1. Introduction
The dispersal of propagules by animals (zoochory) is essential for many plants, fungi, and microorganisms like fruit-bearing trees, multiple salvia species, or mycorrhizal fungi [
1,
2,
3]. Moving vertebrates and invertebrates can thereby shape and connects ecosystems, communities, and populations. The mobile link concept integrates, among other mechanisms, zoochory and the movement behavior of the dispersing individual and empathizes the effects this causes on other species [
4].
The spatial-temporal movement of vectors for phytopathogens is increasingly recognized as a crucial component of understanding disease patterns in many cropping systems [
5,
6,
7]. Unraveling the three-way interaction of crop plants, phytopathogenic fungi, and an arthropod vector requires an interdisciplinary approach. Nevertheless, the enhancing effects of arthropod activity on plant pathogens’ load are well studied for several plant diseases, like the laurel wilt disease of avocados (
Persea americana Mill), or the kernel rot of maize (
Zea mays L.) or Fusarium head blight (FHB) in wheat (
Triticum aestivum L.) [
8,
9,
10]. Although these three-way interactions seem to be omnipresent, the knowledge about the involved mechanisms is still sparse, especially the role of non-pest arthropods.
Besides fungivory, fungal propagules like spores are ingested (endozoochory) accidentally by arthropods while feeding on plant material that is colonized by fungi [
11,
12]. Predatory arthropods like spiders and centipedes ingest fungal propagules via their contaminated prey animals. Therefore, fungal propagules can be found in the digestive system or the feces of these animals of different trophic levels [
5,
13]. Furthermore, the propagules can adhere to the exoskeleton (ectozoochory) of the arthropods while they move between infected plant material [
14,
15]. Moyo et al. [
5] found on herbivory and on predatory arthropods phytopathogenic fungi and showed that herbivore species transmitted the pathogen to healthy plants and that their feces are a source for inoculation. This shows that numerous arthropod species can act as vectors for plant diseases.
Fusarium (F.) spp. and
Alternaria (Al.) spp. (Table Abbreviations and Definitions) are phytopathogenic filamentous fungi that cause immense economic losses worldwide when they infect several crop plants, including wheat [
16]. Both genera produce mycotoxins that harm humans and livestock, making their management, and therefore their dispersal mechanisms to one of the greatest concerns in agriculture [
17,
18]. Wind and rain play major roles in the dispersal of the microscopic spores produced by these two pathogens, but both fungi are also often associated with arthropods [
19,
20,
21]. Especially different
Fusarium species like
F. avenaceum (Fries.) Sacc
, F. oxysporum Schlechtendahl emend. Snyder and Hansen,
F. verticillioides (Sacc) Nirenberg are vectored by various insect species [
9,
15,
22]. Furthermore,
Alternaria spp. is frequently isolated from arthropods, like the red flour beetle, leaf cutter ants, or mites [
23,
24,
25]. Therefore, arthropods may play an important role in the dispersal of
Alternaria spp. as well as of many other fungal pathogens.
Additionally, natural vegetation like grasses and arable weeds, plant debris, or organic matter in the soil are alternative hosts for phytopathogenic fungi like
Fusarium and
Alternaria and are frequent sources for new infections of crop plants [
26,
27,
28]. Arthropods can not only move frequently between crop plants but also between alternative inoculum sources and crop plants. Therefore, it is possible that arthropods regularly disseminate fungal propagules between different hosts and can therefore affect the disease pattern in the environment.
The ground-dwelling carabid beetles are very likely to get into contact with
Fusarium spp. and
Alternaria spp. and other fungi on weeds, plant debris, or crop plants. Their ecology is very diverse and most carabid beetles are very mobile insectivores and considered beneficial for agriculture [
29,
30,
31]. Nevertheless, some species are granivore or food specialists [
32,
33]. These beetles are very common in agricultural landscapes and are well studied, except for their contribution to the microbial community and their potential to disseminate pathogens [
34]. In general, ground-dwelling arthropods are a promising group when investigating supplementary pathways for pathogen vectors since they are very abundant, highly mobile, and move frequently between semi-natural and agricultural habitats.
