Monitoring Spore Dispersal and Early Infections of Diplocarpon coronariae Causing Apple Blotch Using Spore Traps and a New qPCR Method.

Apple blotch (AB) is a major disease of apple in Asia and recently emerged in Europe and the United States. It is caused by the fungus Diplocarpon coronariae (formerly Marssonina coronaria; teleomorph: Diplocarpon mali) and leads to severe defoliation of apple trees in late summer, resulting in reduced yield and fruit quality. To develop effective disease management strategies, a sound knowledge of the pathogen's biology is crucial. Data on the early phase of disease development are scarce: No data on spore dispersal in Europe are available. We developed a highly sensitive TaqMan qPCR method to quantify D. coronariae conidia in spore trap samples. We monitored temporal and spatial dispersal of conidia of D. coronariae and the progress of AB in spring and early summer in an extensively managed apple orchard in Switzerland in 2019 and 2020. Our results show that D. coronariae overwinters in leaf litter, and spore dispersal and primary infections occur in late April and early May. We provide the first results describing early-season dispersal of conidia of D. coronariae, which, combined with the observed disease progress, helps to understand the disease dynamics and will be a basis for improved disease forecast models. Using the new qPCR method, we detected D. coronariae in buds, on bark, and on fruit mummies, suggesting that several apple tissues might serve as overwintering habitats for the fungus, in addition to fallen leaves. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The secret life of plant-beneficial rhizosphere bacteria: insects as alternative hosts.

Root-colonizing bacteria have been intensively investigated for their intimate relationship with plants and their manifold plant-beneficial activities. They can inhibit growth and activity of pathogens or induce defence responses. In recent years, evidence has emerged that several plant-beneficial rhizosphere bacteria do not only associate with plants but also with insects. Their relationships with insects range from pathogenic to mutualistic and some rhizobacteria can use insects as vectors for dispersal to new host plants. Thus, the interactions of these bacteria with their environment are even more complex than previously thought and can extend far beyond the rhizosphere. The discovery of this secret life of rhizobacteria represents an exciting new field of research that should link the fields of plant-microbe and insect-microbe interactions. In this review, we provide examples of plant-beneficial rhizosphere bacteria that use insects as alternative hosts, and of potentially rhizosphere-competent insect symbionts. We discuss the bacterial traits that may enable a host-switch between plants and insects and further set the multi-host lifestyle of rhizobacteria into an evolutionary and ecological context. Finally, we identify important open research questions and discuss perspectives on the use of these rhizobacteria in agriculture.

Transcriptome plasticity underlying plant root colonization and insect invasion by Pseudomonas protegens.

Pseudomonas protegens shows a high degree of lifestyle plasticity since it can establish both plant-beneficial and insect-pathogenic interactions. While P. protegens protects plants against soilborne pathogens, it can also invade insects when orally ingested leading to the death of susceptible pest insects. The mechanism whereby pseudomonads effectively switch between lifestyles, plant-beneficial or insecticidal, and the specific factors enabling plant or insect colonization are poorly understood. We generated a large-scale transcriptomics dataset of the model P. protegens strain CHA0 which includes data from the colonization of wheat roots, the gut of Plutella xylostella after oral uptake and the Galleria mellonella hemolymph after injection. We identified extensive plasticity in transcriptomic profiles depending on the environment and specific factors associated to different hosts or different stages of insect infection. Specifically, motor-activity and Reb toxin-related genes were highly expressed on wheat roots but showed low expression within insects, while certain antimicrobial compounds (pyoluteorin), exoenzymes (a chitinase and a polyphosphate kinase), and a transposase exhibited insect-specific expression. We further identified two-partner secretion systems as novel factors contributing to pest insect invasion. Finally, we use genus-wide comparative genomics to retrace the evolutionary origins of cross-kingdom colonization.

Persistence of root-colonizing Pseudomonas protegens in herbivorous insects throughout different developmental stages and dispersal to new host plants.

