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Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties worldwide. In the United States, Asia, and South America, the causal agent is ‘ Candidatus Liberibacter asiaticus’ ( CLas), a phloem-limited, uncultured alphaproteobacterium. The hemipteran insect vector Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied, but specific roles in D. citri biology are unknown. Using well-established quantitative PCR methods, we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a subset of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, whereas a higher wDi titer was correlated with noninfected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi, and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

Abstract Insects engage in manifold interactions with bacteria that can shift along the parasitism–mutualism continuum. However, only a small number of bacterial taxa managed to successfully colonize a wide diversity of insects, by evolving mechanisms for host-cell entry, immune evasion, germline tropism, reproductive manipulation, and/or by providing benefits to the host that stabilize the symbiotic association. Here, we report on the discovery of an Enterobacterales endosymbiont (Symbiodolus, type species Symbiodolus clandestinus) that is widespread across at least six insect orders and occurs at high prevalence within host populations. Fluorescence in situ hybridization in several Coleopteran and one Dipteran species revealed Symbiodolus’ intracellular presence in all host life stages and across tissues, with a high abundance in female ovaries, indicating transovarial vertical transmission. Symbiont genome sequencing across 16 host taxa revealed a high degree of functional conservation in the eroding and transposon-rich genomes. All sequenced Symbiodolus genomes encode for multiple secretion systems, alongside effectors and toxin-antitoxin systems, which likely facilitate host-cell entry and interactions with the host. However, Symbiodolus-infected insects show no obvious signs of disease, and biosynthetic pathways for several amino acids and cofactors encoded by the bacterial genomes suggest that the symbionts may also be able to provide benefits to the hosts. A lack of host-symbiont cospeciation provides evidence for occasional horizontal transmission, so Symbiodolus’ success is likely based on a mixed transmission mode. Our findings uncover a hitherto undescribed and widespread insect endosymbiont that may present valuable opportunities to unravel the molecular underpinnings of symbiosis establishment and maintenance.

AbstractMany insects engage in stable nutritional symbioses with bacteria that supplement limiting essential nutrients to their host. While several plant sap-feeding Hemipteran lineages are known to be simultaneously associated with two or more endosymbionts with complementary biosynthetic pathways to synthesize amino acids or vitamins, such co-obligate symbioses have not been functionally characterized in other insect orders. Here, we report on the characterization of a dual co-obligate, bacteriome-localized symbiosis in a family of xylophagous beetles using comparative genomics, fluorescence microscopy, and phylogenetic analyses. Across the beetle family Bostrichidae, most investigated species harbored the Bacteroidota symbiont Shikimatogenerans bostrichidophilus that encodes the shikimate pathway to produce tyrosine precursors in its severely reduced genome, likely supplementing the beetles’ cuticle biosynthesis, sclerotisation, and melanisation. One clade of Bostrichid beetles additionally housed the co-obligate symbiont Bostrichicola ureolyticus that is inferred to complement the function of Shikimatogenerans by recycling urea and provisioning the essential amino acid lysine, thereby providing additional benefits on nitrogen-poor diets. Both symbionts represent ancient associations within the Bostrichidae that have subsequently experienced genome erosion and co-speciation with their hosts. While Bostrichicola was repeatedly lost, Shikimatogenerans has been retained throughout the family and exhibits a perfect pattern of co-speciation. Our results reveal that co-obligate symbioses with complementary metabolic capabilities occur beyond the well-known sap-feeding Hemiptera and highlight the importance of symbiont-mediated cuticle supplementation and nitrogen recycling for herbivorous beetles.

Abstract The Rio Grande Valley (RGV) in southern Texas is well-suited for vegetable production due to its relatively mild/warm weather conditions in the fall and winter. Consequently, insects inflict year-round, persistent damage to crops in the RGV and regions with similar climate. Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), commonly known as the potato psyllid, is a known vector of Candidatus Liberibacter solanacearum (CLso) (Hyphomicrobiales: Rhizobiaceae), a fastidious phloem-limited bacterium associated to vein-greening in tomatoes and Zebra Chip in potatoes. Vector control is the primary approach of integrated pest management (IPM) strategies that aim to prevent plant diseases in commercial agricultural systems. However, resistance-selective pressures that decrease the effectiveness of chemical control (insecticide) applications over time are of increasing concern. Therefore, we explore an ecological approach to devising alternative IPM methodologies to manage the psyllid-transmitted CLso pathogen to supplement existing chemical products and application schedules without increasing resistance. In this study, our objective was to examine the effects of plant-growth promoting rhizobacteria (PGPR) on host-vector-pathogen interactions. Soil-drench applications of PGPRs to Solanum lycopersicum (Solanales: Solanaceae) seedlings revealed structural and possible physiological changes to the plant host and indirect changes on psyllid behavior: host plants had increased length and biomass of roots and exhibited delayed colonization by CLso, while psyllids displayed changes in parental (F0) psyllid behavior (orientation and oviposition) in response to treated hosts and in the sex ratio of their progeny (F1). Based on our results, we suggest that PGPR may have practical use in commercial tomato production.

