Microbial and Plant Derived Low Risk Pesticides Having Nematocidal Activity

Microorganisms, virus, weeds, parasitic plants, insects, and nematodes are among the enemies that induce severe economic losses to agrarian production. Farmers have been forced to combat these enemies using different methods, including mechanical and agronomic strategies, since the beginning of agriculture. The development of agriculture, due to an increased request for food production, which is a consequence to the rapid and noteworthy growth of the world’s population, requires the use of more efficient methods to strongly elevate the yield production. Thus, in the last five-to-six decades, a massive and extensive use of chemicals has occurred in agriculture, resulting in heavy negative consequences, such as the increase in environmental pollution and risks for human and animal health. These problems increased with the repetition of treatments, which is due to resistance that natural enemies developed against this massive use of pesticides. There are new control strategies under investigation to develop products, namely biopesticides, with high efficacy and selectivity but based on natural products which are not toxic, and which are biodegradable in a short time. This review is focused on the microbial and plant metabolites with nematocidal activity with potential applications in suitable formulations in greenhouses and fields.


Introduction
Since ancient times, agriculture has been developed to produce food to satisfy the human needs. This request became an emergency in parallel with the increasing world population, which could reach to almost 10 billion by 2050 [1,2]. Unfortunately, this request was negatively affected by a strong reduction in natural resources, the diffused environmental pollution, and noteworthy climate changes [3,4]. In addition to these factors, farmers are forced to combat the natural enemies of agrarian plants, such as pathogens, bacteria, fungi, viruses, weeds, parasitic plants, dangerous insect, and nematodes [5][6][7]. The spread and survival of these enemies has been controlled using different methods, including mechanic and agronomy strategies. However, in the last five-to-six decades, a massive and extensive use of chemicals has occurred, with heavy consequences in terms of environmental pollution and risks to human and animal health. These problems have increased with the repetition of treatments, which are due to the resistance that enemies have developed against the pesticides used for a long time [5,8]. Thus, several multidisciplinary research groups have investigated new control strategies to develop products with high efficacy and selectivity against agrarian pests but based on natural products which are not toxic, and which are biodegradable in a short time [8][9][10][11]. Regarding the previous reviews on metabolites with nematocidal activity isolated from natural sources, SciFinder research was used to locate the article of Lorenzen and Anke (1998) [12], which describes some metabolites with insecticidal and nematocidal activities together with several natural compounds with cytotoxic, antitumoral, antiviral, and phytotoxic activities. Another review extensively describes the insecticidal activity of fungal metabolites, while only a short paragraph is dedicated to the fungal compounds with nematocidal activity. In particular, the peptides produced by Omphalotus spp. [13] are reported. In addition, a review only describes the fungal metabolites belonging to the azophilone family, with several different biological activities with very few compounds showing nematocidal activity [14]. Similar content is reported by Shen et al. (2015) [15], but in terms treating benzenediol lactones with a variable structures and belonging to family of fungal polyketides. Mao et al. (2014) [16] describes natural dibenzo-α-pyrones produced by fungi, mycobionts, plants, and animal feces which exhibit a variety of biological activities including nematocidal properties. Similar content was previously described by Ghisalberti (2002) [17]. Another review only describes the saponins isolated from Medicago sativa L., alfalfa, which is the most known plant species within the Medicago genus, and their nematocidal activity against different nematodes species [18]. Another review deals with extracts or secondary metabolites of the Mexican flora that showed biological activity against dangerous insects or parasitic nematodes [19]. A review about the metabolites produced by extremophilic fungi and belonging to different classes of natural compounds reported them as having different biological activities, including a nematocidal one [20]. One of the last biocontrol methods reported to combat nematodes is based on the changes in soil microbial community, the release of nematocidal compounds, and the induction of plant defenses. This strategy essentially uses biochar-based soil amendments, which is ecofriendly and compatible with a circular economy. However, as biochars induce complex and distinct modes of action, their nature and application regimes should be studied for particular pathogens and their effects must be locally checked [21]. Thus, all previous reviews only partially report about natural compounds with nematocidal activity, as some are restricted to one family of fungal or plant metabolites, some others report metabolites from different natural sources, and they also describe a lot of diverse natural compounds with different biological activities. Others extensively discuss fungal metabolites with insecticidal activity and few compounds with nematocidal activity, and some also cover a short time-span within the literature (2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020).