The discovery of insecticidal activity in root-colonizing pseudomonads, best-known for their plant-beneficial effects, raised fundamental questions about the ecological relevance of insects as alternative hosts for these bacteria. Since soil bacteria are limited in their inherent abilities of dispersal, insects as vectors might be welcome vehicles to overcome large distances. Here, we report on the transmission of the root-colonizing, plant-beneficial and insecticidal bacterium Pseudomonas protegens CHA0 from root to root by the cabbage root fly, Delia radicum. Following ingestion by root-feeding D. radicum larvae, CHA0 persisted inside the insect until the pupal and adult stages. The emerging flies were then able to transmit CHA0 to a new plant host initiating bacterial colonization of the roots. CHA0 did not reduce root damages caused by D. radicum and had only small effects on Delia development suggesting a rather commensal than pathogenic relationship. Interestingly, when the bacterium was fed to two highly susceptible lepidopteran species, most of the insects died, but CHA0 could persist throughout different life stages in surviving individuals. In summary, this study investigated for the first time the interaction of P. protegens CHA0 and related strains with an insect present in their rhizosphere habitat. Our results suggest that plant-colonizing pseudomonads have different strategies for interaction with insects. They either cause lethal infections and use insects as food source or they live inside insect hosts without causing obvious damages and might use insects as vectors for dispersal, which implies a greater ecological versatility of these bacteria than previously thought.

Antimicrobial and Insecticidal: Cyclic Lipopeptides and Hydrogen Cyanide Produced by Plant-Beneficial Pseudomonas Strains CHA0, CMR12a, and PCL1391 Contribute to Insect Killing.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host.

Looking BAK again: Is an old acquaintance of innate immunity involved in the detection of herbivores?

The membrane-based receptor-like kinase BAK1 has been reported to interact with a number of other membrane-based receptors to contribute to a variety of signaling responses to exogenous and endogenous cues. These include brassinosteroid hormones as well as conserved microbe-derived and endogenous patterns. More recently, several lines of evidence have been reported to expand this concept also to the detection and deterrence of insect herbivores. We hereby present results that further support this hypothesis as they show that in Arabidopsis thaliana, herbivore oral secretions trigger two hallmark responses of plant innate immunity and that these responses are significantly reduced in plants that lack functional BAK1 receptors.

Specific surface glycan decorations enable antimicrobial peptide resistance in plant-beneficial pseudomonads with insect-pathogenic properties.

Some plant-beneficial pseudomonads can invade and kill pest insects in addition to their ability to protect plants from phytopathogens. We explored the genetic basis of O-polysaccharide (O-PS, O-antigen) biosynthesis in the representative insecticidal strains Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 and investigated its role in insect pathogenicity. Both strains produce two distinct forms of O-PS, but differ in the organization of their O-PS biosynthesis clusters. Biosynthesis of the dominant O-PS in both strains depends on a gene cluster similar to the O-specific antigen (OSA) cluster of Pseudomonas aeruginosa. In CHA0 and other P. protegens strains, the OSA cluster is extensively reduced and new clusters were acquired, resulting in high diversity of O-PS structures, possibly reflecting adaptation to different hosts. CHA0 mutants lacking the short OSA form of O-PS were significantly impaired in insect virulence in Galleria injection and Plutella feeding assays. CHA0, PCL1391, and other insecticidal pseudomonads exhibited high resistance to antimicrobial peptides, including cecropins that are central to insect immune defense. Resistance of both model strains depended on the dominant OSA-type O-PS. Our results suggest that O-antigen is essential for successful insect infection and illustrate, for the first time, its importance in resistance of Pseudomonas to antimicrobial peptides.

Insect pathogenicity in plant-beneficial pseudomonads: phylogenetic distribution and comparative genomics.

Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.

The Arabidopsis Pep-PEPR system is induced by herbivore feeding and contributes to JA-mediated plant defence against herbivory.

A number of plant endogenous elicitors have been identified that induce pattern-triggered immunity upon perception. In Arabidopsis thaliana eight small precursor proteins, called PROPEPs, are thought to be cleaved upon danger to release eight peptides known as the plant elicitor peptides Peps. As the expression of some PROPEPs is induced upon biotic stress and perception of any of the eight Peps triggers a defence response, they are regarded as amplifiers of immunity. Besides the induction of defences directed against microbial colonization Peps have also been connected with herbivore deterrence as they share certain similarities to systemins, known mediators of defence signalling against herbivores in solanaceous plants, and they positively interact with the phytohormone jasmonic acid. A recent study using maize indicated that the application of ZmPep3, a maize AtPep-orthologue, elicits anti-herbivore responses. However, as this study only assessed the responses triggered by the exogenous application of Peps, the biological significance of these findings remained open. By using Arabidopsis GUS-reporter lines, it is now shown that the promoters of both Pep-receptors, PEPR1 and PEPR2, as well as PROPEP3 are strongly activated upon herbivore attack. Moreover, pepr1 pepr2 double mutant plants, which are insensitive to Peps, display a reduced resistance to feeding Spodoptera littoralis larvae and a reduced accumulation of jasmonic acid upon exposure to herbivore oral secretions. Taken together, these lines of evidence extend the role of the AtPep-PEPR system as a danger detection mechanism from microbial pathogens to herbivores and further underline its strong interaction with jasmonic acid signalling.

