Genetic diversity of the invasive gall wasp Leptocybe invasa (Hymenoptera: Eulophidae) and of its Rickettsia endosymbiont, and associated sex-ratio differences

The blue-gum chalcid Leptocybe invasa Fisher & LaSalle (Hymenoptera: Eulophidae) is a gall wasp pest of Eucalyptus species, likely native to Australia. Over the past 15 years it has invaded 39 countries on all continents where eucalypts are grown. The worldwide invasion of the blue gum chalcid was attributed to a single thelytokous morphospecies formally described in 2004. Subsequently, however, males have been recorded in several countries and the sex ratio of field populations has been found to be highly variable in different areas. In order to find an explanation for such sex ratio differences, populations of L. invasa from a broad geographical area were screened for the symbionts currently known as reproductive manipulators, and both wasps and symbionts were genetically characterized using multiple genes. Molecular analyses suggested that L. invasa is in fact a complex of two cryptic species involved in the rapid and efficient spread of the wasp, the first recovered from the Mediterranean region and South America, the latter from China. All screened specimens were infected by endosymbiotic bacteria belonging to the genus Rickettsia. Two closely related Rickettsia strains were found, each infecting one of the two putative cryptic species of L. invasa and associated with different average sex ratios. Rickettsia were found to be localised in the female reproductive tissues and transovarially transmitted, suggesting a possible role of Rickettsia as the causal agent of thelytokous parthenogenesis in L. invasa. Implications for the variation of sex ratio and for the management of L. invasa are discussed. Nugne

Polyketide synthases of Diaporthe helianthi and involvement of DhPKS1 in virulence on sunflower

Background The early phases of Diaporthe helianthi pathogenesis on sunflower are characterized by the production of phytotoxins that may play a role in host colonisation. In previous studies, phytotoxins of a polyketidic nature were isolated and purified from culture filtrates of virulent strains of D. helianthi isolated from sunflower. A highly aggressive isolate (7/96) from France contained a gene fragment of a putative nonaketide synthase (lovB) which was conserved in a virulent D. helianthi population. Results In order to investigate the role of polyketide synthases in D. helianthi 7/96, a draft genome of this isolate was examined. We were able to find and phylogenetically analyse 40 genes putatively coding for polyketide synthases (PKSs). Analysis of their domains revealed that most PKS genes of D. helianthi are reducing PKSs, whereas only eight lacked reducing domains. Most of the identified PKSs have orthologs shown to be virulence factors or genetic determinants for toxin production in other pathogenic fungi. One of the genes (DhPKS1) corresponded to the previously cloned D. helianthi lovB gene fragment and clustered with a nonribosomal peptide synthetase (NRPS) -PKS hybrid/lovastatin nonaketide like A. nidulans LovB. We used DhPKS1 as a case study and carried out its disruption through Agrobacterium-mediated transformation in the isolate 7/96. D. helianthi DhPKS1 deleted mutants were less virulent to sunflower compared to the wild type, indicating a role for this gene in the pathogenesis of the fungus. Conclusion The PKS sequences analysed and reported here constitute a new genomic resource that will be useful for further research on the biology, ecology and evolution of D. helianthi and generally of fungal plant pathogens

Beauveria bassiana rewires molecular mechanisms related to growth and defense in tomato

Plant roots can exploit beneficial associations with soil-inhabiting microbes, promoting growth and expanding the immune capacity of the host plant. In this work, we aimed to provide new information on changes occurring in tomato interacting with the beneficial fungus Beauveria bassiana. The tomato leaf proteome revealed perturbed molecular pathways during the establishment of the plant–fungus relationship. In the early stages of colonization (5–7 d), proteins related to defense responses to the fungus were down-regulated and proteins related to calcium transport were up-regulated. At later time points (12–19 d after colonization), up-regulation of molecular pathways linked to protein/amino acid turnover and to biosynthesis of energy compounds suggests beneficial interaction enhancing plant growth and development. At the later stage, the profile of leaf hormones and related compounds was also investigated, highlighting up-regulation of those related to plant growth and defense. Finally, B. bassiana colonization was found to improve plant resistance to Botrytis cinerea, impacting plant oxidative damage. Overall, our findings further expand current knowledge on the possible mechanisms underlying the beneficial role of B. bassiana in tomato plants

Effects of pentachlorophenol on Drosophila melanogaster transcriptome

ABSTRACT Among largely used synthetic molecules, the chlorinated ones have been extensively applied as pesticides. Pentachlorophenol (PCP) has been one of the most extensively used biocides in the United States and in Europe. In the last decades its use has been restricted in several countries worldwide, due to its toxicity to wildlife, the long persistence in the environment and bioaccumulation in fat tissues. Public increasing interest in an ecologically safe environment remediation, together with the awareness that biotechnology has high potential to satisfy this need, is leading towards different approaches for bioremediation. PCP-degrading bacteria and fungi catabolizing PCP are known, but looking for diverse and not yet characterized efficient degradation pathways can result in improving the available tools. Insects represent a potential and relatively unexplored source of metabolic mechanisms for detoxification of a wide variety of both natural and synthetic compounds due to their genetic plasticity. Drosophila melanogaster was used as a model insect, and a set of differentially expressed genes associated with PCP response in the fruitfly was identified on the basis of genome-wide microarray analysis. Two PCP doses were tested, 20 and 2000 ppm, the latter causing a higher level of response, in terms of significantly differentially expressed genes. The enrichment analysis of the overexpressed 2000 ppm PCP treatment highlighted a strong response in the biogenic amine metabolic pathways, with the induction of genes potentially involved in degradation pathways, such as the tyrosine monooxygenase. The main detoxifying gene families were involved in the PCP response, with overexpression of five CYPs and one GST. Moreover two ABC transporter genes were found to be induced. Two 2000 ppm PCP resistant Drosophila strains were selected, in order to obtain a deeper knowledge of the effects of PCP on Drosophila, and to elucidate PCP metabolic pathaways. For this purpose HPLC analyses were carried out on PCP supplied diet on which resistant larvae fed on, evidencing a reduction of PCP as a consequence of feeding activity