Thus, the present manuscript reports, for the first time, an overview which is focused on the microbial and plant metabolites with nematocidal activity with potential applications in suitable formulations in greenhouses and fields. The results discussed in the different sections were obtained from SciFinder research covering from 1995 to today, and these are chronologically reported in each paragraph.

Bacterial Metabolites
This section chronologically describes the source, structure, and biological activity of the bacterial metabolites, most of which showed nematocidal activity together with other interesting biological activities.
The avermectins and milbemycms are closely related 16-membered macrocyclic lactones produced by actinomycetes from the genus Streptomyces. Streptomyces avermentilis synthesized avermectins while Streptomyces hygroscopicus and Streptomyces cyaneogriseus produced milbemycms. Avermectins and milbemycins, are constituted by the identical 16-membered macrocyclic backbones which is fused with a hexahydrobenzofuran unit from C-2 to C-8a and a spiroketal unit from C-17 to C-25. The main difference diffrences between the two groups is the substituent at C-13 of macrolactone that is a disaccharaide in avermectin, whereas that position is unsubstituted in milbemycin. Avermectin B 1a and milbemycin D (1 and 2, Figure 1), are representative members of the two groups and thus compound 2 appeared to be the aglyvcone of metabolite 1. The avermectins are produced as ues, 380 and 280 µg/mL, respectively). The pigment also exhibited inhibition on nematode egg-hatching ability [31].  Thumolycin (11, Figure 1), which is a lipopeptide, was isolated from Bacillus thuringiensis, which is a bacterium widely used as a bio-insecticide. Thumolycin has a molecular weight of 696.51 Da and a predicted molecular formula of C 38 H 64 N 8 O 4 , but its structure has not yet been determined. Compound 11 showed a broad spectrum of antimicrobial and nematocidal activities. In particular, C. elegans was significantly inhibited by thumolycin (11), which, when tested at a higher concentration, induced a higher mortality in this nematode. When C. elegans was treated with 600 U of thumolycin, less than 10% nematodes survived. These experiments indicated that thumolycin has inhibitory activity against a wide range of bacteria and effective nematocidal activity [32].
Aureothin and alloaureothin (12 and 13, Figure 1) were isolated from an endophytic bacterium strain, which was identified as Streptomyces sp. AE170020, and appeared to be a rich source of bioactive secondary metabolites with potential as environmentally benign agents. In fact, both metabolites 12 and 13 showed a significant nematocidal activity suppressing the growth, reproduction, and behavior of B. xylophilus. Pine trees are one of the most important forest plants in ecosystems, as they are widespread in natural reserves, parks, and urban ornamental landscapes, and are also source of wood with high economic value for different practical uses. Unfortunately, pine is affected by different pests, such as the pathogen fungus Sphaeropsis sanipea, a producer of phytotoxins [33], and by pine wood nematode (PWN

Fungal Metabolites
This section chronologically reports the source, structure, and biological activity of the fungal metabolites, most of which showed nematocidal activity together with other interesting biological activities.
Some studies were carried out on five Arthropotrys strains to examine their ability to produce metabolites with nematocidal activity. In fact, the well-known linoleic acid was isolated from Arthrobotrys conoides and Arthrobotrys oligospora [35]. A lot of fungi belonging to Ascomycetes Pyrenomycetes and Discomycetes genera were the object of a similar investigation. A total of 29 isolates belonging to 18 genera produced metabolites with nematocidal activity against C. elegans, out of a total of 267 extracts of culture filtrates of the different stains examined [35]. Here, 1-Methoxy-8-hydroxynaphthalene, 1,8-dimethoxynaphthalene, and 5-hydroxy-2-methyl chromanone (14-16, Figure 1) were isolated from Daldinia concentrica [36]. Both naphthalene derivatives (14 and 15) showed nematocidal activity against C. elegans with LD 50 values of 10 and 25 µg/mL, respectively, in addition to cytotoxic and antimicrobial effects, while the chromanone 16 had no nematocidal activity [35].
Here, 14-Epi-dihydrocochlioquinone B and 14-epi-cochlioquinone B (17 and 18, Figure 1) were isolated from Neobulgaria pura [37] and showed nematocidal activity towards C. elegans and Meloidogyne incognita [35]. Furthermore, the close cochlioquinone A (19, Figure 1), which was produced by a Helminthosporium species, competed for the ivermectin binding site on the membrane receptor in nematodes [38]. Ivermectin is the didroderivative of averctim, which as abovereported was originally isolated from soil in Japan as a part of a collaborative program to select microorganisms on the basis of novel microbiological characteristics and a wide variety of pharmacological and chemotherapeutic assays [39].
