Publications

4618

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/ cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth. IMPORTANCE Citrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, “ Candidatus Liberibacter asiaticus” (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Huanglongbing (HLB) is a disease caused by the phloem- limited Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry worldwide. To date, only indirect strategies have been implemented to eradicate HLB. Included among these is the population control of the psyllid vector (Diaphorina citri), which usually provides inconsistent results. Even though strategies for direct CLas suppression seem a priori more promising, only a handful of reports have been focused on a confrontation of the pathogen. Recent developments in polymer chemistry have allowed the design of polycationic self-assembled block copolymers with outstanding antibacterial capabilities. Here, we report the use of polymeric nano-sized bactericide particles (PNB) to control CLas directly in the phloem vasculature. The field experiments were performed in Rioverde, San Luis Potosí, and is one of the most important citrusproducing regions in Mexico. An average 52% reduction in the bacterial population was produced when PNB was injected directly into the trunk of 20 infected trees, although, in some cases, reduction levels reached 97%. These results position PNB as a novel and promising nanotechnological tool for citrus crop protection against CLas and other related pathogens.

AbstractFive cyanobacterial strains exhibiting Nostoc‐like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8‐PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11‐PS, RAN‐4C‐PS, and NP‐KLS‐5A‐PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.

Abstract The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d ‐amino acid racemases that produce the universal PG components d ‐glutamate ( d ‐Glu) or d ‐alanine ( d ‐Ala). We also examined moonlighting enzymes that synthesize d ‐Glu or d ‐Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these ‘orphan’ racemases is a predicted Glu racemase (MurI CPR ) from the CPR bacterium Candidatus Saccharimonas aalborgenesis . The expression of MurI CPR restores the growth of a Salmonella d ‐Glu auxotroph lacking its endogenous racemase and results in the substitution of l ‐Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurI CPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d ‐Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, ‘Candidatus Liberibacter asiaticus’ (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.