The sky is not the limit: Successful foliar application of Steinernema spp. entomopathogenic nematodes to control Lepidopteran caterpillars.

Entomopathogenic nematodes (EPNs) are ubiquitous soil-thriving organisms that use chemical cues to seek and infect soil-dwelling arthropods, yielding various levels of biological control. Going beyond soil application, scientists and practitioners started exploring the option of applying EPNs onto the foliage of crops in attempts to manage leaf-dwelling insect pests as well. Despite some success, particularly with protective formulations, it remains uncertain whether EPNs could indeed survive the phyllospheric environment, and successfully control foliar insect pests. In this context, we tested the potential of commercially produced Steinernema feltiae and S. carpocapsae, two of the most commonly used EPNs in the field of biological control, in controlling Lepidopteran foliar pests of economic importance, i.e. Tuta absoluta and Spodoptera spp. caterpillars as models. We first tested the survival and efficacy of both EPN species against the Lepidopteran caterpillars when applied onto tomato, sweet pepper and lettuce leaves, under controlled conditions and in commercial greenhouse conditions, respectively. Subsequently, we explored the behavioural responses of the EPNs to environmental cues typically encountered in the phyllosphere, and analysed plant volatile organic compounds (VOCs). Our results show that both S. feltiae and S. carpocapsae successfully survived and infected the foliar caterpillars, reaching similar level of control to a standard chemical pesticide in commercial practices. Remarkably, both EPN species survived and remained effective up to four days in the phyllosphere, and needed only a few hours to successfully penetrate the caterpillars. Interestingly, S. feltiae was attracted to VOCs from tomato plants, and tended to prefer those from caterpillar-induced plants, suggesting that the nematodes may actively forage toward its host, although it has never been exposed to leaf-borne volatiles during its evolution. The present study shows the high potential of steinernematids in managing major foliar pests in greenhouses and in becoming a key player in foliar biological control. In particular, the discovery that EPNs use foliar VOCs to locate caterpillar hosts opens up new opportunities in terms of application techniques and affordable effective doses.

Volatiles from soil-borne fungi affect directional growth of roots.

Volatiles play major roles in mediating ecological interactions between soil (micro)organisms and plants. It is well-established that microbial volatiles can increase root biomass and lateral root formation. To date, however, it is unknown whether microbial volatiles can affect directional root growth. Here, we present a novel method to study belowground volatile-mediated interactions. As proof-of-concept, we designed a root Y-tube olfactometer, and tested the effects of volatiles from four different soil-borne fungi on directional growth of Brassica rapa roots in soil. Subsequently, we compared the fungal volatile organic compounds (VOCs) previously profiled with Gas Chromatography-Mass Spectrometry (GC-MS). Using our newly designed setup, we show that directional root growth in soil is differentially affected by fungal volatiles. Roots grew more frequently toward volatiles from the root pathogen Rhizoctonia solani, whereas volatiles from the other three saprophytic fungi did not impact directional root growth. GC-MS profiling showed that six VOCs were exclusively emitted by R. solani. These findings verify that this novel method is suitable to unravel the intriguing chemical cross-talk between roots and soil-borne fungi and its impact on root growth.

Volatiles of pathogenic and non-pathogenic soil-borne fungi affect plant development and resistance to insects.

Plants are ubiquitously exposed to a wide diversity of (micro)organisms, including mutualists and antagonists. Prior to direct contact, plants can perceive microbial organic and inorganic volatile compounds (hereafter: volatiles) from a distance that, in turn, may affect plant development and resistance. To date, however, the specificity of plant responses to volatiles emitted by pathogenic and non-pathogenic fungi and the ecological consequences of such responses remain largely elusive. We investigated whether Arabidopsis thaliana plants can differentiate between volatiles of pathogenic and non-pathogenic soil-borne fungi. We profiled volatile organic compounds (VOCs) and measured CO2 emission of 11 fungi. We assessed the main effects of fungal volatiles on plant development and insect resistance. Despite distinct differences in VOC profiles between the pathogenic and non-pathogenic fungi, plants did not discriminate, based on plant phenotypic responses, between pathogenic and non-pathogenic fungi. Overall, plant growth was promoted and flowering was accelerated upon exposure to fungal volatiles, irrespectively of fungal CO2 emission levels. In addition, plants became significantly more susceptible to a generalist insect leaf-chewing herbivore upon exposure to the volatiles of some of the fungi, demonstrating that a prior fungal volatile exposure can negatively affect plant resistance. These data indicate that plant development and resistance can be modulated in response to exposure to fungal volatiles.

Plant Phenotypic and Transcriptional Changes Induced by Volatiles from the Fungal Root Pathogen Rhizoctonia solani.

Beneficial soil microorganisms can affect plant growth and resistance by the production of volatile organic compounds (VOCs). Yet, little is known on how VOCs from soil-borne plant pathogens affect plant growth and resistance. Here we show that VOCs released from mycelium and sclerotia of the fungal root pathogen Rhizoctonia solani enhance growth and accelerate development of Arabidopsis thaliana. Seedlings briefly exposed to the fungal VOCs showed similar phenotypes, suggesting that enhanced biomass and accelerated development are primed already at early developmental stages. Fungal VOCs did not affect plant resistance to infection by the VOC-producing pathogen itself but reduced aboveground resistance to the herbivore Mamestra brassicae. Transcriptomics of A. thaliana revealed that genes involved in auxin signaling were up-regulated, whereas ethylene and jasmonic acid signaling pathways were down-regulated by fungal VOCs. Mutants disrupted in these pathways showed similar VOC-mediated growth responses as the wild-type A. thaliana, suggesting that other yet unknown pathways play a more prominent role. We postulate that R. solani uses VOCs to predispose plants for infection from a distance by altering root architecture and enhancing root biomass. Alternatively, plants may use enhanced root growth upon fungal VOC perception to sacrifice part of the root biomass and accelerate development and reproduction to survive infection.