3. Occurrence
The number of reports concerning endophytic isolates of Lecanicillium and Akanthomyces has increased in recent years. This is due not only to the several taxonomic reassessments introducing new species, but also to the easier access to techniques and databases for DNA sequencing, which in most instances enable one to overcome the intrinsic difficulties of morphological identification. However, more prompts have probably resulted by the awareness of the basic role that endophytic fungi play on plant fitness, introducing applicative perspectives for investigations in the field. For the above genera, literature shows a prevalence of findings concerning natural phytocoenoses (Table 2) over those inherent crops (Table 3); even more so considering that the latter series includes a few cases of endophytic colonization resulting after artificial inoculation in experimental work. Basically connected with the issue of ecosystem simplification characterizing the agricultural contexts, such a difference emphasizes the opportunity to recover the functional role of this component of the plant holobiont in view of improving crop performances.
Table 2. Endophytic occurrence of Lecanicillium/Akanthomyces in wild contexts.
Species |
Host Plant |
Country |
ITS Sequence √ |
Reference |
A. attenuatus |
Astrocaryum sciophilum |
French Guyana |
MK279520 |
[15] |
|
Conifer plant |
China |
MN908945 |
GenBank |
|
Symplocarpus foetidus |
Canada |
KC916681 |
[16] |
A. lecanii |
Ammophila arenaria |
Spain |
- |
[17] |
|
Dactylis glomerata |
Spain |
AM262369 |
[18] |
|
Deschampsia flexuosa |
Finland |
KJ529005 |
[19] |
|
Elymus farctus |
Spain |
AM924163 |
[17] |
|
Laretia acaulis |
Chile |
- |
[20] |
|
Pinus sylvestris |
Italy |
KJ093501 |
[21] |
|
Pinus sylvestris |
Poland |
- |
[22] |
|
Shorea thumbuggaia |
India |
KJ542654 |
GenBank |
|
Taxus baccata |
Iran |
KF573987 |
[23] |
A. muscarius |
Acer campestre |
Italy |
MT230457 |
This paper |
|
Laurus nobilis |
Italy |
- |
[24] |
|
Myrtus communis |
Italy |
MT230435 |
This paper |
|
Nypa fruticans |
Thailand |
MH497223 |
[25] |
|
Quercus robur |
Italy |
MT230463 |
This paper |
Akanthomyces sp. * |
Arctostaphylos uva-ursi |
Switzerland |
- |
[26] |
|
Carpinus caroliniana |
USA |
- |
[27] |
L. aphanocladii |
Ageratina adenophora |
China |
MK304090 MK304173 MK304418 |
[28] |
|
Hemidesmus indicus |
India |
MH594215 |
[29] |
|
Huperzia serrata |
China |
KP689216 KP689173 |
[30] |
|
Picea mariana |
Canada |
- |
[31] |
L. fungicola |
Phragmites australis |
Korea |
KP017880 |
[32] |
L. kalimantanense |
Zingiber officinale |
Indonesia |
- |
[33] |
L. psalliotae |
Cerastium fischerianum |
Korea |
JX238776 |
[34] |
|
Coix lachryma-jobi |
China |
KJ572167 |
GenBank |
|
Magnolia officinalis |
China |
|
GenBank |
|
Phoradendron perrottettii |
Brazil |
- |
[35] |
|
Pinus radiata |
New Zealand |
- |
[36] |
|
Sedum oryzifolium |
Korea |
KU556134 |
[37] |
|
Tapirira guianensis |
Brazil |
- |
[35] |
|
Triticum dicoccoides |
Israel |
- |
[38] |
Lecanicillium sp. |
Artocarpus lacucha |
India |
MH700423 MH700428 |
GenBank |
|
Bupleurum chinense |
China |
MG561939 |
GenBank |
|
Huperzia serrata |
China |
KM513600 |
[30] |
|
Liparis japonica |
China |
KT719186 KT719187 KT719188 KT719189 KT719192 |
GenBank |
|
Micrandra spruceana |
Peru |
MH267985 |
[39] |
|
Microthlaspi perfoliatum |
Greece |
KT269776 |
[40] |
|
Quassia indica |
India |
MH910098 |
GenBank |
|
Sandwithia guyanensis |
French Guyana |
MN514023 |
[41] |
|
Theobroma gileri |
Ecuador |
- |
[42] |
Table 3. Endophytic occurrence of Lecanicillium/Akanthomyces in crops.
