Molecular Biology

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas) brings a great concern about the phloem nutrient transport in diseased plants. There is an urgent need to find the best management strategies to reduce the losses in the citrus industry worldwide. Endophytic bacteria are negatively affected by CLas pathogen, and these endophytes are associated with improved availability of nutrients and pathogen resistance. This study underpins the relationship between CLas pathogen, endophyte population and nutrients availability in citrus plants. The citrus plants were treated with Bacillus subtilis L1-21 and Hoagland solution to find out synergism efficacy to mitigate citrus HLB. We showed that citrus shoots in the presence of 50% Hoagland solution displayed maximum number of endophytes with 6.28 × 103 to 3.04 × 105 CFU/g. Among 50 candidate strains, B. subtilis L1-21 emerged as potential antagonist against surrogate strain Xanthomonas citri subsp. citri. The citrus half-leaf method identified that application of endophyte L1-21 with 50% Hoagland solution successfully reduces the CLas abundance. We point out that this combination results in a higher number of endophytes population with 2.52 × 104 to 9.11 × 106 CFU/g after 60 days, and reduces CLas pathogen abundance in asymptomatic HLB plants. In HLB symptomatic citrus plants, B. subtilis L1-21 potentially increases the endophyte population from 1.11 × 104 to 5.26 × 107 CFU/g in the presence of Hoagland solution, and pathogen abundance was reduced from 9.51 × 105 to 1.06 × 104 copies/g. Altogether, we suggested that the presence of endophyte L1-21 with Hoagland solution is more effective in HLB asymptomatic citrus plants, but a slight reduction of pathogen was observed in symptomatic plants. The findings revealed the role of indigenous citrus endophyte B. subtilis L1-21 along with other nutrients in the reduction of CLas pathogen abundance inside symptomatic and asymptomatic plants in citrus endophyte–nutrient–pathogen interplay.

Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites , Parvarchaeota , Aenigmarchaeota , Nanoarchaeota , and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community.

ABSTRACT A mesophilic filamentous anoxygenic phototrophic bacterium, designated M50-1, was isolated from a microbial mat of the Chukhyn Nur soda lake (northeastern Mongolia) with salinity of 5−14 g/L and pH 8.0−9.3. The organism is a strictly anaerobic phototrophic bacterium, which required sulfide for phototrophic growth. The cells formed short undulate trichomes surrounded by a thin sheath and containing gas vesicles. Motility of the trichomes was not observed. The cells contained chlorosomes. The antenna pigments were bacteriochlorophyll d and β- and γ-carotenes. Analysis of the genome assembled from the metagenome of the enrichment culture revealed all the enzymes of the 3-hydroxypropionate bi-cycle for autotrophic CO2 assimilation. The genome also contained the genes encoding a type IV sulfide:quinone oxidoreductase (sqrX). The organism had no nifHDBK genes, encoding the proteins of the nitrogenase complex responsible for dinitrogen fixation. The DNA G + C content was 58.6%. The values for in silico DNA‒DNA hybridization and average nucleotide identity between M50-1 and a closely related bacterium ‘Ca. Chloroploca asiatica’ B7-9 containing bacteriochlorophyll c were 53.4% and 94.0%, respectively, which corresponds to interspecies differences. Classification of the filamentous anoxygenic phototrophic bacterium M50-1 as a new ‘Ca. Chloroploca’ species was proposed, with the species name ‘Candidatus Chloroploca mongolica’ sp. nov.

Understanding how phytoplasmas move and multiply within the host plant is fundamental for plant–pathogen interaction studies. In recent years, the tomato has been used as a model plant to study this type of interaction. In the present work, we investigated the distribution and multiplication dynamics of one strain of ‘Candidatus Phytoplasma (Ca. P.) solani’ (16SrXII-A) in tomato (Solanum lycopersicum L., cv. Micro-Tom) plants. We obtained infected plants by grafting, a fast and effective method to maintain phytoplasma infection. In planta spread and multiplication of ‘Ca. P. solani’ was monitored over time using qualitative and quantitative qPCR. Root, apical shoot, lower leaves, and upper leaves were sampled at each sampling time. We hypothesized that ‘Ca. P. solani’ from the grafting site reached firstly the highest leaf, the apex and the roots; subsequently, the phytoplasmas spread to the rest of the upper leaves and then progressively to the lower leaves. Significant differences were found in ‘Ca. P. solani’ titer among different plant tissues. In particular, the concentration of phytoplasma in the roots was significantly higher than that in the other plant compartments in almost all the sampling dates. Since the roots show rapid colonization and the highest concentration of phytoplasmas, they represent the ideal tissue to sample for an early, sensitive and robust diagnosis.