Publications

4851

AbstractIn order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

Abstract Background ‘Candidatus Phytoplasma solani’ is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of ‘Candidatus Phytoplasma solani’ infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes. Results Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots. Conclusions ‘Candidatus Phytoplasma solani’ infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H2O2 accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants.

Disease caused by the bacterial pathogen “Candidatus Liberibacter solanacearum” (Lso) represents a serious threat to solanaceous crop production. Insecticide applications to control the psyllid vector, Bactericera cockerelli Šulc (Hemiptera: Triozidae) has led to the emergence of resistance in psyllids populations. Efforts to select natural resistant cultivars have been marginally successful and have been complicated by the presence of distinct Lso haplotypes (LsoA, LsoB) differing in symptoms severity on potato and tomato. A potentially promising management tool is to boost host resistance to the pathogen and/or the insect vector by promoting mycorrhization. Here we tested the hypothesis that mycorrhizal fungi can mitigate the effect of Lso infection on tomato plants. The presence of mycorrhizal fungi substantially delayed and reduced the incidence of Lso-induced symptoms on tomato as compared to non-mycorrhized plants. However, PCR with specific Lso primers revealed that mycorrhization did not prevent Lso transmission or translocation to newly formed leaves. Mycorrhization significantly reduced oviposition by psyllids harboring LsoA and survival of nymphs from these eggs. However, mycorrhization had no effect on oviposition by psyllids harboring LsoB or the survival of nymphs from parents harboring LsoB. These findings indicate the use of mycorrhizal fungi is a promising strategy for the mitigation of disease caused by both LsoA and LsoB and warrants additional field testing.

Abstract Heritable symbionts are common in terrestrial arthropods and often provide beneficial services to hosts. Unlike obligate, nutritional symbionts that largely persist under strict host control within specialized host cells, heritable facultative symbionts exhibit large variation in within-host lifestyles and services rendered with many retaining the capacity to transition among roles. One enigmatic symbiont, Candidatus Fukatsuia symbiotica, frequently infects aphids with reported roles ranging from pathogen, defensive symbiont, mutualism exploiter and nutritional co-obligate symbiont. Here we used an in vitro culture-assisted protocol to sequence the genome of a facultative strain of Fukatsuia from pea aphids (Acyrthosiphon pisum). Phylogenetic and genomic comparisons indicate that Fukatsuia is an aerobic heterotroph, which together with Regiella insecticola and Hamiltonella defensa form a clade of heritable facultative symbionts within the Yersiniaceae (Enterobacteriales). These three heritable facultative symbionts largely share overlapping inventories of genes associated with housekeeping functions, metabolism, and nutrient acquisition, while varying in complements of mobile DNA. One unusual feature of Fukatsuia is its strong tendency to occur as a co-infection with H. defensa. However, the overall similarity of gene inventories among aphid heritable facultative symbionts suggest that metabolic complementarity is not the basis for co-infection, unless playing out on a H. defensa strain-specific basis. We also compared the pea aphid Fukatsuia with a strain from the aphid Cinara confinis (Lachninae) where it is reported to have transitioned to co-obligate status to support decaying Buchnera function. Overall the two genomes are very similar with no clear genomic signatures consistent with such a transition, which suggests co-obligate status in C. confinis was a recent event.

AbstractPlant pathogenic bacteria interact with their insect host(s)/vector(s) at the cellular and molecular levels. This interaction may alter the physiology of their insect vector, which may also promote the growth and transmission of the bacterium. Here we studied the effect of “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) on physiochemical conditions within its insect vector, the Asian citrus psyllid (ACP), and whether these changes were beneficial for the pathogen. The local microenvironments inside ACPs were quantified using microelectrodes. The average hemolymph pH was significantly higher in infected ACPs (8.13 ± 0.21) than in “Ca. L. asiaticus”-free ACPs (7.29 ± 0.15). The average hemolymph oxygen tension was higher in “Ca. L. asiaticus”-free ACPs than in infected ACPs (67.13% ± 2.11% vs. 35.61% ± 1.26%). Oxygen tension reduction and pH increase were accompanied by “Ca. L. asiaticus” infection. Thus, oxygen tension of the hemolymph is an indicator of infection status, with pH affected by the severity of the infection.