**4. Implications in Crop Protection**

As introduced above, so far there are few observations concerning the effects of endophytic strains of *Lecanicillium* and *Akanthomyces* in crops. Within the limited data available so far, cotton stands out for remarks on the endophytic occurrence of *A. lecanii* from independent cropping areas. In Australia an endophytic isolate was shown to be able to colonize cotton plants ensuring protection against the cotton aphid (*Aphis gossypii*) after artificial inoculation. Besides evidence from direct microscopic examination, the ability to colonize plant tissues was confirmed by re-isolation from leaves of the treated plants, which was successful up to 35 days after inoculation. This persistence can be taken as an indication of an endophytic life strategy, considering that endophytic colonization enables the fungus to become resident in a stable and nutritious insect-attracting environment. High humidity enhanced colonization of both plants and aphids; this expected effect is relevant for the management of the cotton aphid, which is most commonly found in the lower canopy, where humidity is high and the fungus is more protected against the adverse effects of UV radiation from sun [63]. Moreover, contact with conidia of *A. lecanii* significantly reduced the rate and period of reproduction of *A. gossypii*. The culture filtrate of the fungus significantly increased mortality and reduced reproduction, while feeding-choice experiments indicated that the aphids might be able to detect the fungal metabolites. The ethyl acetate and methanolic fractions of culture filtrate and mycelia also caused significant mortality and reduced fecundity [64]. Besides cotton, the same strain displayed the ability to colonize plants of wheat, corn, tomato, bean and pumpkin after artificial inoculation of leaves, while soil inoculation was ineffective [44].

Additional reports from cotton come from Texas [47] and Brazil, where the endophytic occurrence of *A. lecanii* was detected in leaves and roots of both normal and *Bt*-transgenic plants [46]. Although no aspects concerning interactions with pests were evaluated in these cases, it is meaningful that several strains of *A. lecanii* were recovered in each of these three contexts, indicating a possible common association of this species with cotton, which deserves to be more thoroughly verified.

The adaptation of *A. lecanii* to exert entomopathogenicity in association with plants is well attested by the finding that the fungus responds to volatile compounds produced by the plant during insect feeding. Particularly, in a model based on thale cress (*Arabidopsis thaliana*) and the mustard aphid (*Lipaphis erysimi*), compounds such as methyl salicylate and menthol were found to promote spore germination and pathogenicity of the fungus [65,66].

Besides aphids, protective effects after systemic colonization have been demonstrated against the red spider mite (*Tetranychus urticae*) in bean plants. In this case a strain of *A. lecanii* was reported to spread within the plant tissues after artificial inoculation of seeds, promoting growth and impairing survival and fecundity of the mites. These effects were even carried over the following generation of mites fed on fresh plants [48].

Pathogenicity of *A. lecanii* against a wide array of noxious arthropods is integrated by antagonism towards plant pathogenic fungi. In addition to a general antifungal activity demonstrated in vitro against polyphagous species such as *Sclerotinia sclerotiorum, Rhizoctonia solani* and *Aspergillus flavus* [49], possible exploitation of this double functionality has been conceived on several crops, such as coffee where *A. lecanii* behaves as both a parasite of the leaf rust (*Hemileia vastatrix*) and a pathogen of the green scale (*Coccus viridis*) [67]. The same role can be considered in crops where powdery mildews can represent a major phytosanitary problem, such as cucurbits [68,69].

Moreover, antifungal effects could derive from stimulation of the plant defense response, as reported for an endophytic strain able to promote such reaction against *Pythium ultimum* in transformed cucumber plants [52]. Additional experimental evidence in this regard is provided by observations carried out on the date palm (*Phoenix dactylifera*) where the inoculation of endophytic strains of *L. dimorphum* and *L.* cf. *psalliotae,* previously reported for entomopathogenicity against the red palm scale (*Phoenicococcus marlatti*) [56], induced proteins involved in plant defense or stress response. Proteins related with photosynthesis and energy metabolism were also upregulated, along with accumulation of a heavy chain myosin-like protein [70].