Abstract“Huanglongbing” (HLB) is one of the most devastating diseases of citrus orchards worldwide. Samples from 183 citrus plants of different cultivars and rootstock/cultivar combinations, showing HLB symptoms in three Caribbean countries (Cuba, Jamaica, and Guadeloupe-France), were collected to verify the possible co-infection of ‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’ species. The 64% of the samples resulted positive to the ‘Ca. L. asiaticus’ and the 27% to diverse ‘Ca. Phytoplasma’-related species, moreover about the 14% of the samples infected with ‘Ca. Liberibacter’ were also found positive to phytoplasmas, indicating the presence of mixed infection especially in the orchards located in Cuba. Moreover, in one of the samples from Jamaica mixed phytoplasma infection was detected. Moreover the detection of only phytoplasmas in 11 symptomatic citrus samples collected from Cuba and Guadeloupe without ‘Ca. Liberibacter’ detection, confirmed that the symptomatology cannot be the sole criterium to discriminate between the presence of the two pathogens, and molecular detection is necessary to identify single or mixed infections. Diaphorina citri insects collected from Cuba and Guadeloupe resulted infected with ‘Ca. L. asiaticus’ confirming its active role in the dissemination of the pathogen. Only one insect of the Cicadidae family, collected in Guadeloupe, was found positive for phytoplasma presence. Considering that the phytoplasmas belonging to some ‘Candidatus species’ were detected in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute to citrus orchards.

Aster Yellows phytoplasma (AYp; ‘Candidatus Phytoplasma asteris’) is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant’s phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

AbstractPhytoplasmas are specialized small bacteria restricted to the phloem tissue and spread by hemipterans feeding on plant sieve tube elements. As for many other plant pathogens, it is known that phytoplasmas alter the chemistry of their hosts. Most research on phytoplasma-plant interactions focused on the induction of plant volatiles and phytohormones. Little is known about the influence of phytoplasma infections on the nutritional composition of phloem and consequences on vector behavior and development. The plum psyllid Cacopsylla pruni transmits ‘Candidatus Phytoplasma prunorum’, the causing agent of European Stone Fruit Yellows (ESFY). While several Prunus species are susceptible for psyllid feeding, they show different responses to the pathogen. We studied the possible modulation of plant-insect interactions by bacteria-induced changes in phloem sap chemistry. Therefore, we sampled phloem sap from phytoplasma-infected and non-infected Prunus persica and Prunus insititia plants, which differ in their susceptibility to ESFY and psyllid feeding. Furthermore, the feeding behavior and development of C. pruni nymphs was compared on infected and non-infected P. persica and P. insititia plants. Phytoplasma infection did not affect phloem consumption by C. pruni nymphs nor their development time. In contrast, the study revealed significant differences between P. insititia and P. persica in terms of both phloem chemistry and feeding behavior of C. pruni nymphs. Phloem feeding phases were four times longer on P. insititia than on P. persica, resulting in a decreased development time and higher mortality of vector insects on P. persica plants. These findings explain the low infestation rates of peach cultivars with plum psyllids commonly found in field surveys.

Abstract Many insects depend on obligate mutualistic bacteria to provide essential nutrients lacking from their diet. Most aphids, whose diet consists of phloem, rely on the bacterial endosymbiont Buchnera aphidicola to supply essential amino acids and B vitamins. However, in some aphid species, provision of these nutrients is partitioned between Buchnera and a younger bacterial partner, whose identity varies across aphid lineages. Little is known about the origin and the evolutionary stability of these di-symbiotic systems. It is also unclear whether the novel symbionts merely compensate for losses in Buchnera or carry new nutritional functions. Using whole-genome endosymbiont sequences of nine Cinara aphids that harbour an Erwinia-related symbiont to complement Buchnera, we show that the Erwinia association arose from a single event of symbiont lifestyle shift, from a free-living to an obligate intracellular one. This event resulted in drastic genome reduction, long-term genome stasis, and co-divergence with aphids. Fluorescence in situ hybridisation reveals that Erwinia inhabits its own bacteriocytes near Buchnera’s. Altogether these results depict a scenario for the establishment of Erwinia as an obligate symbiont that mirrors Buchnera’s. Additionally, we found that the Erwinia vitamin-biosynthetic genes not only compensate for Buchnera’s deficiencies, but also provide a new nutritional function; whose genes have been horizontally acquired from a Sodalis-related bacterium. A subset of these genes have been subsequently transferred to a new Hamiltonella co-obligate symbiont in one specific Cinara lineage. These results show that the establishment and dynamics of multi-partner endosymbioses can be mediated by lateral gene transfers between co-ocurring symbionts.

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.