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1 I. Introduction

In our last review (Anke and Sterner 2002), published in the first edition of this volume, we focused on the chemical diversity of nematicidal and insecticidal fungal metabolites and the producing organisms. Since then, only a few novel compounds have been described. However, many publications have appeared which describe the biological activities of some of the described compounds (other than insecticidal/nematicidal) or their mode of action. In addition, the ecological significance of some of the compounds has been partly elucidated, e.g. their role in insect pathogens during the colonization of the host. Therefore this chapter also includes some of these aspects.

2 II. Novel Compounds and Their Producers

2.1 A. Peptides, Cyclic Peptides, and Cyclic Depsipeptides

Cyclic peptides and depsipeptides produced by fungi, among them insect pathogenic fungi (e.g. members of the genera Aschersonia, Beauveria, Isaria, Metarhizium, Paecilomyces, Verticillium), and their occurrence have been summarized in four comprehensive review articles (Anke and Sterner 2002; Zimmermann 2007a, b; Anke and Antelo 2009).

Since 2000, new producers of bioactive depsipeptides have been reported, for example, Beauveria fellina strains of marine origin (Lira et al. 2006), Verticillium sp. FKI-1033 (Monma et al. 2006), Aspergillus carneus (Capon et al. 2003), Torrubiella luteorostrata and its anamorph Paecilomyces cinnamomeus (both isolated from a scale insect; Isaka et al. 2007), Verticillium hemipterigenum (Nilanonta et al. 2003; Supothina et al. 2004), an Aureobasidium species from the tropical rain forest (Boros et al. 2006), an unidentified endophytic fungus (Huang et al. 2007), and a soil-borne Phoma species (Aoyagi et al. 2007). For a compilation of beauvericins and enniatins produced by Cordyceps species and their anamorphs as well as other insect pathogens, see Isaka et al. (2005a, b). Pseudodestruxins were found in Nigrosabulum globosum (Che et al. 2001) and reviews on destruxins and the producing organisms were published by Pedras et al. (2002) and Zimmermann (2007b). Some of the relevant compounds are listed in Table 7.1.

Table 7.1. Examples of insecticidal and nematicidal compounds reported lately. The structures are given in Figs. 7.1–7.5
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Chemical screening by HPLC-MS techniques led to the identification of five novel beauverolides from Beauveria bassiana and Paecilomyces spp. (Kuzma et al. 2001; Jegorov et al. 2004). Specific protocols for the detection and quantification of insecticidal cyclodepsipeptides in fungal cultures are now available (Jegorov et al. 2003).

Besides cyclopeptides and depsipeptides, fungi can also produce linear peptides with insecticidal activity; recently reported examples are efrapeptin G and methylated peptides from a marine fungus associated with a sponge, neoefrapeptins from Geotrichum candidium (Nagaraj et al. 2001; Fredenhagen et al. 2006) and the Aib-containing cicadapeptins I and II from Cordyceps heteropoda (Krasnoff et al. 2005). Structures of some linear peptides are shown in Fig. 7.1, cyclic peptides and depsipeptides in Figs. 7.2 and 7.3. The neoefrapeptins comprise a large group of ten 16-residue and two 13-residue peptides containing also non-proteinogenic amino acids (Fredenhagen et al. 2006).

Fig. 7.1.
figure 1

Neofrapeptin A (produced by Geotrichum candidium). Efrapeptin G (from a marine isolate). Cicadapeptin I (from Cordyceps heteropoda). leucinostatin B (from Paecilomyces lilacinus)

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Fig. 7.2.
figure 2

Omphalontins E–I (from Omphalotus olearius). Pseudodestruxin A (from Nigrosabulum globosum). Beauverolide N (from Beauveria bassiana). Verticilide (from Verticillium sp. FKI-1033)

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Fig. 7.3.
figure 3

Argifin (from Gliocladium sp. FTD-0668). Sirodesmin PL (from Laeptosphaeria maculans). Serinocyclin (from Metarhizium anisopliae). Epichlicin (from Epichloe typhina). Apicidin (from Fusarium sp.)

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From protoplasts of a monokaryotic strain of the basidiomycete Omphalotus olearius, five novel hydroxylated omphalotin derivatives (omphalotin E–I) were isolated, complementing the already known omphalotins.

