Metabolites from nematophagous fungi and nematicidal natural products from fungi as alternatives for biological control. Part II: metabolites from nematophagous basidiomycetes and non-nematophagous fungi

In this second section of a two-part mini-review article, we introduce 101 further nematicidal and non-nematicidal secondary metabolites biosynthesized by nematophagous basidiomycetes or non-nematophagous ascomycetes and basidiomycetes. Several of these compounds have promising nematicidal activity and deserve further and more detailed analysis. Thermolides A and B, omphalotins, ophiobolins, bursaphelocides A and B, illinitone A, pseudohalonectrins A and B, dichomitin B, and caryopsomycins A–C are excellent candidates or lead compounds for the development of biocontrol strategies for phytopathogenic nematodes. Paraherquamides, clonostachydiol, and nafuredins offer promising leads for the development of formulations against the intestinal nematodes of ruminants.


General remarks
The chemical ecology of nematophagous fungi is still far from understood. Little has been done to screen for metabolites in nematophagous fungi, or nematicidal metabolites in other fungi, since the pioneering studies by Stadler and colleagues published in the 1990s (Stadler et al. 1993a(Stadler et al. , b, 1994a. In the first part of this review, we discussed 83 primary and secondary metabolites from nematophagous ascomycetes (Degenkolb and Vilcinskas, in press). In this second installment, we consider nematicidal metabolites from nematophagous basidiomycetes and from those fungi that are currently regarded as non-nematophagous species. The numbering system for the compounds introduced here begins at 84 to provide continuity with the first part of the review.
Given that species parasitizing nematodes or their eggs are found in all major fungal phyla including Chytridiomycota, Ascomycota, Basidiomycota, and also the Zoopagomycotina and Mucormycotina, 1 multiple and independent evolution of nematophagy was hypothesized (Barron 1977). The scenario of nematode-fungus associations may be far more complex than previously thought. This was recently exemplified by Morris and Hajek (2014) who reported on the parasitic nematode Deladenus siricidicola (Tylenchida: Neotylenchidae), which is used for biocontrol of the invasive pine-killing woodwasp Sirex noctilio (Hymenoptera: Siricidae). In its mycophagous phase, D. siricidicola feeds exclusively on the growing hyphal tips of its basidiomycete host Amylostereum areolatum (Russulales: Amylostereaceae). The presence of woodwasp larvae triggers the nematode to change its life style-it invades the wasp larvae and sterilizes most of them. Notably, the white-rot fungus, which has so far been thought to serve as food source for Deladenus sp., was also shown to (1) invade the vulva of adult female mycophagous nematodes and (2) to kill and invade nematode eggs. Eggs were parasitized by the hyphal tips of the fungus whereas cystidia seemed to colonize the vulva of adults. It remains to be clarified if a toxin is also involved in the infection process, Authors are aware of the fact that missing evidence does not necessarily imply a non-nematophagous life style of a fungus. However, for reasons of convenience and consistency with literature, we prefer to retain the terminus Bnon-nematophagous^for those associations without evidence for nematophagy.

Metabolites from the genus Pleurotus
The small but monophyletic family Pleurotaceae comprises nematophagous white-rot fungi (Thorn et al. 2000;Kirk et al. 2008). Members of the genus Pleurotus, such as the oyster mushroom Pleurotus ostreatus, have been shown to secrete tiny toxin-containing droplets, which effectively paralyze a nematode without killing it within 30 s of contact. The prey is subsequently penetrated by the fungal trophic hyphae and digested within 24 h (Thorn and Barron 1984; Barron and Thorn 1987).
The first nematicidal compound isolated from the genus Pleurotus was (E)-2-decenedioic acid (84). P. ostreatus NRRL 3526 (= ATCC 90520) was grown for 30 days at room temperature (21-23°C) on autoclaved, damp wheat straw. Thereafter, an aqueous extract of the colonized substrate was filtered, and the filtrate was freeze-dried. After reconstitution of the lyophilizate in water, the organic fraction of the extract was further purified, finally by HPLC of the acetone-soluble fraction. The nematicidal principle, compound 84, which eluted as a single peak, was characterized by MS and NMR. An aqueous solution of pure 84 at a concentration of 300 μg/ml caused the immobilization of 95 % of a test population of the nematode Panagrellus redivivus within 1 h. Notably, this effect could not be reversed by rinsing the treated nematodes with deionized water. Organic extracts of a static straw culture have not been prepared and investigated for possible nematicidal activity (Kwok et al. 1992).