Here, 5-Pentyl-2-furaldehyde (21, Figure 1), was isolated as a metabolite with nematocidal activity from the culture filtrates of an unidentified species of the Dermateaceae family. The strain was collected in Australia. Compound 21 was also isolated from Irpex lacteus, which is a wood-inhabiting basidiomycete [40], and showed moderate activity against Aphelenchoides besseyi, M. incognita, and C. elegans with LD 50 values 60 of and 75 µg/mL when tested on the penultimate and last nematode, respectively [40].
Thermolides A-F (65-70, Figure 3), which belong to a class of PKSNRPS hybrid metabolites constituted by a 13-membered lactam-bearing macrolactone, were isolated from a thermophilic fungus Talaromyces thermophilus. Macrocyclic PKS-NRPS hybrid metabolites are a unique family of natural products, essentially produced by bacteria with broad and outstanding biological activities. All the metabolites 65-68 were assayed against three types of nematodes, including the root-knot nematode M. incognita, pine-wood nematode B. siylopilus, and free-living nematode Panagrellus redivevus [61]. Compounds 65 and 66 showed the strongest activities against all the worms, with LC 50 values ranging from 0.5-1.0 µg/mL, similar to those of the avermectin used as control, while compound 67 and 68 had, respectively, moderate and weak inhibitory effect on the same organisms [62]. The gene ThmABCE from this fungus is fundamental for thermolide synthesis. Furthermore, a heterologous and engineered expression of the Thm genes in Aspergillus nidulans and E. coli induced a strongly increased yield not only in thermolide production, but also in that of different esterified analogues, such as butyryl-(thermolides J and K) hexanoyl-, and octanyl-derivatives or mixed thermolides. In addition, thermolides L and M were also obtained via genome mining-based combinatorial biosynthesis, and represent the first L-phenylalanine-based thermolides [63].
Grammicin (78, Figure 4), which is a dihydrofuranone, was isolated from Xylaria grammica KCTC 13121BP, showing a strong nematocidal activity against M. incognita. The fungus was isolated from a lichen, Menegazzia sp., which was collected on Giri Mountain in Korea [67]. Compound 78, which was also previously isolated from the same fungus collected from wood in Cameroon and Peru [68], is a structural isomer of the well-known mycotoxin patulin (79, Figure 4). The latter compound (79) was first isolated in 1943 from Penicillium griseofulvum and Penicillium expansum [69] and then, as recently reviewed [70], from several species belonging to not less 30 genera including Penicillium, Aspergillus, Paecilomyces, and Byssochlamys. Compound 79 is the most common mycotoxin found in apples and apple-derived products and other food, and is associated with immunological, neurological and gastrointestinal outcomes with high human health risks [71]. Grammicin (78) showed strong nematocidal activity against M. incognita in J2 juvenile mortality and eggs-hatching inhibition with EC 50 values of 15.95 and 5.87 µg/mL, respectively, compared to trans-cinnamaldehyde used as positive control, which showed in both assay EC 50 values of 18.34 and 10.50 µg/mL, respectively. The same compound exhibited weak antibacterial effects against several microorganisms responsible for severe crop diseases [67]. Furthermore, it exhibited very low or no cytotoxic activity when assayed against a human first-trimester trophoblast cell line SW.71. Instead, patulin (79), in the same bioassays, showed a weak nematocidal EC 50 /72 h value of 115.67 µg/mL and strong antibacterial and cytotoxic activities. In addition, compared with trans-cinnamaldehyde, grammicin (78) showed comparable J2 killing activity but a stronger egg-hatching inhibitory effect. These results suggest that grammicin and its fungal producer have potential for biocontrol of root-knot nematode disease in crops [67].

Plant Metabolites
This section chronologically describes the source, structure, and biological activity of plant metabolites, most of which showed nematocidal activity together with other interesting biological activities.