This study investigates if and how carabids contribute to the dispersal of Fusarium spp., Alternaria spp., and other fungi. We wanted to quantify the fungal, and the Fusarium and Alternaria load (number of propagules or genomes) of different carabid species. Despite species-specific impacts, we searched for more generic traits explaining fungal loads in carabids, like carabid’s body size and weight. Furthermore, we wanted to identify Fusarium and Alternaria species and their abundances on the body surface of the carabid beetles.
Hypothesis 1 (H1). We hypothesize that carabids frequently get in contact with fungi including Fusarium and Alternaria fungi, and hence we expect to find whose DNA or propagules on and in most of the carabid beetles.
Hypothesis 2 (H2). Additionally, we expect in general a high percentage of Fusarium and Alternaria in the total fungal load, and a higher species number and load of Fusarium than Alternaria fungi because many Fusarium species are known to be dispersed by arthropods.
Hypothesis 3 (H3). Furthermore, we expect that certain traits of the carabids, here the body size and body weight, affect the total fungal, Fusarium, and Alternaria load and that these are higher in larger carabid species.
We collected carabids with pitfall traps in wheat fields close to semi-natural small water bodies (kettle holes). These pond-like habitats are suspected to be a source for phytopathogenic fungi since they provide moisture and alternative hosts plants for phytopathogenic fungi [
35,
36]. Fungal propagules on the body surface (exogenous) of the carabid beetles were quantified and
Fusarium spp. and
Alternaria spp. were identified with a culture-dependent approach. Additionally, exogenous and endogenous fungal,
Fusarium, and
Alternaria DNA, from the body surface and the inner body parts of the carabid beetles were quantified using qPCR-based methods. This paper provides the first insights into the role of a common agricultural non-pest insect in the dispersal of devastating plant pathogens.
4. Discussion
We explored the loads and frequencies of different fungal taxa, on the body surface and in the entire bodies of different carabid species with a molecular and a culture-dependent method. With a special focus on the phytopathogenic and mycotoxigenic fungal taxa Fusarium and Alternaria, we identified multiple species of both genera on the body surface of the carabids. We related the total fungal, Fusarium and Alternaria, load to the body size and weight, and compared the carabid species to identify traits affecting the fungal loads. Interactions between the ground-dwelling carabids and different fungal genera, including Fusarium and Alternaria, are very frequent in crop fields, are positively affected by the body size and weight, and differ between the carabid species.
The culture-dependent and the qPCR method showed similar trends regarding the total fungal load, but also the differences regarding Fusarium and Alternaria fungi provided relevant insights. In general, the culture-dependent method quantified only viable and potentially infectious fungal propagules that were washed off the body surface of the carabids. In contrast, exogenous and endogenous fungal DNA of viable or dead propagules was detected together from the body surface and the guts of the carabid beetles in the qPCR approach.
Horizontal transmission of propagules or DNA between individual carabids during the collecting within the pitfall traps could not be excluded with the used methods. Furthermore, in the culture-dependent method, contamination of the body surface with fungal material from the feces or another body secretes is to some extent possible. Both issues should be addressed in further studies in more detail.
The DNA of different fungal genera were detected in 100% of the investigated carabid beetles with the qPCR method. The microbiota in the guts of insects consists of bacteria, protists, archaea, and a few fungi [
50,
51]. Digestive fungi are common in the digestive tract of insects that feed on detritus or wood, but they probably play a minor role for the investigated carabids since their diet is not wood or detritus-based [
50,
52]. Furthermore, gut mycobiota are necessary for the immune response and protection against pathogens [
53]. In general, the gut mycobiota of insects could either closely relate to the fungi in the food and the environment of the insects, or fungal species composition in the guts could change independently from the environmental mycobiota which suggests a filtering mechanism [
11,
51]. Commonly found fungal genera in the guts of insects are
Aspergillus,
Mucor,
Cladosporium,
Fusarium,
Penicillium, yeasts like basidiomycetes, or ascomycetes [
51,
54]. The feces of arthropods can be a relevant inoculum source since the propagules of many fungal species stay viable after digestion, like
Fusarium oxysporum, the Grapevine Trunk Disease Pathogens
Phaeomoniella chlamydospora, or
Fusarium proliferatum [
5,
15,
55].