Species |
Host Plant |
Country |
ITS Sequence √ |
Reference |
A. attenuatus |
Brachiaria sp. |
Kenya |
KU574698 |
[43] |
|
Salvia miltiorrhiza |
China |
JX406555 |
GenBank |
A. lecanii |
Cucurbita maxima |
Australia |
- |
[44] |
|
Gossypium hirsutum |
Australia |
- |
[45] |
|
Gossypium hirsutum |
Brazil |
- |
[46] |
|
Gossypium hirsutum |
Texas, USA |
KP407570 |
[47] |
|
Solanum lycopersicum |
Australia |
- |
[44] |
|
Phaseolus vulgaris |
Australia |
- |
[44] |
|
Phaseolus vulgaris |
China |
- |
[48] |
|
Pistacia vera |
Iran |
MF000354 |
[49] |
|
Triticum aestivum |
Australia |
- |
[44] |
|
Vitis vinifera |
Spain |
- |
[50] |
|
Zea mays |
Australia |
- |
[44] |
A. muscarius |
Brassica oleracea |
New Zealand |
- |
[51] |
|
Cucumis sativus |
Canada |
- |
[52] |
|
Cucumis sativus |
Japan |
- |
[53] |
|
Prunus cerasus |
Iran |
KY472303 |
[54] |
L. aphanocladii |
Zea mays |
Slovenia |
- |
[55] |
L. dimorphum |
Phoenix dactylifera |
Spain |
- |
[56] |
L. psalliotae |
Phoenix dactylifera |
Spain |
- |
[56] |
Lecanicillium sp. |
Citrus limon |
Iran |
MN448344 |
GenBank |
|
Vitis vinifera |
China |
MT123107 |
GenBank |
|
Zea mays |
India |
- |
[57] |
Overall, Table 2 and Table 3 include 65 citations of endophytic strains belonging to these two genera as a result of a search considering literature in the field and the GenBank database. A widespread capacity to colonize plants from heterogeneous ecological contexts is evident considering that these citations refer to 54 species belonging to 35 botanical families. With 10 species Poaceae is the most represented family, followed by Arecaceae and Pinaceae with three species each, and Anacardiaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Euphorbiaceae and Malvaceae with two species. The rest of the families (Apocynaceae, Araceae, Asteraceae, Betulaceae, Caryophyllaceae, Crassulaceae, Dipterocarpaceae, Ericaceae, Fabaceae, Fagaceae, Lamiaceae, Lauraceae, Lycopodiaceae, Magnoliaceae, Moraceae, Myrtaceae, Orchidaceae, Rosaceae, Rutaceae, Santalaceae, Sapindaceae, Simaroubaceae, Solanaceae, Taxaceae, Vitaceae and Zingiberaceae) are represented by a single species.
Such a variety of hosts seems to contrast any hypothesis of host specialization, and is rather indicative of a possible tendency to spread horizontally within the phytocoenoses. In this respect, the recovery of
A. muscarius from four woody species (
Acer campestre,
Laurus nobilis, Quercus robur and
Myrtus communis in two separate stands) at the Astroni Nature Reserve near Napoli, Italy (
[24] and in this paper), appears to support this ability, which may as well imply a permanent functional role in natural ecosystems. On the other hand, indications of a constant association with crop species could be favorable for possible applications in IPM. The limited available data only support preliminary clues in the case of cotton (
Gossypium hirsutum) where, considering the economic impact of insect pests, the endophytic occurrence of strains of
A. lecanii reported from distant countries such as Australia, Brazil and the United States might deserve further attention.
Phylogenetic Relationships of Endophytic Strains
In the evolving taxonomic scheme outlined above, the endophytic isolates provisionally classified as Lecanicillium sp. are to be further considered for a more definite taxonomic assignment. In this perspective, we propose a phylogenetic analysis (Figure 1) considering strains whose sequences of internal transcribed spacers of ribosomal DNA (rDNA-ITS) are deposited in GenBank (Table 2 and Table 3), along with official reference strains for the currently accepted species of Lecanicillium and Akanthomyces (Table 1).
Figure 1. Phylogenetic tree based on maximum likelihood (ML) analysis of the rDNA-ITS sequences deposited in GenBank for the known species (
Table 1) and the endophytic strains of
Lecanicillium and
Akanthomyces (in bold,
Table 2 and
Table 3). Multiple sequence alignment comprised 592 nucleotide positions, including gaps. The analysis was carried out using RAxML software (version 8.2.12;
https://cme.h-its.org/exelixis/web/software/raxml) for ML, PAUP (version 4.0a166;
https://paup.phylosolutions.com) for maximum parsimony (MP), and MrBayes (version 3.2.7a;
https://nbisweden.github.io/MrBayes/download.html) for Bayesian analysis. Phylogenetic tree was drawn using FigTree software (version 1.4.4;
http://tree.bio.ed.ac.uk/software/figtree). Details and complete references are specified in a recent paper
[58]. Bootstrap support values ≥60% for ML and MP are presented above branches as follows: ML/MP, bootstrap support values <50% are marked with ‘-’. Branches in bold are supported by Bayesian analysis (posterior probability ≥95%).