Twenty-four secondary metabolites were isolated from Bupleurum salicifolium [75], which is a plant native to the western Canary Islands from Gran Canaria to El Hierro, where it is frequently found up to 1000 m above sea level [76]. The plant is highly specialized in biosynthesizing secondary metabolites, principally lignans, coumarins, and flavonols, which all derive from shikimic acid and belong to different classes of natural compounds (Dewick, 2002) [77]. All the metabolites were tested against viruses, grampositive and gram-negative bacteria, the yeast Candida albicans, the nematodes G. pallida and G. rostochiensis, the insect Spodoptera littoralis, and the crustacean Artemia salina. These compounds were also tested against tumoral and non-tumoral cell lines. In particular, considering the limited amount available, only dibenzyl-butyrolactone, lignans, such as guayarol, buplerol, matairesinol and its dimethyl ether, bursehernin, pliviatolide, thujaplicatin, methyl ether (96-102, Figure 5) and 2-chloro-matairesinol, nortrachelogenin, nortrachelogenin triacetate, and 2-hydroxy-thujaplicatin-methyl ether (103-106, Figure 5) were tested on potato cyst nematode hatching using G. pallida and G. rostochiensis. After 14 days, all the compounds assayed stimulated the hatching of more juveniles than distilled water (negative control). In particular, matairesinol and bursehernin (98 and 100) significantly reduced hatching by 70% and 55%, respectively, when compared to the positive control agent. The HID recorded for bursehernin (100) was 16.42 µg/mL, while no differences were observed in the inhibition of G. pallida or G. rostochiensis. These results suggested that the presence of a methylene-dioxy group in the aromatic ring B of the dibenzyl-butyrolactone skeleton plays a significant role to impart nematostatic activity. When the methylene-dioxy group was substituted by a methoxy and a hydroxyl group, as in buplerol (97), or two hydroxy groups, as in guayarol (96), a significant reduction in the activity was observed. Furthermore, in compounds lacking the methylenedioxy group, the activity increased according to the number of free hydroxy groups present, as observed in compounds 96 > 98 > 97 > 105. Nortrachelogenin triacetate (105) bears an acetyl group at position 2 in the lactone ring, which could be a consistent steric hindrance between this compound and the receptor on the nematode eggshell whose existence was hypothesized by Atkinson and Taylor (1980;1983) [78,79]. None of the compounds tested showed nematocidal activity when tested on second-stage juveniles of G. pallida and G. rostochiensis [75]. Successively, some of the same authors tested 22 aromatic derivatives and the conjugated carbonyl compound t-3-penten-2-one for nematocidal activity against the same 2 nematodes, namely G. pallida and G. rostochiensis. Among all the compounds assayed, nine showed high toxicity on the infective stages (second instar juveniles) of the nematodes, with a LC 50 ranking from 2 × 10 −6 to 1.26 × 10 −3 M. As expected, the toxicity is due to the presence of a conjugated carbonyl system [80].  The cyclic hydroxamic acids are common secondary metabolites found in plants of the Poaceae family, such as corn, wheat, and rye, and known for the allelopathy of rye (Secale cereale). The latter plant is well-known for its allelopathic activity. Some of cyclic hydroxamic acids, such as DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one), DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one) (107 and 108, Figure 5), and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxybenzoxazolin-2(3H)-one) (109 and 110, Figure 5) are commercially available and, thus, were used to test their toxicity against M. incognita second-stage juveniles (J2) and eggs and mixed-stages of Xiphinema americanum (X. americanum). The LC 50 value of 74.3 µg/mL for DIBOA was recorded when assayed against M. incognita eggs after 168 h exposure, while there was no possible recorded any value for the other compounds. In the assay on M. incognita J2 mortality, the LD 50 values were 20.9, 46.1, and 49.2 µg/mL for compound 107, 108, and 110, respectively. For compound 108, no value was measured. In the assay against X. americanum, the LD 50 values recorded after 24 h of exposure were 18.4 and 48.3 µg/mL for compounds 107 and 108, respectively, while compounds 109 and 110 had no effect on nematode mortality. These results showed that X. americanum was more sensitive to DIBOA and DIMBOA (107 and 108) than M. incognita J2, while eggs of M. incognita were less sensitive to the hydroxamic acids than J2. Only DIBOA (107) resulted in a 50% reduction in egg hatching; MBOA (110) was not toxic to X. americanum or M. incognita eggs but was toxic to M. incognita J2. Furthermore, BOA (109) was the least toxic hydroxamic acid tested and did not reduce M. incognita egg hatching after 1 week of exposure or increase X. americanum mortality after 24 h of exposure. These results showed that the presence of 4-hydroxy-2H-1,4-oxazin-3(4H)-one is determinant of the imparted toxicity, as this activity was strongly reduced or lost when this residue was substituted by oxazol-2(3H)-one [81].