Viable fungal propagules of different genera were detected with the culture-dependent method on the body surface of 74% of the investigated carabids. A considerably lower frequency was detected by plating the appendages and guts of fungivore Collembola [
11]. Common fungal genera on insects are
Fusarium,
Epicoccum,
Penicillium,
Aspergillus,
Cladosporium, and yeasts like
Candida [
23,
56,
57]. Fungal propagules of different genera attached very frequently to the body surface of carabid beetles and were possibly transported. In general, propagules can attach to different body parts like the hairs on legs or antennae, or stick to the wings or mouthparts of the insects when the insect is foraging, or moving between infected plant material and get in contact with fruit bodies, mycelia, or spores of the fungi [
58,
59]. The exogenous acquisition of fungal propagules increases with exposure time to an inoculum source. The transmission of propagule decreases with time after the exposure to the inoculum source [
58].
The fungal genera
Fusarium and
Alternaria were detected in 49% (propagules, exogenous) and 98% (DNA, exogenous and endogenous), respectively. Higher frequencies for
Fusarium propagules on the body surface of insects like bark beetles and pigweed weevil (
Hypolixus haerens) were detected previously [
56,
57]. However, studies on the banana weevil (
Cosmopolites sordidus), a known vector for
F. oxysporum, detected a lower frequency on the body surface [
60].
Trunk disease pathogens were isolated from two arthropod species in frequencies similar to the here detected frequencies for
Fusarium propagules, which were based on these considered effective vectors [
5]. Therefore, carabid beetles are probably vectors for
Fusarium fungi, transporting propagules on the body surface.
Studies investigating the arthropod-mediated dispersal for
Alternaria fungi, especially studies quantifying endogenous and exogenous fungi separately with molecular methods, are very sparse. With the culture-dependent method, viable propagules of
Al. brassicicola were often detected in the feces of the flea beetle (
Phyllotreta cruciferae) and
Al. spp. were found in the guts of mites [
11,
59]. On the body surface, viable propagules of
Al. infectoria,
Al. arborescence, and
Al. alternata were recently isolated from Leaf-cutting ants, different collembolan, and the red flour beetle (
Tribolium castaneum), respectively [
11,
23,
24]. Based on the very frequent detection of
Alternaria DNA in this study, we suggest that carabids could also be considered a vector for
Alternaria fungi.
Previous studies showed that
Fusarium and
Alternaria propagules stay viable in the feces or the gut of arthropods [
15,
59]. However, the proportion of viable propagules or the amount of DNA detected in the feces and therefore the actual infection potential should be investigated in further studies. The quantification of transferred fungal propagules and the effect of disease development would be the next step to estimate the relevance of this dispersal mechanism and should be the target of further studies.
Carabids move frequently between semi-natural breeding habitats which are suggested to be a source for phytopathogenic fungi and adjacent crop fields and disperse further into new habitats [
61]. Carabid beetles are very mobile and can cover distances of several meters in a random pattern like a correlated random walk and much longer distances in a directed movement pattern, e.g.,
P. versicolor was observed to walk 87 m per day [
62,
63]. Many species, especially the smaller ones, can fly too or are drifted by wind [
34]. These mobile insects can exchange microorganisms and link different habitat types by covering shorter distances very frequently and longer distances from time to time. This movement behavior makes them potentially relevant vectors for several microorganisms. Additionally, carabid species vary in their spatial-temporal load, larger carabid species are usually less common than smaller ones [
34]. Therefore, to evaluate the impact of a carabid species on the dissemination of fungi, their species-specific load and movement behavior has to be considered too.
In general, our second hypothesis regarding the higher load of
Fusarium compared to
Alternaria fungi was only partly confirmed. We detected frequent and abundant viable
Fusarium propagules on the body surface of the carabids.
