Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

Root-Associated Entomopathogenic Fungi Modulate Their Host Plant's Photosystem II Photochemistry and Response to Herbivorous Insects.

The escalating food demand and loss to herbivores has led to increasing interest in using resistance-inducing microbes for pest control. Here, we evaluated whether root-inoculation with fungi that are otherwise known as entomopathogens improves tomato (Solanum lycopersicum) leaflets' reaction to herbivory by Spodoptera exigua (beet armyworm) larvae using chlorophyll fluorescence imaging. Plants were inoculated with Metarhizium brunneum or Beauveria bassiana, and photosystem II reactions were evaluated before and after larval feeding. Before herbivory, the fraction of absorbed light energy used for photochemistry (ΦPSII) was lower in M. brunneum-inoculated than in control plants, but not in B. bassiana-inoculated plants. After herbivory, however, ΦPSII increased in the fungal-inoculated plants compared with that before herbivory, similar to the reaction of control plants. At the same time, the fraction of energy dissipated as heat (ΦNPQ) decreased in the inoculated plants, resulting in an increased fraction of nonregulated energy loss (ΦNO) in M. brunneum. This indicates an increased singlet oxygen (1O2) formation not detected in B. bassiana-inoculated plants, showing that the two entomopathogenic fungi differentially modulate the leaflets' response to herbivory. Overall, our results show that M. brunneum inoculation had a negative effect on the photosynthetic efficiency before herbivory, while B. bassiana inoculation had no significant effect. However, S. exigua leaf biting activated the same compensatory PSII response mechanism in tomato plants of both fungal-inoculated treatments as in control plants.

Timing of sub-lethal insecticide exposure determines parasite establishment success in an insect-helminth model.

Environmental toxicants are pervasive in nature, but sub-lethal effects on non-target organisms and their parasites are often overlooked. Particularly, studies on terrestrial hosts and their parasites exposed to agricultural toxicants are lacking. Here, we studied the effect of sequence and timing of sub-lethal exposures of the pyrethroid insecticide alpha-cypermethrin on parasite establishment using the tapeworm Hymenolepis diminuta and its intermediate insect host Tenebrio molitor as a model system. We exposed T. molitor to alpha-cypermethrin (LD20) before and after experimental H. diminuta infection and measured the establishment success of larval tapeworms. Also, we conducted in vitro studies quantifying the direct effect of the insecticide on parasite viability. Our results showed that there was no direct lethal effect of alpha-cypermethrin on H. diminuta cysticercoids at relevant concentrations (LD10 to LD90 of the intermediate host). However, we observed a significantly increased establishment of H. diminuta in beetles exposed to alpha-cypermethrin (LD20) after parasite infection. In contrast, parasite establishment was significantly lower in beetles exposed to the insecticide before parasite infection. Thus, our results indicate that environmental toxicants potentially impact host-parasite interactions in terrestrial systems, but that the outcome is context-dependent by enhancing or reducing parasite establishment depending on timing and sequence of exposure.

Differences in life stage sensitivity of the beetle Tenebrio molitor towards a pyrethroid insecticide explained by stage-specific variations in uptake, elimination and activity of detoxifying enzymes.