Simplicillium lanosoniveum CBS 704.86 (GenBank: AJ292396) was used as outgroup reference. Main clades are indicated by colored boxes A, B, C, D, E and F.
Although more DNA sequences, such as the translation elongation factor 1 alpha (
TEF) and RNA polymerase II largest subunits 1 (
RPB1) and 2 (
RPB2), are considered in taxonomic assessments concerning genera in the Cordycipitaceae
[12][13][25][59], provisional identification of isolates recovered in the course of biodiversity studies is routinely done on account of ITS. Therefore only these kinds of sequences are usually deposited in GenBank for such strains, representing the only possible marker available for phylogenetic reconstructions.
In the absence of opportunities for a direct examination of these isolates, the phylogenetic tree proposed in
Figure 1 provides an indication for their provisional assimilation to any of the accepted taxa in the genera
Lecanicillium and
Akanthomyces. A major cluster in the upper part of the tree includes the type strains of the species of
Cordyceps,
Akanthomyces (except
A. aranearum), and of
L. nodulosum and
L. uredinophilum, which are also credited for ascription to
Akanthomyces, along with all the endophytic strains ascribed to the species
A. lecanii, A. muscarius and
A. attenuatus (clades A, B and C, respectively). However, just two out of seven endophytic isolates ascribed to
A. lecanii are next to the type strain of this species, while five more isolates rather group with
A. muscarius. Confirming evidence from previous phylogenetic analyses
[25][59],
A. attenuatus is very close to
A. muscarius, but an isolate from the palm
Astrocaryum sciophilum is displaced in clade B. Another isolate from
Brachiaria sp. reported as
A. attenuatus is more distant, having
L. uredinophilum as the closest relative. While these remarks cannot be taken as an evidence of a more common endophytic occurrence of
A. muscarius, they represent an indication that at least some isolates of this species might have been misidentified as
A. lecanii. This is not surprising, considering that a previous study pointed out the difficulty of resolving species ascription of strains previously ascribed to
V. lecanii by using ITS sequences only
[60].
Interestingly, no endophytic isolates provisionally identified as
Lecanicillium sp. belong to the above major
Akanthomyces cluster. Three of them are part of clade D, corresponding to the species
L. aphanocladii, which also includes two strains identified as
L. psalliotae. This is acceptable since these species and
L. dimorphum have been reported in a close phylogenetic relationship in previous analyses
[6][59]. However,
L. psalliotae seems somehow problematic with reference to the resolution power of ITS, considering that it was reported as the closest relative (99.65% sequence identity at 100% query cover) of another isolate from
Microthlaspi perfoliatum [40], which is in a quite distant position in our phylogenetic tree.
As many as seven unidentified strains cluster with
L. fungicola, prevalently with the type strain of var.
aleophilum (clade E), indicating a relevant endophytic occurrence of this species, which was not recognized so far. Another isolate reported as
L. fungicola [32], deserves a more careful consideration with reference to its basal placement. In fact, BLAST search in GenBank indicated a 100% identity with ten strains of this species and several strains of the unrelated
Simplicillium aogashimaense. The latter was characterized in 2013 with the support of a phylogenetic analysis based on ITS only, which anyway showed a consistent distance from
L. fungicola [61]. Quite meaningfully, in our analysis the isolate in question was placed in proximity to the outgroup (
Simplicillium lanosoniveum) on which our tree was rooted. Considering that sequences of six out of this group of ten
L. fungicola strains were deposited in GenBank before 2013, it is quite possible that original misidentification of those that might rather have been
Simplicillium strains could have determined the incorrect assignment of the more recent isolates.
Finally, three isolates (two Indian from
Artocarpus lacucha and one Chinese from
Vitis vinifera) are grouped in clade F together with the type strains of the recently described
L. coprophilum [11],
L. restrictum and
L. testudineum [62]. A BLAST search in the GenBank database shows the first species as the closest relative, with 100% and 99.81% ITS sequence identity for the Chinese and the Indian isolates, respectively.
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].