Interestingly the monokaryotic strain grew faster and produced higher amounts of these compounds than the dikaryotic parental strain from which it was obtained (Liermann et al. 2009). All O. olearius strains, irrespective of their geographical origin, seem to produce omphalotin derivatives (Anke et al., unpublished data). In fruiting bodies, omphalotins could not be detected. Omphalotins E-I exhibited similar nematicidal activities against Meloidogyne incognita (Kofoid & White) as omphalotin A. Antibacterial or antifungal activities were not detected and none of the compounds showed cytotoxic effects towards mouse leukemia cells (L1012 cells) or human colon adenocarcinoma cells (Colo 320 cells) at concentrations up to 50 μg/ml. The novel compounds were seemingly produced at the expense of the known omphalotins, including omphalontin A, the amount of which decreased drastically towards the end of the fermentation (Liermann et al. 2009).

2.2 B. Novel Metabolites and New Derivatives of Insecticidal or Nematicidal Metabolites

A screening of some 500 endophytic fungi for the production of nematicidal metabolites led to the selection of 17 strains with selective activity towards Meloidogyne incognita, while Caenorhabditis elegans was less affected (Schwarz et al. 2004). In five strains identified as Phomposis phaseoli and Melanconium betulinum (four strains), the nematicidal principle was found to be the simple metabolite 3-hydroxypropionic acid (Schwarz et al. 2004).

In the Sarcosomataceae (teleomorphs of the genera Galiella, Urnula, Strumella), the production of the galiellalactone precursors pregaliellalactone (Fig. 7.4) and structurally related hexaketides accounted for the nematicidal activity. Interestingly an endophytic strain of Cistus salviifolius, which according to its 18S rDNA sequences also belongs to the Sarcosomataceae, produced the same compounds (Köpcke et al. 2002a, b). A further nematicidal metabolite, MK7924 (Fig. 7.5), with weak activity against Aspergillus niger and Caenorhabditis elegans was isolated from Coronophora gregaria (Kumazawa et al. 2003).

Fig. 7.4.
figure 4

Paraherquamides H, I (from Penicillium cluniae). Pregaliellalactone (from Galiella rufa). Quinolonone B, yaequinolones D, F (from Penicillium sp. FKI-2140). Penipratynolene and 2,6-pyridinedicarboxylic acid (from Penicillium bilaiae). Quinolactacide (from Penicillium citrinum). Peniprequinolone (from Penicllium simplicissium). Nafuredin-γ (from Aspergillus niger)

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Fig. 7.5.
figure 5

Nodulisporic acids B2, C (from Nodulisporium sp.). MK7924 (from Coronophora gregaria). Communesins C, D, E (from Penicillium expansum)

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Quinolactacide (Fig. 7.4), an insecticidal quinolone, was isolated from solid-state cultures of Penicillium citrinum (Abe et al. 2005). Using Artemia salina as test organism, seven novel yaequinolones were isolated from Penicillium sp. FKI-2140, together with nine known and structurally related compounds, among them quinolinones A and B, penigequinolones A and B, and peniprequinolone (Uchida et al. 2006a, b).

Quinolone derivatives were also obtained from other Penicillium species, e.g. P. janczewskii (He et al. 2005) and P. simplicissimum (Kusano et al. 2000). Peniprequinolone, in addition to its strong insecticidal properties, inhibits the root-lesion nematode Pratylenchus penetrans (Kusano et al. 2000). Some of the quinolones and their producers are listed in Table 7.1, and their structures are given in Fig. 7.4. Another Penicillium metabolite active against the nematode Pratylenchus penetrans is penipratynolene (Nakahara et al. 2004). The compound was isolated together with 6-methoxy-carbonylpicolinic acid and 2,6-pyridinedicarboxylic acid (Fig. 7.4) from Penicillium bilaiae.

New nodulisporic acids were isolated from a Nodulisporium sp., namely nodulisporic acids B, B1, B2, and C, C1, C2, and Δ23 nodulisporic acid C4 (Ondeyka et al. 2002, 2003; Singh et al. 2004). The compounds are active against fleas on dogs (Shoop et al. 2001) and comprise complex chemical structures (see Fig. 7.5). From Penicillium expansum Link MK-5, communesins C, D, and E (Fig. 7.5) were obtained, along with two known communesins (A, B). All novel communesins showed selective insecticidal activity against silkworms (Hayashi et al. 2004).