Volatile Organic Compound (VOC) Profiles of Different Trichoderma Species and Their Potential Application

Fungi emit a broad spectrum of volatile organic compounds (VOCs), sometimes producing species-specific volatile profiles. Volatilomes have received over the last decade increasing attention in ecological, environmental and agricultural studies due to their potential to be used in the biocontrol of plant pathogens and pests and as plant growth-promoting factors. In the present study, we characterised and compared the volatilomes from four different Trichoderma species: T. asperellum B6; T. atroviride P1; T. afroharzianum T22; and T. longibrachiatum MK1. VOCs were collected from each strain grown both on PDA and in soil and analysed using proton transfer reaction quadrupole interface time-of-flight mass spectrometry (PTR-Qi-TOF-MS). Analysis of the detected volatiles highlighted a clear separation of the volatilomes of all the four species grown on PDA whereas the volatilomes of the soil-grown fungi could be only partially separated. Moreover, a limited number of species-specific peaks were found and putatively identified. In particular, each of the four Trichoderma species over-emitted somevolatiles involved in resistance induction, promotion of plant seed germination and seedling development and antimicrobial activity, as 2-pentyl-furan, 6PP, acetophenone and p-cymene by T. asperellum B6, T. atroviride P1, T. afroharzianum T22 and T. longibrachiatum MK1, respectively. Their potential role in interspecific interactions from the perspective of biological control is briefly discussed

<i>Trichoderma harzianum</i> Volatile Organic Compounds Regulated by the THCTF1 Transcription Factor Are Involved in Antifungal Activity and Beneficial Plant Responses

The transcription factor THCTF1 from Trichoderma harzianum, previously linked to the production of 6-pentyl-2H-pyran-2-one (6-PP) derivatives and antifungal activity against Fusarium oxysporum, has been related in this study to conidiation, production of an array of volatile organic compounds (VOCs) and expression of methyltransferase genes. VOCs emitted by three T. harzianum strains (wild type T34, transformant ΔD1-38 that is disrupted in the Thctf1 gene encoding the transcription factor THCTF1, and ectopic integration transformant ΔJ3-16) were characterized by Proton Transfer Reaction-Quadrupole interface-Time-Of-Flight-Mass Spectrometry (PTR-Qi-TOF-MS). Thctf1 disruption affected the production of numerous VOCs such as the antifungal volatiles 2-pentyl furan and benzaldehyde which were under-emitted, and acetoine, a plant systemic defense inductor, which was over-emitted. Biological assays show that VOCs regulated by THCTF1 are involved in the T. harzianum antifungal activity against Botrytis cinerea and in the beneficial effects leading to Arabidopsis plant development. The VOC blend from the disruptant ΔD1-38: (i) inhibited Arabidopsis seed germination for at least 26 days and (ii) when applied to Arabidopsis seedlings resulted in increased jasmonic acid- and salicylic acid-dependent defenses

Context-Dependent Effects of Trichoderma Seed Inoculation on Anthracnose Disease and Seed Yield of Bean (Phaseolus vulgaris): Ambient Conditions Override Cultivar-Specific Differences

Root colonizing Trichoderma fungi can stimulate plant immunity, but net effects are strain × cultivar-specific and changing ambient conditions further contribute to variable outcomes. Here, we used four Trichoderma spp. to inoculate seeds of four common bean (Phaseolus vulgaris) cultivars and explored in three different experimental setups the effects on fungal anthracnose after leaf inoculation with Colletotrichum lindemuthianum. Plants growing in pots with field soil under greenhouse conditions exhibited the highest and those in the open field the lowest overall levels of disease. Among 48 Trichoderma strain × bean cultivar × setup combinations, Trichoderma-inoculation enhanced disease in six and decreased disease in ten cases, but with the exception of T. asperellum B6-inoculated Negro San Luis beans, the strain × cultivar-specific effects on anthracnose severity differed among the setups, and anthracnose severity did not predict seed yield in the open field. In the case of Flor de Mayo beans, Trichoderma even reduced yield in anthracnose-free field plots, although this effect was counterbalanced in anthracnose-infected plots. We consider our work as a case study that calls for stronger emphasis on field experiments in the early phases of screenings of Trichoderma inoculants as plant biostimulants

Species delimitation of <i>Leptocybe invasa</i> specimens based on bPTP analysis on the COI dataset.

<p>Clades highlighted with coloured boxes and names in bold after a | symbol correspond to recognised putative species of <i>L</i>. <i>invasa</i>. Posterior delimitation probabilities values are reported above branches.</p

FISH on <i>L</i>. <i>invasa</i> ovarioles and eggs.

<p>Distribution of <i>Rickettsia</i> in the ovary (A and B), germaria (C) and the eggs (D, E, and F) of <i>Leptocybe invasa</i>. In the ovary <i>Rickettsia</i> bacteria (bright red spots) are inside the developing oocytes (A) and are densely clustered in the germaria (C). Merged image of ovarioles simultaneously stained with the <i>Rickettsia</i> specific probe RickPn-Cy3 and the universal bacterial probe EUB338-Cy5, showing <i>Rickettsia</i> cells in orange (B). Within the egg, bacteria are distributed in the head (E and F) and in the peduncle (D). DAPI-stained nuclei are blue. Bars, 20 μm.</p