Publications

4535

Phytoplasma, a potentially hazardous pathogen associated with witches’ broom, is an economically harmful disease-producing bacteria that damages chilli cultivation. Phytoplasma-infected plants display various symptoms that indicate significant disruptions in normal plant physiology and behaviour. Diseases caused by phytoplasma are widespread and have a major economic impact on crop quality and yield. This work focuses on identifying and examining chilli microRNAs (miRNAs) as potential targets against the 16S rRNA and secA gene of “Candidatus Phytoplasma trifolii” (“Ca. P. trifolii”) through plant miRNA prediction algorithms. Mature chilli miRNAs (CA-miRNAs) were collected and used to hybridise the 16S rRNA and secA genes. A total of four common CA-miRNAs were picked according to genetic consensus. Three algorithms applied in the present study suggested that the physiologically relevant, top-ranked miR169b_2 has a possibly specific site at nucleotide position 1,006 for targeting the ‘Ca. P. trifolii’ 16S rRNA gene. The circos algorithm was then utilised to create the miRNA-mRNA regulatory network. The free energy between the miRNA:mRNA duplex was also computed, and the best value of −17.46 kcal/mol was obtained for CA-miR166c_2. Currently, there are no suitable commercial ‘Ca. P. trifolii’-resistant chilli crops. As a result, the expected biological data provide useful evidence for developing ‘Ca. P. trifolii’-resistant chilli plants.

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium ‘Candidatus Liberibacter spp.’, among which ‘Ca. L. africanus’ (CLaf) has posed a significant threat to citrus production in Africa for nearly a century. CLaf is closely related to the globally prevalent ‘Ca. L. asiaticus’ (CLas), whereas little is known about the virulence of CLaf, primarily because of limited genome resources. In this study, we completed the whole-genome assembly and annotation of the CLaf strain Zim (from Zimbabwe). Compared with CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effector (SDE) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that the CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of ‘Ca. L. solanacearum’, the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, V9J15_02010, V9J15_02015, V9J15_02025, and V9J15_02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

El maíz (Zea mays L.) se ha constituido como el cultivo de mayor importancia económica a nivel mundial; esté cereal perteneciente a la familia Poaceae, es originario de México y utilizado tanto para consumo humano como ganadero. En México, su importancia va mucho mas allá de lo económico, su influencia a nivel social y cultural se ha visto plasmada en los vestigios de civilizaciones antiguas como la establecida por los mayas, para quienes el maíz tenía un significado religioso; sin embargo, algunos factores bióticos como bacterias, virus, hongos y fitoplasmas pueden limitar la producción de las diversas variedades de maíz cultivadas en territorio mexicano. Durante el ciclo de cultivo 2021, se colectaron plantas de maíz presentes en parcelas comerciales en el municipio de Valparaiso, Zacatecas, México las cuales presentaban una sintomatología relacionada con la presencia de fitoplasmas (enanismo, cambios de color en hojas de verde a blanco, declive general y muerte); además, se muestrearon plantas de maiz asintomáticas como testigos e insectos de la especie Dalbulus maidis. Las muestras se analizaron mediante la reacción en cadena de la polimerasa (PCR) anidada utilizando la combinación de oligonucleótidos universales P1/Tint, seguida por el par de oligonucleótidos R16F2n/R16R2 con lo que se logró obtener secuencias del gen 16S rDNA de fitoplasmas. Los análisis filogenético y RFLP virtual de las secuencias de 16S rDNA obtenidas a partir de las muestras analizadas mostraron una identidad de secuencia del 100% entre sí y una identidad de secuencia del 99.7 % con el gen homólogo de Candidatus Phytoplasma asteris (accesión del GenBank M30790). La incidencia de esta nueva enfermedad fue del 15 % en los cultivos muestreados. Estos resultados relacionan a Dalbulus maidis y a Candidatus Phytoplasma asteris como pobible vector y agente causal de una nueva sintomatología en maiz.

Abstract Anoxic and hypoxic environments serve as habitats for diverse microorganisms, including unicellular eukaryotes (protists) and prokaryotes. To thrive in low-oxygen environments, protists and prokaryotes often establish specialized metabolic cross-feeding associations, such as syntrophy, with other microorganisms. Previous studies show that the breviate protist Lenisia limosa engages in a mutualistic association with a denitrifying Arcobacter bacterium based on hydrogen exchange. Here, we investigate if the ability to form metabolic interactions is conserved in other breviates by studying five diverse breviate microcosms and their associated bacteria. We show that five laboratory microcosms of marine breviates live with multiple hydrogen-consuming prokaryotes that are predicted to have different preferences for terminal electron acceptors using genome-resolved metagenomics. Protist growth rates vary in response to electron acceptors depending on the make-up of the prokaryotic community. We find that the metabolic capabilities of the bacteria and not their taxonomic affiliations determine protist growth and survival and present new potential protist-interacting bacteria from the Arcobacteraceae, Desulfovibrionaceae, and Terasakiella lineages. This investigation uncovers potential nitrogen and sulfur cycling pathways within these bacterial populations, hinting at their roles in syntrophic interactions with the protists via hydrogen exchange.