The concurrent role against plant pests and pathogens is known to operate for other *Lecanicillium* and *Akanthomyces* species, and for non-endophytic strains of various origin, as more in detail discussed in dedicated papers [71,72]. The need to combat multiple adversities has also prompted the evaluation of a possible combined use of these fungi with chemical pesticides. In this respect, it has been observed that the spread of *A. lecanii* in plant tissues is not affected by treatments with insecticides belonging to several classes [73]. Moreover, substantial safety of insecticides has been reported in in vitro assays carried out on *A. muscarius*, while several herbicides and fungicides were responsible for negative effects or even suppression of mycelial growth [74]. For the latter species, in vivo observations on the sweet potato whitefly (*Bemisia tabaci*) demonstrated the positive effects of association with chemical insecticides in view of reducing their use, particularly in the greenhouse [75]. Again with reference to application of *A. muscarius* for the control of *B. tabaci*, it is worth mentioning the synergistic effects resulting in combined treatments with matrine, a plant-derived quinolizidine alkaloid [76].

In addition to the indirect side effects deriving from protection against biotic and abiotic adversities, many endophytes have been reported to promote plant growth through essentially two mechanisms; that is the release of plant hormones, or the improvement of nutritional conditions. Of course, strains possessing both properties are likely to contribute in an additive manner, as observed for an isolate of *L. psalliotae* from cardamom (*Elettaria cardamomum*). Besides producing indole-3-acetic acid, this strain enhanced chlorophyll content of leaves as a likely result of release of siderophores, and increased availability of zinc and inorganic phosphate by promoting their solubilization [77]. Release of siderophore has also been reported for an endophytic isolate of *A. lecanii* from *Pistacia vera* [49].

## **5. Biochemical Factors Involved in the Tritrophic Interaction with Plants and Pests**

It has been previously introduced that, at least in part, the antagonistic/pathogenic ability by *Lecanicillium* and *Akanthomyces* strains is mediated by biochemical factors, such as enzymes and secondary metabolites. Endophytic fungi are regarded as a goldmine of undescribed chemodiversity, and even diffusely reported as capable to synthesize bioactive products originally characterized from their host plants [78]. Although it is quite reasonable that they exploit this biosynthetic potential in the natural environment, more rigid opinions occasionally question a real role by these compounds until their production is demonstrated in plants. Pending a solution of this diatribe through the development of methods for ascertaining their effective release and bioactivity in plant tissues, so far research in the field has disclosed interesting properties by species of *Lecanicillium* and *Akanthomyces*, too.

The first metabolomic studies concerning these fungi were carried out with strains of *V. lecanii* before the taxonomic revision. Two isolates were found to produce 2,6-dimethoxy-*p*-benzoquinone, phenylalanine anhydride, aphidicolin and dipicolinic acid, with the latter showing insecticidal effects in bioassays on the blowfly *Calliphora erythrocephala* [79]. Afterwards, two more triterpenoid carboxylic acids with alleged insecticidal properties were reported from the same source [80]. Incompletely identified toxic products, possibly phospholipids, were extracted from another strain showing activity against *B. tabaci*, the western flower thrips (*Frankliniella occidentalis*) and a few aphid species [81]. Anti-insectan effects against the corn earworm (*Helicoverpa zea*) were later reported for vertilecanin A, the most abundant component in a group of five new phenopicolinic acid analogues [82]. Moreover, two structurally unidentified products were extracted from two Chinese strains, displaying toxic, ovicidal and antifeedant properties against *B. tabaci* [83]. Finally, the novel indolosesquiterpenes lecanindoles A-D, with quite peculiar structures and bioactivities, were characterized from another aphidiculous strain [84].

