Publications

4482

AbstractPlacozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3–6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host’s internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11–13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan’s nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

AbstractHuanglongbing (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogenCandidatus Liberibacter asiaticus(CLas) is an alphapro-teobacterium of theRhizobiaceaefamily that has been identified as the cause. The virulence of CLas has been attributed, in part, to prophage encoded genes. Prophage and prophage like elements have been identified in 12 of the 15 CLas available genomes, and are classified into three prophage types. Here, we re-examined all 15 CLas genomes using ade novoprediction approach and expanded the number of prophage like elements from 16 to 33. Further, we find that all CLas contain at least one prophage-like sequence. Comparative analysis reveals a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 CLas strain that had previously been reported as lacking prophages. Our findings provide both a resource and new insights into the evolutionary relationship between phage and CLas pathogenicity.

Phytoplasmas are cell-wall-less bacteria that cause diseases in approximately 1,000 plant species. ‘Candidatus Phytoplasma pyri’, the causal agent of pear decline, induces various symptoms on its hosts, leading to weakening and dieback of the plants, reduced fruit size and yield, and, consequently, considerable financial losses in all pear-growing areas. Fighting this disease requires a reliable and inexpensive method for pathogen detection in propagation material as well as plant stocks in orchards and breeding facilities. Here, we present a field-suitable detection protocol for ‘Ca. P. pyri’ based on loop-mediated isothermal amplification (LAMP) targeting the phytoplasmal 16S ribosomal DNA sequence. The combination of a simplified sample preparation method based on sodium hydroxide and colorimetric visualization of LAMP results enables a laboratory-independent pathogen detection. The detection limit is comparable with analysis by polymerase chain reaction; however, the pear decline LAMP detection method is superior in terms of ease of use, cost, and time effectiveness for obtaining results.

There is accumulating evidence that root system collapse is a primary symptom associated with Huanglongbing (HLB)-induced tree decline, especially for commercial sweet orange and grapefruit trees on Swingle and Carrizo rootstocks. Maintaining root health is imperative to keep trees productive in an HLB-endemic environment. Preliminary greenhouse and field studies have shown that HLB-impacted trees had secondary and micronutrient deficiencies that were much greater in the roots than in the leaves, and that treatments containing three-times the recommended dose of manganese (Mn) improved tree health and growth and increased feeder root density in greenhouse trees. These results suggested that trees in an HLB-endemic environment have higher specific micronutrient requirements than those currently recommended. To test this hypothesis, established Vernia sweet orange grafted onto rough lemon rootstock trees were divided into eight supplemental CRF nutrition treatments (including two-times and four-times the recommended doses of Mn and boron) using a randomized complete block design in a commercial grove in St. Cloud, FL. The following supplemental nutrition treatments were used: no extra nutrition (control); Harrell’s–St. Helena mix 0.9 kg per tree; Harrell’s with 32 g of Florikan polycoated sodium borate (PSB) per tree; Harrell’s with 90 g of TigerSul® Mn sulfate (MS) per tree; Harrell’s with 32 g of PSB and 90 g of MS per tree; 180 g of MS per tree; 64 g of PSB per tree; and 180 g of MS plus 64 g of PSB per tree applied every 6 months since Fall 2015. Leaf and soil nutritional analyses were performed in Mar. 2017, Sept. 2017, and May 2018; a quantitative polymerase chain reaction was performed for Candidatus Liberibacter asiaticus (CLas) titer estimation in Nov. 2017. Significantly higher cycle threshold (Ct) values indicating reduced CLas bacterial populations were observed in trees that received the higher doses of Mn, especially those receiving four-times the recommended dosage of Mn (180 g Mn). Many trees exhibited Ct values of 32 or more, indicating a nonactive infection. Fruit yields of these trees were also increased. No significant differences in juice characteristics, canopy volume, and trunk section area were found between control plants and plants treated with 180 g Mn. Soil and leaf nutrients B, K, Mn, and Zn were significantly different among treatments at various times during the study. Our results strongly suggest that overdoses of Mn can suppress CLas bacterial titers in sweet orange trees on rough lemon rootstock, thus providing a therapeutic effect that can help restore tree health and fruit yields. This response was not observed when Mn and B were combined in the overdose, suggesting an antagonistic effect from B on Mn metabolism. When an overdose of Mn is used, biological functions and tree tolerance lost due to nutritional imbalances caused by HLB might be restored. Further studies are needed to elucidate which metabolic pathways are altered by comparing overdosed and conventionally fertilized HLB-impacted trees and to determine if the observed therapeutic effects can be achieved in trees grafted to other important commercial rootstocks.

‘Candidatus Liberibacter asiaticus’ is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of ‘Ca. L. asiaticus’ genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 ‘Ca. L. asiaticus’ strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that ‘Ca. L. asiaticus’ populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ‘Candidatus Liberibacter asiaticus’ and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ‘Ca. L. asiaticus’ inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ‘Ca. L. asiaticus’. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ‘Ca. L. asiaticus’ transmission by ACP. In this study, we report a new method for targeted early detection of ‘Ca. L. asiaticus’ in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that ‘Ca. L. asiaticus’ remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ‘Ca. L. asiaticus’ in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ‘Ca. L. asiaticus’ inoculum removal and preventing HLB from spreading.