Abstract Phytoplankton are the base of marine food webs. They form intricate interactions with heterotrophic bacteria ranging from mutualistic to pathogenic that together impact oceanic carbon and nutrient cycling. Our understanding of these interactions in marine environments remains primarily limited to laboratory-based studies of model organisms. Here, we report the discovery and characterization of Ekhidna algicida sp. nov. strain To15, isolated from the oligotrophic Pacific Ocean (16°N, 140°W) based on its algicidal effect on the pelagic diatom Thalassiosira oceanica. Subsequent co-culture experiments demonstrate that E. algicida is lethal within days to a diverse array of diatoms, including diatoms isolated from similar locations, with the effect mediated by bacterial exudates produced during co-culture with susceptible diatoms. Exudates of E. algicida monoculture are not algicidal, suggesting a pathogenic shift upon interaction with susceptible diatoms. The genome of E. algicida To15 encodes for a type IX secretion system (T9SS), together with candidate secreted proteases, suggesting a potential protein-mediated pathogenicity. Twenty additional algicidal Ekhidna strains were subsequently isolated from the Pacific Ocean. All these algicidal bacteria pass through 0.2 μm pore-size filters, highlighting the importance of the often-overlooked group of “filterable” marine bacteria. Our findings reveal E. algicida as a Pacific Ocean diatom pathogen, with potential impacts on microbial community composition dynamics in pelagic ecosystems.

Abstract Analysing the nitrogen (15ε) and oxygen (18ε) isotope effects of anaerobic ammonium oxidation (anammox) is essential for accurately assessing its potential contribution to fixed-N losses in the ocean, yet the 18ε of anammox remains unexplored. Here, we determined the previously unexplored 18ε of anammox using a highly enriched culture of the marine anammox species “Ca. Scalindua sp”. Because Scalindua significantly accelerated oxygen isotope exchange between NO2− and H2O, we introduced a new rate constant for anammox-mediated oxygen isotope exchange (keq, AMX = 8.44 ~ 13.56 × 10−2 h−1), which is substantially faster than abiotic oxygen isotope exchange (keq, abio = 1.13 × 10−2 h−1), into a numerical model to estimate the 18ε during anammox. Based on our experimental results, we successfully determined the 18ε associated with: (1) conversion of NO2− to N2 (18εNO2- → N2 = 10.6 ~ 16.1‰), (2) NO2− oxidation to NO3− (18εNO2- → NO3- = −2.9 ~ −11.0‰, inverse fractionation), (3) incorporation of oxygen from water during NO2− oxidation to NO3− (18εH2O = 16.4 ~ 19.2‰). Our study underscores the possibility that unique anammox oxygen isotope signals may be masked due to substantial anammox-mediated oxygen isotope exchange between NO2− and H2O. Therefore, careful consideration is required when utilizing δ18ONO3- and δ18ONO2- as geochemical markers to assess the potential contribution of anammox to fixed-N losses in the ocean.

The ongoing spread of X-disease, which is associated with ‘ Candidatus Phytoplasma pruni’, has resulted in severe economic losses for cherry and stone fruit growers in the U.S. Pacific Northwest in the last decade. Given that this pathogen is transmitted by polyphagous leafhopper species, primarily Colladonus montanus ssp. reductus and C. geminatus in the Pacific Northwest, alternative plant hosts present a significant management concern. Here, we surveyed phytoplasma incidence in non- Prunus plants found in and around stone fruit orchard borders and compared these results to the leafhopper feeding patterns through gut content analysis. We confirmed ‘ Ca. P. pruni’ infection in 21 plant species from 15 families. Of these, 15 species were commonly found in the diets of phytoplasma-carrying leafhoppers. Due to the abundance of common plant species, including Taraxacum, Malva, and Trifolium spp., alternative hosts were found across all growing seasons, although ‘ Ca. P. pruni’ titer was low (<102 cells) in most of these hosts. No geographic patterns were identified; instead, phytoplasma incidence and spread at individual orchards was related to the management of annual and biennial host species, thus removing preferred leafhopper feeding hosts. These data suggest that alternative host removal, along with the removal of infected trees, is an effective means of slowing disease spread.