Metabolites from the genus Nematoctonus
Nematoctonus robustus, the anamorph of Hohenbuehelia grisea 2 (Agaricales, Pleurotaceae), is able to trap nematodes conidia, which form sticky knobs upon germination (Dowe 1987). N. robustus CBS 945.69 was grown in a fermenter at 24°C for 11 days until the antimicrobial activities of the extracts did not increase any further. The bioactive principle consisted of dihydropleurotinic acid (103) and pleurotin (104), two 1,4-naphthoquinone antibiotics, and leucopleurotin (105), a precursor thereof. Biosynthesis of pleurotin involves a farnesylhydroquinone intermediate which is further cyclized, rearranged, and oxidized (Gill and Steglich 1987). Compounds 103-105 displayed weak antifungal activities and medium-toweak activities against bacteria and yeasts. None of the three quinones was nematicidal for C. elegans (Stadler et al. 1994b); however, effects toward other nematode species have not been reported so far. Notably, pleurotin was shown to act as an inhibitor of the thioredoxin-thioreductase system (Welsh et al. 2003). Subsequently, different species of pleurotin-producing basidiomycetes were investigated, and a fermentation protocol was developed to obtain this anticancer lead metabolite in concentrations >300 mg/l (Shipley et al. 2006). A total synthesis of 104 and 105 was also reported (Hart and Hunag 1988).
Nematicidal metabolites from nematophagous basidiomycetes as well as compounds 103-105 are illustrated in Fig. 1.

Bulgarialactones from Bulgaria inquinans A 40-94
The black bulgar (Bulgaria inquinans), a saprotrophic ascomycete (Phacidiales, Phacidiaceae), grows on the bark of decaying deciduous trees and logs, preferably on oak. An organic extract of fruiting bodies yielded three azaphilones, named bulgarialactones A, B, and C (131-133), but only compound 132 could be isolated in sufficient quantities for further analysis. The mycelia of an 11-day submerged culture of strain A 40-94 yielded compounds 131-133 as dark red oils, whereas organic extracts of the culture filtrate yielded only compound 132 in preparative amounts. The LD 50 values of compounds 131 and 132 against C. elegans were 5 and 10-25 μg/ml, respectively, whereas compound (133) could not be tested due to its instability and low yield (Stadler et al. 1995f).

Thermolides from Talaromyces thermophilus 3 YM 3-4
Submerged cultures of the thermophilc fungus Talaromyces thermophilus YM 3-4 were grown for 21 days at 45°C, yielding six colorless oils, named thermolides A-F (134-139). These provided the first evidence for a hybrid polyketide synthase non-ribosomal peptide synthetase (PKS-NRPS) of fungal origin (Niu et al. 2014). 4 All thermolides feature an unusual 13-membered lactam-bearing macrolactone ring system. Thermolides A (134) and B (135) exhibited strong nematicidal activities with LC 50 values against M. incognita, Bursaphelenchus xylophilus 5 and P. redivivus as high as those of the avermectin standard, ranging from 0.5 to 1.0 μg/ml. Thus, thermolides A and B but also the less bioactive thermolides C (136) and D (137) 6 may serve as lead candidates for the development of new biological nematicides (Guo et al. 2012).

