Publications

4364

Abstract In 2022, EFSA was mandated by the European Commission's Directorate‐General for Health and Food Safety (M‐2022‐00070) to provide technical assistance on the list of Union quarantine pests qualifying as priority pests, as specified in Article 6(2) of Regulation (EU) 2016/2031 on protective measures against plant pests. As part of Task C, EFSA conducted comprehensive expert knowledge elicitations for candidate priority pests on the lag period, rate of expansion and impact on production (yield and quality losses) and the environment. This report provides the rationale for the dataset on the three Candidatus Liberibacter species associated with citrus greening disease, delivered to the European Commission's Joint Research Centre, to feed into the Impact Indicator for Priority Pest (I2P2) model and complete the pest prioritisation ranking exercise.

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

Opinion 131 addresses a Request for an Opinion asking the Judicial Commission to conserve the genus name Proteus Hauser 1885 (Approved Lists 1980) over its earlier homonym, the protozoan genus name Proteus Müller 1786. The Judicial Commission agrees that the later homonym is illegitimate and that the replacement of the prokaryotic name Proteus would be undesirable. It is also concluded that Proteus Müller 1786 is an objectively invalid name under the International Code of Zoological Nomenclature. Judicial Opinions 9, 12 and 130 serve as precedents for the conservation of Proteus Hauser 1885 (Approved Lists 1980) over Proteus Müller 1786. This action is taken here and makes the prokaryotic name Proteus legitimate.

The anammox bacteria “Candidatus Kuenenia stuttgartiensis” (Ca. Kuenenia) are able to gain energy by combining ammonium and nitrite to produce nitrogen gas, which is an ecologically and technically significant activity process. In this reaction, nitric oxide serves as a recognized intermediate in the reduction of nitrite, which is subsequently combined with ammonium to produce hydrazine. However, the enzyme that converts nitrite to nitric oxide remains elusive. In this study, we investigated the nitrite-reducing activity in “Ca. Kuenenia stuttgartiensis” strain CSTR1 to identify candidates for such an enzyme. An optimized in vitro assay was established to measure nitrite-reducing activities, with which we followed the activity in protein fractions obtained from various fractionation methods. Separation of the cell extract of strain CSTR1 with size exclusion chromatography yielded active fractions corresponding to a molecular size range of 150–200 kDa. Several proteins coeluted with the nitrite-reducing activity, including the hydroxylamine dehydrogenase HOX, an NADP-dependent isopropanol dehydrogenase (Adh), an electron-transfer 4Fe-4S subunit protein (Fcp), and a nitric oxide detoxifying flavorubredoxin (NorVW). However, further separation of the cell extract with anion exchange chromatography, resulted in much lower activity yields, and activities were distributed among several fractions. In addition, fractionation of cell extracts using ultracentrifugation and ultrafiltration linked the activity to HOX, but could not exclude the involvement of other proteins in the activity. Overall, our results suggest that the molecular mechanism for nitrite reduction in “Ca. Kuenenia” strains is more complex than that currently described in the literature. Nitrite reduction appears to be strongly associated with HOX but may additionally require the participation of other proteins.