It is widely accepted that sensitivity towards pesticides varies significantly between species. Much less is known about the potential differences in pesticide sensitivity and its biological mechanism throughout the lifecycle of a single species. In the present study we used three life-stages (larvae, pupae and adult) of the holometabolous insect Tenebrio molitor to investigate: i) Life-stage specific differences in sensitivity towards the pyrethroid insecticide α-cypermethrin after topical exposure, and ii) whether these differences can be explained by the degree of uptake and/or excretion. Finally, we investigated if an efficient excretion coincided with higher activities of the detoxifying enzymes cytochrome P450 (P450), esterases (EST) and glutathione-S-transferease (GST). We found that mobility of adults of T. molitor was more affected by α-cypermethrin treatment than larvae and pupae. Mortality was relatively low for all life stages and did not vary significantly with dose within the duration of the experiment, which indicated that death was (at least partly) due to starvation (indirect effect of paralysis) rather that direct effects of the insecticide. Insecticide treatment during the pupal stage further impaired normal development from pupa to adult. Toxicokinetic measurements showed that cuticle penetration of α-cypermethrin differed significantly between life-stages. Approximately 50% of the applied insecticide had penetrated the adult cuticle after 1 h, whereas a maximum of 30% and 16% had penetrated the waxier cuticle of larvae and pupae. Further, the pupal stage lacked the ability to excrete compounds, and hence internal insecticide concentrations in pupae increased or stagnated until emergence of the adult. Finally, quantification of detoxification enzymes showed a markedly higher activity of P450 in adults and larvae compared to pupae. These findings suggest that assessing toxicity and/or risk of pesticides collectively for a species may not be adequate without taking into account the potential sensitivity differences between life stages.

Factors affecting vegetable growers' exposure to fungal bioaerosols and airborne dust.

We have quantified vegetable growers' exposure to fungal bioaerosol components including (1→3)-ß-d-glucan (ß-glucan), total fungal spores, and culturable fungal units. Furthermore, we have evaluated factors that might affect vegetable growers' exposure to fungal bioaerosols and airborne dust. Investigated environments included greenhouses producing cucumbers and tomatoes, open fields producing cabbage, broccoli, and celery, and packing facilities. Measurements were performed at different times during the growth season and during execution of different work tasks. Bioaerosols were collected with personal and stationary filter samplers. Selected fungal species (Beauveria spp., Trichoderma spp., Penicillium olsonii, and Penicillium brevicompactum) were identified using different polymerase chain reaction-based methods and sequencing. We found that the factors (i) work task, (ii) crop, including growth stage of handled plant material, and (iii) open field versus greenhouse significantly affected the workers' exposure to bioaerosols. Packing of vegetables and working in open fields caused significantly lower exposure to bioaerosols, e.g. mesophilic fungi and dust, than harvesting in greenhouses and clearing of senescent greenhouse plants. Also removing strings in cucumber greenhouses caused a lower exposure to bioaerosols than harvest of cucumbers while removal of old plants caused the highest exposure. In general, the exposure was higher in greenhouses than in open fields. The exposures to ß-glucan during harvest and clearing of senescent greenhouse plants were very high (median values ranging between 50 and 1500 ng m(-3)) compared to exposures reported from other occupational environments. In conclusion, vegetable growers' exposure to bioaerosols was related to the environment, in which they worked, the investigated work tasks, and the vegetable crop.

Induction of a Compensatory Photosynthetic Response Mechanism in Tomato Leaves upon Short Time Feeding by the Chewing Insect Spodoptera exigua.

In addition to direct tissue consumption, herbivory may affect other important plant processes. Here, we evaluated the effects of short-time leaf feeding by Spodoptera exigua larvae on the photosynthetic efficiency of tomato plants, using chlorophyll a fluorescence imaging analysis. After 15 min of feeding, the light used for photochemistry at photosystem II (PSII) (ΦPSII), and the regulated heat loss at PSII (ΦNPQ) decreased locally at the feeding zones, accompanied by increased non-regulated energy losses (ΦNO) that indicated increased singlet oxygen (1O2) formation. In contrast, in zones neighboring the feeding zones and in the rest of the leaf, ΦPSII increased due to a decreased ΦNPQ. This suggests that leaf areas not directly affected by herbivory compensate for the photosynthetic losses by increasing the fraction of open PSII reaction centers (qp) and the efficiency of these centers (Fv'/Fm'), because of decreased non-photochemical quenching (NPQ). This compensatory reaction mechanism may be signaled by singlet oxygen formed at the feeding zone. PSII functionality at the feeding zones began to balance with the rest of the leaf 3 h after feeding, in parallel with decreased compensatory responses. Thus, 3 h after feeding, PSII efficiency at the whole-leaf level was the same as before feeding, indicating that the plant managed to overcome the feeding effects with no or minor photosynthetic costs.