Ruixianglangdusu B, umbelliferone, chamaejasmenin C, daphnoretin 7-methoxyneoch aejasmin A, (+)-chamaejasmine, chamaechromone, and isosikokianin A (114-120, Figure 5) were isolated from the organic extract of Stellera chamaejasme L. roots, which showed significant nematocidal activity against B. xylophilus and Bursaphelenchus mucronatus [83].  compound 114, 118 and 121 showed weak activity when tested in the same conditions. The nematocidal activities of the eight purified compounds against B. mucronatus were similarly observed at 72 h after treatment but the toxicity values of compounds 117 and 120 were highest at a concentration of 400 µM. The nematocidal activity of compound 119 was strongest against B. mucronatus at the lowest test concentration, while the most toxic compounds were 114, 116, and 120, with LC 50 values ranging from 0.003 to 0.6 µM, which were comparable with that of the lambda cyhalothrin (LC 50 = 1.1 µM) used as the positive commercial control [83].
Medium-chain fatty acids and phenolic acids were the main component of the organic extract of Picria fel-terrae. The plant extract showed toxicity against free-living nematode C. elegans and the parasitic nematode Haemonchus contortus, killing C. elegans adults and inhibiting the motility of 48 exsheathed L3 of H. contortus. The same extract had minimal cytotoxic activity in mammalian cell 49 culture [84].
A screening was carried out carried out for 790 plant metabolites, including those obtained from Tagetes spp., Azadirachta indica, and Capsicum frutescens, and involved testing their nematocidal activity against C. elegans. A total of 10 compounds proved to be toxic, 3 of which were further evaluated for their inhibitory activities against egg hatching of C. elegans and J2 M. incognita and the wild nematode N2 L4 eggs. Only 1,4-naphthoquinone (122, Figure 5) appeared to be an active compound that could not only kill N2 L4 nematodes (LC 50 42.26 ± 2.53 µg/mL), and inhibit egg hatching of N2 (LC 50 34.83 ± 0.58 µg/mL), but also showed toxicity on more than 50% of M. incognita at a concentration of less than 50 µg/mL (LC 50 33.51 ± 0.21 µg/mL). The results obtained using C. elegans demonstrated that compound 122 could influence reactive oxygen production, superoxide dismutase activity, and the heat-shock transcription factor (HSF)-1 pathway, suggesting that compound 122 stimulated significant oxidative stress [85].
Additionally, 3β-Angeloyloxy-6β-hydroxyfuranoeremophil-1(10)-ene (134, Figure 6), the main secondary metabolite extracted from the roots of Senecio sinuatos, showed nematocidal activity against the second-stage juveniles (J2) of M. incognita and N. aberrans. Compound 134 was alkaline hydrolyzed to produce a derivative which, in turn, was differently esterified with anhydride acetic, benzoic acid, 2-nitrobenzoic acid, 2-bromobenzoic acid, 4-nitrobenzoic acid, 4-bromobenzoic acid, and 4-methoxybenzoic acid to produce the corresponding 6-O-acetyl ester and benzoyl esters. All compounds and the corresponding benzoic acids were tested for nematocidal activity against M. incognita and N. aberrans J2 using fluopyram as a positive control. In particular, the benzoyl esters possess more nematocidal activity than the corresponding free benzoic acids, while compound 134 had nematocidal activity against M. incognita when assayed at 10 µg/mL, and this effect was more nematostatic as the concentration decreased at the most effective time of 72 h [99].

Conclusions
Nematodes are one of several enemies that induce severe economic losses in agrarian production, forcing farmers to use different methods to prevent their growth and diffusion. Among these methods, the last five-to-six decades have seen a massive and extensive use of chemicals, with heavy negative consequences, such as an increase in environmental pollution and risks for human and animal health. A negative effect of the use of chemicals in agriculture is also their noteworthy contribution to climate change. The development of new control strategies based on natural products with high efficacy and selectivity has become an emergency. This review reports, for the first time, a complete overview of the microbial and plant metabolites with nematocidal activity and, thus, they are a potential applications in suitable formulations in greenhouses and fields. All the results described are summarized in Table 1. The compounds selected for their efficacy and specific nematocidal activity should be investigated firstly for their human, animal, and environmental toxicological effects. Then, for the promising compounds, a total, convenient, and ecofriendly synthesis should be developed for their large production at an industrial level.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Conflicts of Interest:
The author declares no conflict of interest.