Evolutionary divergence of the plant elicitor peptides (Peps) and their receptors: interfamily incompatibility of perception but compatibility of downstream signalling.

Plant elicitor peptides (Peps) are potent inducers of pattern-triggered immunity and amplify the immune response against diverse pathogens. Peps have been discovered and studied extensively in Arabidopsis and only recently orthologues in maize were also identified and characterized in more detail.Here, the presence of PROPEPs, the Pep precursors, and PEPRs, the Pep receptors, was investigated within the plant kingdom. PROPEPs and PEPRs were identified in most sequenced species of the angiosperms. The conservation and compatibility of the Pep-PEPR-system was analysed by using plants of two distantly related dicot families, Brassicaceae and Solanaceae, and a representative family of monocot plants, the Poaceae. All three plant families contain important crop plants, including maize, rice, tomato, potato, and canola. Peps were not recognized by species outside of their plant family of origin, apparently because of a divergence of the Pep sequences. Three family-specific Pep motifs were defined and the integration of such a motif into the Pep sequence of an unrelated Pep enabled its perception. Transient transformation of Nicotiana benthamiana with the coding sequences of the AtPEPR1 and ZmPEPR1a led to the recognition of Pep peptides of Brassicaceae or Poaceae origin, respectively, and to the proper activation of downstream signalling. It was concluded that signalling machinery downstream of the PEPRs is highly conserved whereas the leucine-rich repeat domains of the PEPRs co-evolved with the Peps, leading to distinct motifs and, with it, interfamily incompatibility.

Several MAMPs, including chitin fragments, enhance AtPep-triggered oxidative burst independently of wounding.

AtPeps are a family of small peptides in Arabidopsis that are believed to act as endogenous amplifiers of the plant's innate immune response. In our recent study, (10) we showed that in Arabidopsis leaf disks, bacterial MAMPs (microbe-associated molecular patterns) such as the flagellin derived elicitor flg22, greatly enhanced the release of reactive oxygen species (ROS) triggered by a subsequent AtPep-perception. This enhanced ROS production could be a hallmark either of improved local defense or of the initiation of ROS-based systemic signaling. Here, we established a superior ROS detection system based on a new derivative of luminol (L-012). With this sensitive system we were able to show that chitin, too, acts as an enhancer of AtPep-triggered ROS, linking this specific defense response amplification also to the recognition of fungal pathogens. In addition we used the more sensitive ROS assay to transfer the experimental setup from cut leaf disks to unwounded seedlings. Thereby we revealed that wounding is not a prerequisite to enable the MAMP-induced enhancement of the AtPep-triggered ROS response.

The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst.

The endogenous Arabidopsis (Arabidopsis thaliana) peptides, AtPeps, elicit an innate immune response reminiscent of pattern-triggered immunity. Detection of various danger signals, including microbe-associated molecular patterns (MAMPs), leads to elevated transcription of PROPEPs, the AtPep precursors, and PEPRs, the AtPep receptors. It has been hypothesized that AtPeps are involved in enhancing pattern-triggered immunity. Following this idea, we analyzed the relationship between MAMP- and AtPep-elicited signaling. We found that the perception of MAMPs enhanced a subsequent AtPep-triggered production of reactive oxygen species (ROS). Intriguingly, other components of AtPep-triggered immunity like Ca(2+) influx, mitogen-activated protein kinase phosphorylation, ethylene production, and expression of early defense genes, as well as ROS-activated genes, remained unchanged. By contrast, treatment with methyl jasmonate promoted an increase of all analyzed AtPep-triggered responses. We positively correlated the intensities of generic AtPep-triggered responses with the abundance of the two AtPep receptors by generating constitutively expressing PEPR1 and PEPR2 transgenic lines and by analyzing pepr1 and pepr2 mutants. Further, we show that enhanced, as well as basal, ROS production triggered by AtPeps is absent in the double mutant of the respiratory burst oxidase homologs D and F (rbohD rbohF). We present evidence that the enhancement of AtPep-triggered ROS is not based on changes in the ROS detoxification machinery and is independent of mitogen-activated protein kinase and Ca(2+) signaling pathways. Taken together, these results indicate an additional level of regulation besides receptor abundance for the RbohD/RbohF-dependent production of AtPep-elicited ROS, which is specifically operated by MAMP-triggered pathways.