Publications

3807

AbstractA previously unknown haplotype of the plant pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) was found in cultivated carrots and parsnips in eastern Finland. That same haplotype was found in western Finland, over 300 km away, in the family Polygonaceae, the species Fallopia convolvulus (wild buckwheat) and Persicaria lapathifolia (pale persicaria) growing as weeds within carrot and parsnip fields. The infected plants, both apiaceous and polygonaceous, showed symptoms of foliar discolouration. This is the first report of Lso bacteria in plants of the family Polygonaceae. The finding that the polygonaceous plants infected with a previously unknown haplotype of Lso were growing among the apiaceous plants infected with Lso haplotype C suggests that these two haplotypes might be transmitted by different vectors. Phylogenetic analyses showed that the new haplotype, called haplotype H, is distinct from the previously characterized haplotypes and appears to have diverged early from their common ancestor. Multi-locus sequence analysis revealed four different sequence types (strains) within the haplotype H. These findings suggest that the haplotype H is likely to be endemic in northern Europe and that the genetic diversity within the Lso species is higher than previously assumed.

Abstract Background Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. Results Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. Conclusions Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, ‘Candidatus Leucobacter sulfamidivorax‘.

Abstract Huanglongbing, a highly destructive disease of citrus species, is associated with a fastidious, gram-negative, phloem-limited bacteria (Candidatus Liberibacter spp.). In Florida, the causative agent of Huanglongbing (HLB) is C. Liberibacter asiaticus (CLas) and it is transmitted by the insect vector, Asian citrus psyllid (Diaphorina citri Kuwayama). Previous investigations have revealed systemic infection of CLas with an erratic and uneven distribution of pathogen in tree phloem. However, previous investigations did not consider the potential impact of plant vegetative growth on presence/absence of CLas in planta. Our objectives were to determine: 1) the effect of vegetative growth of Citrus sinensis (L.) Osbeck cv Valencia on detection of CLas in mature leaves, and 2) the impact of CLas inoculation frequency on progression of CLas titer in citrus leaves through the first year of infection. Temporal dynamics of CLas detection were associated with vegetative flush growth. Surprisingly, there was no difference in CLas titer detected between plants exposed to infected vectors for a one-time 7 d inoculation access period, as compared with plants exposed to continuously breeding CLas-infected insects over the course of an entire year of plant infection. Our results suggest that the CLas bacterium is transported through phloem during annual movement of carbon compounds needed for vegetative plant growth, including transportation from roots to mature leaves. These results highlight the importance of vegetative growth on temporal dynamics of CLas in citrus, and suggest a critical role of the sink-source interaction on presence/absence of CLas in leaves.

‘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.