Fusarium DNA was very rare in the analysis of the entire body and considerably less than
Alternaria DNA. This is in line with other studies which found
Fusarium propagules frequently on the body surface of insects, but not in the digestive tract [
15,
60]. Furthermore,
Fusarium fungi produce mycotoxins effect for humans and animals [
64]. In known insect vectors for different phytopathogenic fungi, reduced survival, fecundity, biomass, and a slower development were observed in the insect species [
9,
65,
66]. These findings suggest that carabids may avoid ingesting food that contains high levels of
Fusarium and/or its mycotoxins. Nevertheless, carabids beetles and probably other ground-dwelling arthropods move regularly in environments where
Fusarium fungi are frequent so that propagules can attach to the body surface.
In contrast,
Alternaria DNA was detected very frequently in the analysis of the entire bodies but viable propagules on the body surface of the carabids were rarely found. In contrast, propagules of
Al. brassicicola were frequently detected by culture-dependent method on the body surface of flea beetles (
Phyllotreta cruciferae), and viable propagules were detected in the feces of the flea beetles but this frequency was not given by the authors [
59].
Alternaria is occasionally detected on the body surface or feces of different arthropods, but seldom in a comparative approach. Nevertheless, the results of this study suggest, that carabid beetles ingest
Alternaria fungi very frequently and the fungal DNA accumulates in the digestive tract. However, the propagules either do not attach to the body surface of the carabid beetles, or they don’t stay viable as effectively as
Fusarium propagules since they are more sensitive towards drought stress and UV radiation [
67,
68].
The fungal load of insects is also affected by the ability of propagules to attach to the body surface, which is mediated by fungal species-specific traits. This includes the spore-bearing structures, physical and chemical properties of the propagules like enzymes, or glycoproteins, or electrostatic recognition systems. Terrestrial spore types can range from dry hydrophobic to sticky hydrophilic conidia [
69]. Additionally, our results showed that the production of microconidia did not increase the number of detected CFU and has probably no advantage for the dispersal by the carabids.
Disease-induced plant volatile chemical emissions, caused by a
Fusarium infection, change the behavior of arthropods. They can be repellent for grain aphids or attractive for sap beetles [
66,
70]. Carabid beetles also change their behavior according to volatiles send out by plants [
71].
In our fieldwork, the data sampling for both methods differed slightly in time, the collection of carabids for the molecular approach were six weeks earlier. We cannot fully exclude that the sampling period and the development state of the vegetation might also affect the fungal community. In further experiments, sampling should be done at the same time.
We identified a complex fungal community on the body surface of the carabids consisting of various
Fusarium and
Alternaria species in different frequencies and abundances, including relevant phytopathogens.
F. culmorum is one of the main agents of Fusarium Head Blight [
17] and made up 47% of all detected
Fusarium CFU.
F. sambucinum,
F. equiseti, and
F. sporotrichioides are regularly associated with FHB and were also detected here in lower frequencies [
72,
73].
Al. infectoria made up half of the detected
Alternaria CFU on the body surface of the beetles and produces low amounts of mycotoxins [
44]. The other three detected
Alternaria species
Al. alternata,
Al. arborescence, and
Al. tenuissima are pathogens that generate higher levels of mycotoxins and induce diseases like the black point disease, black kernel, and leaf blight [
44,
74].
In general, arable weeds are an inoculum source for
Fusarium species, next to different crops like maize and wheat [
26]. Most
Fusarium species can survive on crop residuals, soil, and dead plant matter where they easily interact with ground-dwelling arthropods [
75].
Alternaria fungi are ubiquitous saprotrophs or opportunistic pathogens and colonize a wide range of plant species, like different types of crops such as small-grain cereals, fruit, and vegetables [
76]. Both fungal genera are often found together on wheat plants and compete for the same resources [
77].
Competition shapes the microbial community and differences in the saprotrophic capacity of fungal species can affect the species composition [
78].
F. solani,
F. oxysporum,
F. poae, and
F. sporotrichioides have a better saprotrophic capacity in crop residues or soil than
F. graminearum [
78]. Furthermore,
Fusarium and
Alternaria are known antagonists that affect the growth and mycotoxin production of each other [
79,
80]. Competitive and antagonistic interactions affect the production of primary inoculum and the growth of fungi on the plant residuals and soil and therefore the potential fungal load of insects that share the same habitat [
78].