The large family of paraherquamide metabolites has gained new members from P. cluniae Quintanilla: paraherquamide H and I (Fig. 7.4) together with five known derivatives. Structure–activity relationships revealed paraherqueamide E to be the most potent member, with a LD50 of 0.089 μg/nymph of the milkweed bug Oncopeltus fasciatus Dallas (López-Gresa et al. 2006).

Nafuredin is one of the few insecticidal metabolites that does not contain nitrogen, i.e. it is not an alkaloid. The substance was isolated from a marine-derived Aspergillus niger strain using mitochondria of Ascaris suum as test system with the aim to find inhibitors of NADH-fumarate reductase from helminths (Omura et al. 2001). By weak alkaline treatment the compound was converted to its γ-lactone (Fig. 7.4) which also inhibits NADH-fumarate reductase (Shiomi et al. 2005).

Several reports on insecticidal activities of extracts obtained from fungal culture filtrates have been published without identification of the active components (Meyer et al. 2004; Mohanty and Prakash 2008). Thus, Chen et al. (2003) reported on an extract obtained from an endophytic fungus with high activities against Heliothis armigera; the metabolites isolated, however, were not insecticidal (Yasui et al. 2006).

Many insecticidal or nematicidal metabolites, despite their complex structures, have been chemically synthesized during the last few years, for example, 9-prenylpaxilline (Smith and Cui 2003), marcfortine A, and structurally related metabolites of the paraherquamide group, many of which have potent insecticidal and antihelmintic properties (Williams 2002; Williams et al. 2003; Trost et al. 2007): quinolactacide (Abe et al. 2006), nafuredin-γ (Nagamitsu et al. 2003), and verticilide (Momna et al. 2006).

The successful in vitro synthesis of enniatins with the multienzyme enniatin synthetase may open the way to novel derivatives, since the substrate binding pocket for the α-hydroxy carboxylic acid accepts chemically different acids (Glinski et al. 2001; Feifel et al. 2007).

3 III. Biological Activities and Mode of Action

Cyclic depsipeptides and their biological activities were thoroughly reviewed by Sarabia et al. (2004), insecticidal and other biological activities of destruxins, isariins, enniatins, and beauverolides by Anke and Sterner (2002), by Anke and Antelo (2009), and by Zimmermann (2007a, b).

Sirodesmin PL produced by Leptosphaeria maculans was found to have phytotoxic, antibacterial, and insecticidal properties (Rouxel et al. 1988; Boudart 1989). Serinocyclin A isolated from Metarhizium anisopliae condia produced a sublethal locomotory defect in mosquito larvae (Krasnoff et al. 2007). Argifin and argadin, two cyclopentapeptides from a Gliocladium sp. and a Clonostachys sp. turned out to be potent inhibitors of chitinase B from the gram-negativ bacterium Serratia marcescens (Arai et al. 2000). When injected into cockroach larvae, their moult was arrested (Houston et al. 2002).

Like many peptaibols, the efrapeptins act as channel-forming ionophores and have insecticidal, antimalarial, and antiprotozoal activities (Nagaraj et al. 2001). In addition, they inhibit exocytosis but not endocytosis in eukaryotic cells (Muroi et al. 1996) and exhibit antibacterial and antifungal activities (Bandani et al. 2000).

Efrapeptins bind strongly to V-ATPases in the brush border membrane of the mid-gut of the wax moth Galleria mellonella (Bandani et al. 2001). Leucinostatins A and B originally isolated from Penicillium lilacinum [now Paecilomyces lilacinus (Thom) Samson] were found to have nematicidal activities (Arai et al. 1973; Park et al. 2004). Like other peptaibols they act on bacteria, are uncouplers in the inner mitochondria membrane and inhibit mitochondrial ATP synthesis. Recently interesting and potent in vivo and in vitro antitrypanosomal activities of these compounds have been reported. Despite its toxicity, the strong in vivo antitrypanosomal activity of leucinostatin B makes the compound a promising lead structure for the development of drugs with a new type of action mode (Ishiyama et al. 2009).

Among nine beauverolides tested for acyl-CoA:cholesterol acyltransferase (ACAT) inhibitory activity in CHO-cells expressing ACAT1 or ACAT2, beauverolides I and III inhibited ACAT1 rather selectively, and no antimicrobial or cytotoxic activities were detected.