Paraherquamides from Penicillium charlesii ATCC 20841
Seven oxindole alkaloids, paraherquamides A-G (141-147), were isolated from 7-or 14-day static cultures of Penicillium charlesii ATCC 20841 grown at 25°C. The major compound paraherquamide A (141) was also the most active one, with an LD 50 value of 2.5 μg/ml against C. elegans (141). The LD 50 values of the other compounds ranged from 6 μg/ml (145) to 160 μg/ml (144). Broad-spectrum activity was observed against the three pathogenic nematodes Haemonchus contortus, Trichostrongylus colubriformis, and T. sigmodontis, each of them located in a distinct part of the gastrointestinal tract of the gerbil, Meriones unguiculatus (Ostlind et al.  (Manamgoda et al. 2014). Static cultures in vermiculite-containing medium were incubated for 14 days at 25°C producing the yellow, crystalline pbenzoquinone derivative cochlioquinone A (148). This caused the immobilization of 50 % of a C. elegans population after 16 h at a concentration of 135 μM (Schaeffer et al. 1990). Cochlioquinone A was also obtained from B. leersiae (Barrow and Murphy 1972), which is a pathogen of Leersia and Setaria spp. (Poaceae, Manamgoda et al. 2014).

Nematicidal ophiobolins
Approximately 30 C 25 sesterterpenoids bearing a tricyclic 5-8-5 ring system (ophiobolins) have been isolated from fungi. Most of the producers are members of the genus Bipolaris (Pleosporales, Pleosporaceae), which include economically important phytopathogens such as B. oryzae (syn. Cochliobolus miyabeanus), the brown spot pathogen of rice B. maydis (C. heterostrophus) that causes southern corn leaf blight, and B. sorghicola, which causes leaf spot in sorghum. Even so, ophiobolin K (149) was initially isolated from Aspergillus ustus JP 118 growing in a roller jar on a solid vermiculite-containing medium for 28 days at 25°C. This caused the immobilization of 50 % of a C. elegans population after 16 h at a concentration of 10 μg/ml, whereas 6epiophiobolin K (150) was inactive (Singh et al. 1991 C. elegans, which accounts for an interaction at the ivermectin binding site (Tsipouras et al. 1996). 7 The practical application of ophiobolins may be limited by their instability (Yun et al. 1988) and other diverse bioactivities (Au et al. 2000). For example, some ophiobolins are strongly phytotoxic, whereas others were harmless to plants (Yun et al. 1988;Evidente et al. 2006a, b). No structure-activity data are yet available to evaluate the relationship between the nematicidal and phytotoxic activities of these compounds.

Bursaphelocides from an anamorphic fungus
A taxonomically unidentified, sterile fungus (strain D1084) isolated from plant debris and grown in submerged culture for 6 days at 27°C was shown to produce the cyclodepsipeptides bursaphelocides A and B (155, 156). Both compounds contain 2-hydroxy-3-methylpentanoic acid, isoleucine, N-methylvaline, N-methylalanine and β-alanine, but they differ in that compound 155 also contains proline, whereas in compound 156, this residue is 4-methylproline. 8 The Bcotton ball on fungal mat method^was used for bioassayguided fractionation of the culture broth. Compounds 155 and 156 caused >96 and >98 % mortality, respectively, when added to cultures of B. xylophilus at a concentration of 100 μg/ml per ball. Insecticidal activity was observed against Drosophila melanogaster larvae as well as weak phytotoxic activity in an alfalfa (Medicago sativa) seed germination test (Kawazu et al. 1993).

Endophytic ascomycetes producing 3-hydroxypropionic acid
Submerged cultures of a number of endophytic fungi were screened for potential nematicidal activity against B. xylophilus using bioassay-guided fractionation. Five strains with the highest activities were used for the isolation and structural elucidation of the bioactive principles, including Phomopsis phaseoli (Diaporthaceae, Diaporthales) and Melanconium betulinum (Melanconidaceae, Diaporthales). However, the only nematicidal metabolite in all five isolates was identified as 3-hydroxypropionic acid (158). Notably, both of the species listed above may live either as plant pathogens or harmless endophytes (Schwarz et al. 2004). Because phytotoxic fungal isolates must not be used for integrated pest management, the pure compound should instead be considered for biocontrol applications. The structures of nematicidal metabolites from non-nematophagous ascomycetes are summarized in Fig. 3.
yielded nine nematicidal cyclic dodecapeptides that were not present in the fruiting bodies. The main compound, omphalotin A (165), is a colorless oil that remains stable during isolation and storage. Remarkably, its LD 50 against the plant-parasitic M. incognita was 2 μg/ml, which is ten times more potent than the ivermectin standard. The saprotrophic nematode C. elegans was 35-fold less susceptible. Compound 165 was shown to protect cucumber and lettuce cultures from nematodes, with no evidence of additional phytotoxic, insecticidal, or antimicrobial activities. Cytotoxic effects were comparatively weak Mayer et al. 1997Mayer et al. , 1999. Compound 165 contains a high proportion of methylated L-amino acids including sarcosine (methylglycine), methylvaline, and methylisoleucine Büchel et al. 1998 Fig. 3 Nematicidal metabolites from other non-nematophagous ascomycetes (Büchel et al. 1998). Monokaryotic strains, which have been obtained from O. olearius TA 90170 protoplasts, yielded five additional hydroxylated compounds, omphalotins E-I (169-173), after 9 days of fermentation. Their nematicidal activity against M. incognita was highly selective, with LD 50 values between 0.5 and 2.0 μg/ml (Liermann et al. 2009). One future challenge is to optimize fermentation conditions to improve the low yields of these compounds. In the meantime, a high-yielding method for solid-phase synthesis has been developed for compound 165 and other N-alkylated peptides using racemization-free triphosgene-mediated couplings (Thern et al. 2002).

