Publications

4591

Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.

AbstractXylitol was first applied to enhance nitrogen removal from saline wastewater through “Candidatus Brocadia sinica”‐dominated anammox process under low temperature. The reactor was maintained at 15°C, and the salinity of wastewater was 35 g/L. Ammonium removal rate (ARR) and nitrite removal rate (NRR) were stable at around 0.27 kg/(m3 d) without xylitol addition. As an osmotic pressure regulator and cryoprotective agent, optimal ARR and NRR were 0.51 kg/(m3 d) and 0.63 kg/(m3 d) at 0.3 mM xylitol. At the addition of 1 mM high‐dosage xylitol, there existed dissimilatory reduction in nitrate to ammonium nitrogen and heterotrophic denitrification in the reactor. Remodified logistic model was suitable to simulate removal process with xylitol addition. As a result, xylitol dose should be controlled within 0.3 mM, which greatly promoted the nitrogen removal from saline wastewater under low temperature.Practitioner points Xylitol could be used as osmotic pressure regulator and cryoprotective agent to enhance nitrogen removal. The optimal dose was achieved at 0.3 mM xylitol for “Candidatus Brocadia sinica” in low‐temperature saline wastewater. High‐dosage xylitol could interfere with nitrogen removal efficiency due to the presence of DNAR and HB. Remodified logistic model was suitable for the analysis and prediction of nitrogen removal process with xylitol addition.

‘Candidatus Liberibacter asiaticus’ (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

Abstract Presence of glycogen granules in anaerobic ammonium-oxidizing (anammox) bacteria has been reported so far. However, very little is known about their glycogen metabolism and the exact roles. Here, we studied the glycogen metabolism in “Ca. Brocadia sinica” growing in continuous retentostat cultures with bicarbonate as a carbon source. The effect of the culture growth phase was investigated. During the growing phase, intracellular glycogen content increased up to 32.6 mg-glucose (g-biomass dry wt)−1 while the specific growth rate and ATP/ADP ratio decreased. The accumulated glycogen begun to decrease at the onset of entering the near-zero growth phase and was consumed rapidly when substrates were depleted. This clearly indicates that glycogen was synthesized and utilized as an energy storage. The proteomic analysis revealed that “Ca. B. sinica” synthesized glycogen via three known glycogen biosynthesis pathways and simultaneously degraded during the progress of active anammox, implying that glycogen is being continuously recycled. When cells were starved, a part of stored glycogen was converted to trehalose, a potential stress protectant. This suggests that glycogen serves at least as a primary carbon source of trehalose synthesis for survival. This study provides the first physiological evidence of glycogen metabolism in anammox bacteria and its significance in survival under natural substrate-limited habitat.

Abstract Candidatus Liberibacter solanacearum (Lso) is a phloem-limited, Gram-negative, unculturable bacterium that is primarily spread by psyllid insect vectors. It is considered very invasive due to its ability to be transported primarily in infective psyllids (Munyaneza et al., 2007a; 2010a,b; 2012a,b; Munyaneza, 2012; Alfaro-Fernandez et al., 2012a,b). It has been shown that Lso distribution in the Americas, New Zealand and Europe follows the distribution of its known psyllid vectors (Munyaneza, 2010; 2012).In New Zealand, where Lso was introduced along with Bactericera cockerelli, supposedly from Western USA in early 2000s, the bacterium had already spread to both North and South Island by the time it was first documented in 2006 (Gill, 2006). It is clear that introduction of the psyllid vectors of Lso into new regions is likely to result in the rapid spread of this bacterium. Lso and several of its vectors are already on several alert lists, including the EPPO A1 Regulated Quarantine Plant Pests.

AbstractThe Candidate Phyla Radiation (CPR) constitutes a large group of mostly uncultured bacterial lineages with small cell sizes and limited biosynthetic capabilities. They are thought to be symbionts of other organisms, but the nature of this symbiosis has been ascertained only for cultured Saccharibacteria, which are epibiotic parasites of other bacteria. Here, we study the biology and the genome of Vampirococcus lugosii, which becomes the first described species of Vampirococcus, a genus of epibiotic bacteria morphologically identified decades ago. Vampirococcus belongs to the CPR phylum Absconditabacteria. It feeds on anoxygenic photosynthetic gammaproteobacteria, fully absorbing their cytoplasmic content. The cells divide epibiotically, forming multicellular stalks whose apical cells can reach new hosts. The genome is small (1.3 Mbp) and highly reduced in biosynthetic metabolism genes, but is enriched in genes possibly related to a fibrous cell surface likely involved in interactions with the host. Gene loss has been continuous during the evolution of Absconditabacteria, and generally most CPR bacteria, but this has been compensated by gene acquisition by horizontal gene transfer and de novo evolution. Our findings support parasitism as a widespread lifestyle of CPR bacteria, which probably contribute to the control of bacterial populations in diverse ecosystems.

Abstract Zebra chip, is a potato disease associated with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) and vectored by the potato psyllid, Bactericera cockerelli Šulc. Potato psyllids are native to North America, where four haplotypes have been described. They are able to colonize a wide range of solanaceous species, crops, and weeds. The epidemiology of zebra chip disease is still poorly understood and might involve the different haplotypes of psyllids as well as two haplotypes of Lso. As several perennial weeds have been recognized as potential host for potato psyllids and Lso, a yearly monitoring of several patches of bittersweet nightshade (Solanum dulcamara) and field bindweed (Convolvulus arvensis) located in the potato-growing region of southern Idaho was conducted from 2013 to 2017, to gain insight into psyllid dynamics in non-potato hosts and Lso presence in the fields. Potato psyllids caught on each host were individually tested for Lso, and a subset were haplotyped based on the CO1 gene, along with the haplotyping of Lso in positive samples. On bittersweet nightshade, the Northwestern haplotype was numerically dominant, with around 2.7% of psyllids found to be carrying either Lso haplotype A or B, suggesting a limited role in zebra chip persistence, which has infected Idaho fields at a low occurrence since the 2012 outbreak. Field bindweed was found to be a transient, non-overwintering host for potato psyllid of Northwestern, Western and Central haplotypes late in the season, suggesting minor, if any, role in persistence of Lso and field infestation by potato psyllids.