Both compounds produced by the entomopathogenic fungus Beauveria bassiana exert anti-atherogenic activity in low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects and thus may serve as lead structures for new anti-atherosclerotic agents (Namatame et al. 2004). Contrary to the beauverolides, related beauvericin was clearly cytotoxic (Matsuda et al. 2004; Ohshiro et al. 2007). Cell lines derived from insects, e.g. Spodoptera frugiperda SF-9 cells, were also inhibited by beauvericin (Calo et al. 2003; Fornelli et al. 2004). The compounds induced rapid cell death in Xenopus oocytes via influx of Ca2+ (Tang et al. 2005).

Furthermore, significant effects were detected for destruxin E on insect haemocytes (Vey et al. 2002) and for different destruxins, e.g destruxins A, B, and E, on human and insect cell lines (Skrobek and Butt 2005). Verticilide from moulds of the genus Verticillium inhibits the binding of ryanodine to its receptor (RyR) and, hence, has insecticidal activity (Monma et al. 2006).

The target of PF1022A, a fungal cyclo-octadepsipetide, is a latrophilin-like receptor from the parasitic nematode Haemonchus contortus (Saeger et al. 2001). Emodepsin, a semi-synthetic depsipeptide derived from PF1022A, has already been successfully used against helminths in veterinary medicine (Conder et al. 1995; Dyker et al. 2004; Samson-Himmelstjerna et al. 2005). Due its limited availability and therefore a rather high price, its use is restricted to small pets. PF1022A is a metabolite of an endophytic fungus from the ornamental plant Camellia japonica (Sasaki et al. 1992; Scherkenbeck et al. 2002). Based on its 18S rRNA gene sequence, the endophyte was tentatively identified as a member of the ascomycetous family Xylariaceae close to Xylaria polymorpha and Rosellinia necatrix (Miyado et al. 2000).

Selective nematicidal properties were only reported for the omphalotins with high inhibitory activity towards Meloidogyne incognita and lower activities against Caenorhabditis elegans (Mayer et al. 1999). The nematicidal properties of hydroxylated omphalotins, some of which can be produced by monokaryotic strains generated from dikaryotic parent mycelia, were found to be higher than those of the unsubstituted compound, but unfortunately they were not stable (Büchel et al. 1998; Liermann et al. 2009). Their mode of action has not yet been elucidated.

Paraherquamides, potent anthelmintic agents isolated from various Penicillium species, were reported to possess promising activities against drug-resistant intestinal parasites (Williams et al. 2003).

Quinolactacide was also strongly active against Myzus persicae, and 250 ppm were lethal to 88% of the aphides tested; and, at a concentration of 500 ppm, 42% mortality towards the diamondback moth (Plutella xylostella) was recorded (Abe et al. 2006).

Nafuredin is a selective inhibitor of the helminth complex I (NADH-fumarate reductase). It showed only very weak inhibition of the mammalian complex I (NADH ubiquinone reductase from bovine liver) but was selectively active in vivo against the stomach worm Haemonchus contortus in sheep (Omura et al. 2001). The γ-lactone was almost equally active in the helminth complex I assay but less active in the in vivo tests (Shiomi et al. 2005). The differences between the human and helminth complex I make this an interesting target for the development of novel selective drugs against parasitic nematodes.

4 IV. Ecological Significance

Many secondary metabolites play a crucial role for fungi in their natural habitats. For example, endophytic fungi of grasses belonging to the genera Neotyphodium/Epichloe confer protection from mammalian and insect herbivores, or enhanced resistance to nematodes and phytopathogenic fungi (Schardl et al. 2004; Panaccione et al. 2006). Some of these beneficial effects are due to secondary metabolites.

Loline and peramine have been identified among the fungal metabolites with insecticidal activities in the plant host. Another metabolite from Epichloe typhina, epichlicin, efficiently prevents the germination (IC50 value of 22 nM) of Cladosporium phlei spores, a host plant pathogen (Seto et al. 2007). Likewise, sirodesmin PL or zearalenone and other mycotoxins produced by Leptoshaeria maculans and Fusarium species, respectively, were detected in the plant hosts (Laser et al. 2003; Elliott et al. 2007). Some endophytic fungi produce 3-hydroxypropionic acid as a nematicidal principal (Schwarz et al. 2004). This might be the natural nematicide with the simplest chemical structure; however it remains to be elucidated whether the compound is also produced in planta.

