artificial mixture only inhibited the settling of M. persicae, in the same manner as the saturated methyl hexadecanoate. These data showed that on esterified fatty acids, the pattern for anti-aphid activity is different in comparison with that of the free unsaturated fatty acids8. The difference between the effects on the settling of R. padi caused by the artificial and natural mixtures can be attributed to the presence of other minor unknown constituents in the latter.

The biological activity of fatty acids and their derivatives on pest insects have been previously reported27. For example, 9,12-octadecadienoic acid has shown activity on Aedes aegypti, Callosobruchus maculates, Lymantria dispar, Liposcelis bostrychophila, and Malacosoma disstria; 9-octadecenoic acid on Anopheles aegypti, Anopheles stephensi; 9,12-octadecadienoic, hexadecanoic and octadecanoic acids on S. frugiperda1,18,25. Fatty acids can be extracted from plants and from fungi. In this sense, various studies have found that different species of fungi, such as Aspergillus ustus, Cladosporium sp., Curvularia sp., Penicillium sp., and Rhizopus oryzae produce this type of compounds23,24,31.

The TLC and GC–MS analyses of acetonitrile fraction of G. masseei showed a complex mixture of compounds, where traces of ergosterol, a component of the fungal membrane, were observed. Ergosterol is commonly oxidized in the presence of light into ergosterol endoperoxide. There are various reports on the strong antimicrobial activity of ergosterol endoperoxide, but no data on its effect against insects has been published5,30. Although ergosterol endoperoxide was not detected in the G. masseei extracts, its effect was evaluated on aphid settling, where this compound slightly inhibited the settling of M. persicae and R. padi (Table 5).

It is important to note that the activity of the full extract of G. masseei, compared to that of the fractions, subfractions and commercial standards, suggests that the anti-aphid effect was most likely due to the synergistic effect of the known and unknown minor components; however, further investigation is required in this regard.

The fungus C. rosea is a cosmopolitan species which has shown a wide range of biological activities, in particular as a biocontrol agent against phytopathogenic fungi and nematodes21,34. The activity in this case has been attributed to secondary metabolites, such as gliocladine A-E, verticillin A and 11′-deoxy-verticillin A, Sch52900, and Sch5290111. In previous works, we have found that the EA extract of C. rosea is active against human pathogenic bacteria, plant pathogenic fungi, and plant parasitic nematodes13,14. Surprisingly, in the present work, the EA extract of C. rosea showed no activity on M. javanica.

It is also pertinent to mention that the effect of the most active fungal extracts evaluated in the present work was similar to that of other natural products isolated from microorganisms or plants3.

In summary, the settling inhibition capacity of the fungal extracts screened in this work highlights the potential of these compounds for biorational plant protection. ME extracts of C. rosea and EA extracts of G. masseei showed the highest anti-settling activity on M. persicae and R. padi. Furthermore, we observed that ester derivatives of hexadecanoic, 9-octadecenoic and 9,12-octadecadienoic acids were some of the active principles and the most abundant components in the EA extract of G. masseei, which may have a synergistic effect with other unknown minor constituents. This work also contributes with insights into the biological activity of natural products derived from micromycetes isolated from the tropical regions of Mexico.

The authors declare that there is no conflict of interests regarding the publication of this article.