Outlook and perspectives
More than 30,000 natural products have been isolated from fungi (Bérdy 2012), but fewer than 300 nematicidal compounds have been confirmed, representing just 280 producing species distributed over 150 genera (Laatsch 2014;Li and Zheng 2014). The screening of culture collections for nematicide-producing fungi could therefore yield more useful compounds than libraries of previously isolated natural products. The chemical structures of nematicidal metabolites are highly diverse, ranging from simple fatty acids and other organic acids to pyrones, lactones, benzoquinones, anthraquinones, furans, alkaloids, cyclodepsipeptides, peptaibiotics, and hybrid structures such as lactam-bearing macrolactones. It is therefore impossible to predict whether either a given fungal species or a particular fungal metabolite is likely to be nematicidal, and the activity against different nematode species may also vary. It is therefore essential to screen fungi and their metabolites against multiple economically important nematode species (Table 1), including common phytoparasites and nematodes that parasitize animals (e.g., H. contortus). The established model species C. elegans is often exquisitely sensitive toward nematicides, even primary metabolites such as fatty acids (Stadler et al. 1994a;Anke et al. 1995), although exceptions include oligosporon (2), which is inactive against  (181) trichodermin (184) nafuredin (182) nafuredin- (183) 183a 183b  Fig. 3 continued.
In the second part of this review, 101 substances from nematophagous basidiomycetes and non-nematophagous fungi were introduced, some of which exhibit pronounced nematicidal activity. 10 Thermolides A (134) and B (135) displayed potent nematicidal activity against M. incognita, B. xylophilus, and P. redivivus, comparable to that of the avermectin standard, but it remains difficult to produce large amounts of these compounds because the producers are thermophilic and cannot grow efficiently at temperatures below 45°C, so cultivation conditions will need to be optimized. Other potent fungal nematicides discussed herein have only been isolated in minute quantities. This may reflect suboptimal fermentation conditions, as observed for the omphalotins (165-173), or physicochemical instability, as observed for epoxidized lachnumon (109, 119, 120) and lachnumol derivatives (110), bulgarialactones (131-133), and ophiobolins K (149), M (151), and C (152).
Another challenge that must be addressed is that some nematicide-producing fungi are obligate phytopathogens (e.g.,  Bipolaris spp.), whereas others are facultative phytopathogens that may also exist as endophytes. In these cases, the producers cannot be used as biocontrol agents, and the nematicidal compounds they biosynthesize must be isolated, e.g., cochlioquinone A (148) and 3-hydroxypropionic acid (158). Yang et al. (2010) have even suggested that the nematicidal mycotoxin trichodermin (184) could be isolated from Trichoderma strains producing it, but the use of mycotoxigenic fungi or pure mycotoxins in biocontrol had been discussed and argued against by Degenkolb et al. (2008) and Chaverri et al. (2015). Mycorrhizin A (106) and some of its derivatives from L. papyraceum (107,108,117,118,121) as well as cheimonophyllon A (159) showed at least weak mutagenic activity in the Ames test.