UV-B Radiation Tolerance and Temperature-Dependent Activity Within the Entomopathogenic Fungal Genus Metarhizium in Brazil.

Metarhizium comprises a phylogenetically diverse genus of entomopathogenic fungi. In Brazil, Metarhizium anisopliae s.str. subclade Mani 2 is predominantly isolated from insects, while M. robertsii and M. brunneum mostly occur in the soil environment. Solar radiation and high temperatures are important abiotic factors that can be detrimental to fungal propagules. We hypothesized that among 12 Brazilian isolates of Metarhizium spp., M. anisopliae Mani 2 (n = 6), being adapted to abiotic conditions of the phylloplane, is more tolerant to UV light and high temperatures than M. robertsii (n = 3) and M. brunneum (n = 3). Inoculum of each isolate was exposed to UV-B for up to 8 h and viability evaluated 48 h later. After 8 h under UV-B, most of the isolates had germination rates below 5%. Discs of mycelia were incubated at different temperatures, and diameter of colonies were recorded for 12 days. Mycelia of M. robertsii isolates grew faster at 33 °C, while M. anisopliae and M. brunneum grew most at 25 °C. Dry conidia were incubated at 20, 25 or 40 °C for 12 days, and then viabilities were examined. At 40 °C, conidia of five M. anisopliae isolates were the most tolerant. In the three experiments, considerable intra- and inter-specific variability was detected. The results indicate that conclusions about tolerance to these abiotic factors should be made only at the isolate level.

Associations between carabid beetles and fungi in the light of 200 years of published literature.

Describing and conserving ecological interactions woven into ecosystems is one of the great challenges of the 21st century. Here, we present a unique dataset compiling the biotic interactions between two ecologically and economically important taxa: ground beetles (Coleoptera: Carabidae) and fungi. The resulting dataset contains the carabid-fungus associations collected from 392 scientific publications, 129 countries, mostly from the Palearctic region, published over a period of 200 years. With an updated taxonomy to match the currently accepted nomenclature, 3,378 unique associations among 5,564 records were identified between 1,776 carabid and 676 fungal taxa. Ectoparasitic Laboulbeniales were the most frequent fungal group associated with carabids, especially with Trechinae. The proportion of entomopathogens was low. Three different formats of the data have been provided along with an interactive data digest platform for analytical purposes. Our database summarizes the current knowledge on biotic interactions between insects and fungi, while offering a valuable resource to test large-scale hypotheses on those interactions.

Human exposure to airborne fungi from genera used as biocontrol agents in plant production.

The fungi Trichoderma harzianum, T. polysporum, T. viride, Paeciliomyces fumosoroseus, P. lilacinus, Verticillium/lecanicillium lecanii, Ulocladium oudemansii, U. atrum and Beauveria bassiana are used or considered to be used for biocontrol of pests and plant diseases. Human exposure to these fungi in environments where they may naturally occur or are used as biocontrol agents has not been directly investigated to date. This review aims to provide an overview of the current knowledge of human exposure to fungi from the relevant genera. The subject of fungal taxonomy due to the rapid development of this issue is also discussed. B. bassiana, V. lecanii, T. harzianum, T. polysporum, P. lilacinus and U. oudemansii were infrequently present in the air and thus people in general seem to be seldom exposed to these fungi. However, when V. lecanii was present, high concentrations were measured. Fungi from the genera Trichoderma, Paecilomyces and Ulocladium were rarely identified to the species level and sometimes high concentrations were reported. T. viride and U. atrum were detected frequently in different environments and sometimes with a high frequency of presence in samples. Thus, people seem to be frequently exposed to these fungi. Sequence data have led to recent revisions of fungal taxonomy, and in future studies it is important to specify the taxonomy used for identification, thus making comparisons possible.