Species-specific traits of the carabid species might also affect the endogenous and exogenous fungal load. Fungal propagules can adhere to different structures on the body surface of carabids. The investigated carabid species differed in the number of hairs, bristles, and dimples on the cuticle [
40]. Among the investigated carabid species,
H. signaticornis is densely punctate and pubescent all over its body, however,
B. lampros and
B. properans are nearly hairless with only a few dimples. Furthermore, the diet of the investigated carabid species varies from granivorous like
A. aenea, or a mixed diet part plant-based and part carnivorous like
H. affinis or pure carnivore diet consisting of other arthropods like
B. lampros and or
P. versicolor [
52].
Further studies should aim for a larger sample size to identify underlying patterns in the interaction of carabid beetles and fungi and between fungal species. The previously mentioned propagules’ properties, the diet, the cuticle structure of the carabids should also be paid more attention in subsequent studies as well as the relationship between the beetle-associated fungal population and the fungi in the beetle’s environment.
Nevertheless, carabid beetles do disperse a variety of microorganisms, including fungal species of great economic relevance. This could alter the competitive and antagonistic interactions in the fungal community and affect the growth, the production of primary inoculum, or mycotoxins of economically relevant fungi.
The third hypothesis, regarding the effect of body size and weight on the fungal load, can also be confirmed. Significant differences between the carabid species were detected, regarding endogenous and exogenous total fungi and
Alternaria DNA, and exogenous propagules of total fungi and
Fusarium. Overall, larger or heavier species showed a higher fungal load which is corroborated by Yamoah et al. [
81]. This trend was confirmed by positive moderate correlation coefficients ranging from 0.43 to 0.58. In contrast, Moyo et al. [
5] detected pathogens in similar frequencies in a 3–5 mm large Cocktail Ant (
Crematogaster peringueyi) than in the 20–45 mm large millipede (
Ommatoiulus moreleti) but this aspect was not further analyzed by the authors.
In this study, body size and body weight were important factors. However, this only partly explained the distribution of the fungal load between carabid species as the correlation coefficient between fungal load and body size/weight suggests. In the culture-dependent approach, the comparison of the five investigated carabid species showed a relatively clear pattern where the largest species showed the highest fungal load. The morphological structure of the body surface of the carabids was not considered in the analyses but might be the source of the remaining variance. However, morphology only explains a part of the differences since the intra-specific variance and the variance between the morphologically similar species
B. lampros and
B. properans are considerably high. Other relevant factors, like the ecology of the carabids, should be investigated too. In the qPCR approach, the midsized species
A. aenea and
A. spp. showed a high fungal load, and in the largest species,
P. versicolor, a large intra-species variance was detected. This suggests that next to the body weight of the beetles, diet is also an important trait.
P. versicolor feeds on other insects and ingests the mycobiota of its prey, which could explain the great intra-specific variance [
52]. Species of the genus
Amara feed primarily on seeds and grains, which are common hosts for
Fusarium and
Alternaria [
27,
52,
82]. This diet probably explains the higher fungal load in this species. Overall, the fungal load is strongly affected by body size and weight. However, the diet and the morphological structure of the body surface of the carabids are relevant as well. Individual differences in the behavior of the insects or the fungal community in the habitat are also affecting the fungal load of the carabid beetles.
Carabids show a remarkable potential to disseminate propagules of different fungal genera, including multiple species of the phytopathogenic Fusarium and Alternaria fungi.
On the one hand, this dispersal mechanism could enhance crop diseases by transporting propagules from different inoculum sources to the crop plants. Therefore, the dispersal of fungal propagules by ground-dwelling arthropods should be given greater emphasis in the analysis of crop diseases. On the other hand, based on zoochory, carabid beetles could be mobile linkers and alter the fungal community in (semi-) natural habitats and crop fields by exchanging fungal species or strains and link these habitats via their extensive movement pattern. Unraveling the movement behavior of arthropods and their associations with microorganisms is crucial to understand dynamics and patterns in micro-communities.