Later on more strains were found to produce novel compounds without a direct connection with their entomopathogenicity. Two inactive aromadendrane sesquiterpenes, inonotins M and N, were extracted from a strain of *L. psalliotae* [85]. An unidentified *Lecanicillium* sp. was reported to produce lecanicillolide [86], and lecanicillones A-C, three unusual dimeric spiciferones with an acyclobutane ring displaying moderate cytotoxic effects [87]. More interesting inhibitory effects on tube formation by endothelial cells, implying antiangiogenic properties, were reported for the decalin polyketide 11-norbetaenone, from a strain of *L. antillanum* [88].

Besides novel compounds, investigations on these fungi have also disclosed the production of well-known bioactive metabolites. A strain of *L. psalliotae* was found to produce oosporein, a common product of *Beauveria* spp., which displayed strong inhibitory activity against the potato late blight fungus (*Phytophthora infestans*) [89]. Likewise, several cyclic depsipeptides have been reported from miscellaneous isolates. The list includes eight destruxin analogues, well-known secondary metabolites of *M. anisopliae*, by strain KV71 of *L. longisporum* (the active principle of the mycoinsecticide Vertalec) [90]; bassianolide, previously reported from *B. bassiana*, from *A. lecanii* [91], and the antifungal verlamelins A-B, previously known from *Simplicillium lamellicola*, from an unidentified *Lecanicillium* strain [92]. Finally, stephensiolides C, D, F, G and I, originally characterized from a gram-negative bacterium (*Serratia* sp.) symbiotic with a mosquito (*Anopheles stephensi*), have been recently detected in the culture extract of an endophytic *Lecanicillium* sp. as the bioactive principles responsible for antibacterial activity against methicillin-resistant *S. aureus* [41]. Inhibitory properties against the same bacteria, along with cytotoxicity on human lung fibroblast cells, were ascribed to cyclic depsipeptides contained in the culture extracts of a strain of *A. attenuatus* [15].

Antibiotic effects against *S. aureus* were also reported for akanthomycin, extracted from cultures of *Akanthomyces gracilis* together with the closely related pyridine alkaloids 8-methylpyridoxatin and cordypyridone C [93]. Additional findings from *Akanthomyces novoguineensis* concerning the akanthopyrones [94], akanthol, akanthozine, butanamide and oxodiazanone derivatives [95] are not to be further considered in this review by reason that this species is now classified in the genus *Hevansia* [14].

This concise analysis of the pertinent literature, mostly made of independent or occasional findings, highlights the importance of carrying out more systematic work on the metabolomics of members of *Lecanicillium* and *Akanthomyces*. In fact, a thorough revision could ascertain whether some compounds eventually represent biochemical markers for selected species, and which products are effectively associated with the expression of pathogenicity towards insects, nematodes and spiders, as well as with antagonism/mycoparasitism against plant pathogens. In this respect, an interesting hypothesis has been advanced concerning the above-mentioned dipicolinic acid, which is known to act as a prophenoloxidase inhibitor and an immunosuppressive agent in insects. After its concomitant detection as a product of several entomopathogenic species belonging to the Hypocreales, including *A. muscarius*, it has been advanced that the acquired ability to synthesize this compound might have shaped evolution of these fungi from mere plant associates to the more specialized lifestyle as arthropod pathogens [96].

Literature on enzyme production by endophytic strains of *Lecanicillium* and *Akanthomyces* is more limited. Chitinolytic enzymes are not only necessary to these fungi to penetrate cuticle of insects, nematodes or spiders, but they are also involved in the activation of the disease response by the plant and induction of systemic resistance [97–99]. The same function may also be played by other enzyme complexes, such as proteases and β-glucanases, which are known to integrate the enzymatic profile of many endophytes [100–102]. Besides directly affecting survival and fecundity of the green peach aphid (*Myzus persicae*) in a concentration-dependent manner, a protein characterized from a strain of *A. lecanii* was found to concomitantly induce upregulation in tomato plants of genes associated to the salycilate and jasmonate pathways, which are involved in the systemic response to biotic stress [103].