The function of shearamide A, an insecticidal cyclopeptide isolated from the ascostromata of Eupenicillium shearii (Belofsky et al. 1998), and likewise sclerotiamide from Aspergillus sclerotiorum sklerotia (Whyte et al. 1996), may be to protect the reproductive or survival structures of the fungi against insects or nematodes, similar to ergopeptides in the sclerotia of Claviceps species (Leistner and Steiner 2009).

Investigations on the role of destruxins in the pathogenicity of Metarhizium anisopliae against three species of insects revealed a direct relationship between the titre of destruxins produced by the strains in vitro and their destructive action (Kershaw et al. 1999).

However, in M. anisopliae mutants, incapable of destruxin production, virulence towards Galleria mellonella was unaltered (Amiri-Besheli et al. 2000). In the plant pathogenic mould Alternaria brassicae, destruxin B is a host-specific toxin. In three Brassica species the degree of their sensitivity to destruxin B positively correlated with their degree of susceptibility (Pedras et al. 2002).

From cultures of a number of fungi producing cyclic depsipeptides (e.g. Beauveria bassiana), also dipeptides composed of the same amino acids as the depsipeptides were isolated.

Other insect pathogens like Verticillium species and Metarhizium anisopliae as well as plant pathogenic fungi, e.g. Colletotrichum gloeosporioides, Exserohilum holmi, Gliocladium deliquenscens, Alternaria and Trichoderma spp. were also found to produce dipeptides. An unidentified endophyte from mangrove leafs (Rhizophora spp.) produced two cyclic depsipeptides and three diketopiperazines (Huang et al. 2007).

It might be interesting to elucidate the insecticidal and nematicidal activities exhibited by cocktails of all these secondary metabolites of a pathogen. Such synergistic effects of different fungal metabolites have been largely neglected so far. In general, the role of dipeptides and depsipeptides in insect and plant pathogenicity is not fully understood. Further, it is intriguing that depsipeptides are widespread in phytopathogens (e.g. Cochliobolus with anamorphs Helminthosporium and Bipolaris, Calonectria with its anamorph Cyclindrocladium, as well as Fusarium and Alternaria), insect pathogens (Aschersonia, Beauveria, Cordyceps, Diheterospora, Fusarium, Hirsutella, Isaria, Metharizium, Paecilomyces, Tolypocladium, Verticillium), and others (Zimmermann 2007a, b; Buckingham 2008). As molecular tools become more and more available, this question may be correctly addressed and respectively answered in the near future.

The efrapeptins produced by insect pathogenic Tolypocladium species are also produced in vivo; the amounts, however, were found to be too small to cause the death of insects. Therefore, it was suggested that the compounds act in concert with additional, not yet known, pathogenicity factors (Bandani et al. 2000).

For the function of enzymes in entomopathogenic fungi and their role in disease development, see Khachatourians and Qazi (2008) in this context. The effects of secondary metabolites on the enzymes involved in pathogenesis in plants and insects alike is another innovative field, in which interesting results still wait to be elucidated.

5 V. Conclusions

Natural products derived from plants, animals, and microorganisms constitute only 7.6% of the global insecticides market (Elbert et al. 2007). However, more than 50 000 microbial metabolites are known. Half of them exhibit bioactivities, first and foremost antibiotic activity. Fungal metabolites represent about 40% of these natural products (Bérdy 2005; Buckingham 2008). So far, no secondary metabolite from a fungus has been developed into a marketable insecticidal or nematicidal product, despite the fact that most insect pathogens are fungi and many have successfully been screened for the production of “soft pesticides”. However, as it appears, many of the compounds isolated from these fungi are rather toxic, like the destruxins or efrapeptins. While they fulfil their ecological role very well, i.e. killing the host animal, they do not meet the requirements of modern agricultural pesticides regarding selectiveness, low costs, and environmental safety. Nevertheless, the example of PF1022A shows that fungal metabolites can be developed into drugs useful in agriculture and veterinary medicine. The many synthetic efforts using natural products as lead structures also point in this direction. Taking into account the overall number of fungal species, which is estimated to be around 1.5 million (Hawksworth 2001), and comparing this number with the number of about 50 000 species screened so far, it seems to be only a matter of time until the first insecticide from a fungus is introduced into the market.