Publications

4516

AbstractThe black curry leaf psyllid, Diaphorina communis, is a host of the citrus pathogen, ‘Candidatus Liberibacter asiaticus’ (‘CLas’). However, there is a paucity of information on its biology; hence, this study was conducted to evaluate survival and development on citrus, in this instance mandarin (Citrus reticulata) and curry leaf (Bergera koenigii), and transmission of ‘CLas’. Given its similarity with the Asian citrus psyllid (Diaphorina citri), sequences of the COI and 16S genes were examined to see if they could distinguish molecularly between nymphs of these two species. Field and laboratory experiments showed that D. communis nymphs transferred to mandarin branches failed to survive. Adults could survive on flush growth of ‘CLas’‐infected mandarin trees; however, little oviposition took place, and the single resulting nymph did not survive to the second instar. These observations suggest that D. communis does not develop, or rarely develops on mandarin and possibly other Citrus spp. Curry leaf appears to be its preferred host, and complete development can take place, possibly exclusively, on this species. Studies on acquisition of ‘CLas’ by D. communis and possible transmission assessed in 2014 and 2015 using greenhouse‐grown curry leaf and mandarin seedlings with naturally infected mandarin stumps as a source of ‘CLas’ indicated a low acquisition rate of ‘CLas’. In addition, although D. communis can acquire ‘CLas’, it either cannot transmit it or transmission is limited, and the pathogen may not multiply within the insect. Phylogenetic analyses indicate that differences in COI and 16S regions may prove useful for differentiating between early instar D. communis and D. citri nymphs when fifth instar nymphs and adults are not present on host plants.

Summary Anaerobic oxidation of methane (AOM) is a microbial process degrading ample methane in anoxic environments, and Ca. Methanoperedens mediated nitrate‐ or metal‐reduction linked AOM is believed important in freshwater systems. This work, via 16S rRNA gene diversity survey and 16S rRNA quantification, found abundant Ca. Methanoperedens along with iron in the cold Zoige wetland at Tibetan Plateau. The wetland soil microcosm performed Fe(III) reduction, rather than nitrate‐ nor sulphate‐reduction, coupled methane oxidation (3.87 μmol d −1 ) with 32.33 μmol Fe(II) accumulation per day at 18°C, but not at 30°C. A metagenome‐assembled genome (MAG) recovered from the microcosm exhibits ~74% average nucleotide identity with the reported Ca. Methanoperedens spp. that perform Fe(III) reduction linked AOM, thus a novel species Ca. Methanoperedens psychrophilus was proposed. Ca. M. psychrophilus contains the whole suite of CO 2 reductive methanogenic genes presumably involving in AOM via a reverse direction, and comparative genome analysis revealed its unique gene categories: the multi‐heme clusters (MHCs) cytochromes, the S‐layer proteins highly homologous to those recovered from lower temperature environments and type IV pili, those could confer Ca. M. psychrophilus of cold adaptability. Therefore, this work reports the first methanotroph implementing AOM in an alpine wetland.

AbstractThe Asian citrus psyllid, Diaphorina citri is an invasive insect 1 and a vector of ’Candidatus Liberibacter asiaticus’ (CLas), a bacterium whose growth in Citrus species results in citrus greening disease 2,3. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification 4 and nevertheless contain significant quantities of host DNA 5,6. To overcome these hurdles, we developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to directly sequence CLas and the complete, primarily uncultivable, microbiome from D. citri adults. The PDF protocol yielded CLas abundances upwards of 60% and enabled detection of 156 genetic variants in these strains compared to progenitor strains in Florida, which included prophage encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can inform agricultural management strategies related to pathogen evolution 7, insect microbiome surveillance 8, antibiotic resistance screening 9, and gut content analysis 10.

A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds in boreal and subarctic areas. However, it has probably escaped detection in many previous studies that used 16S rRNA gene amplicon sequencing due to insufficient database coverage, as previously analysed metagenome-assembled genomes (MAGs) affiliated with Ca. Methylumidiphilus do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG data set. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we found 5 MAGs, each containing one 1532 bp sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in 2 unique 16S rRNA gene sequences. These sequences represented 2 different putative species: Ca. Methylumidiphilus alinenensis (GenBank accession OK236221) and another unnamed species of Ca. Methylumidiphilus (GenBank accession OK236220). We suggest that including these 2 sequences in reference databases will enhance 16S rRNA gene-based detection of members of this genus from environmental samples.

Amycolatopsis coloradensis ATCC 53629 is the producer of the glycopeptide antibiotic avoparcin. While setting up the production of the avoparcin complex, in view of its use as analytical standard, we uncovered the production of a to-date not described ristosamynil-avoparcin. Ristosamynil-avoparcin is produced together with α- and β-avoparcin (overall indicated as the avoparcin complex). Selection of one high producer morphological variant within the A. coloradensis population, together with the use of a new fermentation medium, allowed to increase productivity of the avoparcin complex up to 9 g/L in flask fermentations. The selected high producer displayed a non-spore forming phenotype. All the selected phenotypes, as well as the original unselected population, displayed invariably the ability to produce a complex rich in ristosamynil-avoparcin. This suggested that the original strain deposited was not conforming to the description or that long term storage of the lyovials has selected mutants from the original population.

Abstract An attempt was made to characterize the Phytoplasma in suspected cowpea (Vigna unguiculata L.) samples showing symptoms consisting of bud proliferation and stunting at Experimental Farm, ICAR-NBPGR, New Delhi during Kharif 2020. Associated phytoplasma was characterized based on sequence analysis of target 16S rDNA fragments of 1247 bp amplified from symptomatic samples of cowpea by nested-polymerase chain reaction assay using universal phytoplasma specific primers pairs (P1/P7 and R16F2n/ R16R2). The 16S rDNA sequences understudy shared 98.6% similarity with that of the Candidatus Phytoplasma aurantifolia (GenBank accession: U15442). In phylogenetic analysis, the target 16S rDNA fragment from two samples (Cow-1 and Cow-2) clustered with strains of the 16SrII group of Ca. Phytoplasma aurantifolia. The virtual RFLP pattern showed the highest similarity with the reference pattern of the 16SrII-D subgroup (GenBank accession: Y10097) with a coefficient of 0.97. Since obtained coefficient value is equal to the threshold (0.97) for designating a different strain. Therefore, this phytoplasma infecting cowpea is proposed to be a new strain of Ca. P. aurantifolia as 16SrII-Ysubgroup.

Huanglongbing (HLB) has turned into a devastating botanical pandemic of citrus crops, caused by Candidatus Liberibacter asiaticus (CLas). However, until now the disease has remained incurable with very limited control strategies available. Restoration of the affected microbiomes in the diseased host through the introduction of an indigenous endophyte Bacillus subtilis L1-21 isolated from healthy citrus may provide an innovative approach for disease management. A novel half-leaf method was developed in vitro to test the efficacy of the endophyte L1-21 against CLas. Application of B. subtilis L1-21 at 104 colony forming unit (cfu ml−1) resulted in a 1,000-fold reduction in the CLas copies per gram of leaf midrib (107 to 104) in 4 days. In HLB-affected citrus orchards over a period of 2 years, the CLas incidence was reduced to < 3%, and CLas copies declined from 109 to 104 g−1 of diseased leaf midribs in the endophyte L1-21 treated trees. Reduction in disease incidence may corroborate a direct or an indirect biocontrol effect of the endophytes as red fluorescent protein-labeled B. subtilis L1-21 colonized and shared niche (phloem) with CLas. This is the first large-scale study for establishing a sustainable HLB control strategy through citrus endophytic microbiome restructuring using an indigenous endophyte.