Publications

4039

Abstract Uncultured UBA5794 actinobacteria are frequently found in marine and inland water environments by using metagenomic approaches. However, knowledge about these actinobacteria is limited, hindering their isolation and cultivation, and they are always confused with “Candidatus Actinomarinales” based on 16S rRNA gene classification. Here, to conduct genomic characterization of them, we obtained three high-quality UBA5794 metagenome-assembled genomes (MAGs) from a hydrothermal sediment on the Carlsberg Ridge (CR) and retrieved 131 high-quality UBA5794 genomes from public datasets. Phylogenomic analysis confirms UBA5794 as an independent order within the class Acidimicrobiia. Genome-based metabolic predictions reveal that flexible metabolism and diversified energy acquisition, as well as heavy metal(loid) detoxification capacity, are crucial for the ability of UBA5794 to thrive in diverse environments. Moreover, there is separation between sponge-associated and free-living UBA5794 groups in phylogeny and functional potential, which can be attributed to the symbiotic nature of the sponge-associated group and the extensive horizontal gene transfer (HGT) events observed in these bacteria. Ancestral state reconstruction suggests that the UBA5794 clade may have originated from a free-living environment and then some members gradually migrated to the sponge host. Overall, our study sheds light on the ecological adaptation and evolutionary history of the ubiquitous but poorly understood UBA5794 actinobacteria.

‘Candidatus Phytoplasma prunorum’ has been associated with severe disease in Prunus spp., which are commodities of economic importance in the USA. The introduction and establishment of ‘Ca. P. prunorum’ in the USA could result in huge economic losses, thus creating a need for validated diagnostic tools, which are the cornerstone of successful surveillance, quarantine, and eradication measures. Whole-genome comparisons led to the identification of a diagnostic marker gene specific to ‘Ca. P. prunorum’ (PE639). The PE639 assay was duplexed with an 18S rDNA plant internal control and compared to modified 23S (phytoplasmas) and imp (‘Ca. P. mali’) assays. The PE639 assay produced congruent results to 23S and imp assays for all metrics, demonstrating high linearity, repeatability, intermediate precision, and reproducibility. The limit of detection was comparable for all assays tested, and all demonstrated 100% analytical specificity, selectivity, and diagnostic specificity for their respective target species. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™ OPUS. A synthetic gBlocks™ control was designed and validated to work with all assays, as well as conventional PCR assays targeting 16S rDNA and tuf genes. These validated assays and synthetic control represent beneficial tools that support efforts to protect USA agriculture and facilitate safe trade.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an invasive citrus pest that vectors the phloem-dwelling bacterium, Candidatus Liberibacter asiaticus (CLas), which is a putative causal agent of citrus greening disease known as Huanglongbing (HLB). In Florida, the D. citri–CLas pathosystem complex is at present considered the greatest threat the citrus industry must manage. Brassinosteroids (Brs), a relatively new class of plant hormones, are approved for commercial use in Florida citrus and are believed to delay HLB progression. Individual protective covers (IPCs), a type of psyllid exclusion mesh bags, are increasingly being adopted to efficiently protect newly planted citrus trees from HLB infection. However, IPCs typically must be removed after 2–3 years due to tree growth. With the aim of prolonging tree health after IPC removal, we are investigating the efficacy Brs in protecting and/or preventing trees from ACP and CLas infection once they are left exposed. Upon IPC removal from trees covered for 3 years, monthly Br treatments started in trees from three mandarin cultivars (Early Pride, SugarBelle and Tango) on two different rootstocks, sour orange and US-942. The IPCs effectively excluded psyllids, prevented CLas infection, and clearly improved most of the horticultural traits measured. Although this is an on going experiment, we have already found that treatment with Brs reduced CLas bacterial titer by 10-fold on average two months after IPC removal. The long-term efficacy of the treatments still needs to be assessed and will be discussed. 

ABSTRACT We report the draft genomes of five Clostridium isolates from soil and agricultural by-products, four of which are proposed as Candidatus species. Members of the genus Clostridium are of significant industrial interest, and the availability of their genome sequences facilitates the understanding and exploration of their functional potential.

Bacterial strain SL12-8T was characterized and isolated from the skin microbiota of Pelophylax perezi, the Perez’s frog. Strain SL12-8T stained Gram-negative and formed rod-shaped cells that grew optimally at 25 °C and pH 7.0–7.5. The G+C content of the DNA was 66.2 mol%. Ubiquinone 8 was the respiratory quinone identified in the studied strain and the most closely related taxon. The major fatty acids were summed in feature 3 and summed in feature 8 and C16:0, representing 84% of the total fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequence placed strain SL12-8T within the order Burkholderiales in a distinct lineage. The 16S rRNA gene sequence similarities of strain SL12-8T to that of Rubrivivax albus, Scleromatobacter humisilvae, Piscinibacter aquaticus, Azohydromonas caseinilytica and Aquincola agrisoli were 94.41, 94.08, 93.72, 93.72 and 93.64%, respectively. The draft genome sequence of strain SL12-8T comprises 3,115,197 bases with a 313-fold mapped genome coverage. The assembled genome consists of 53 large contigs with more than 500 bp, and the genome encodes 2,814 putative coding sequences. The analysis of the available genomes from the closest genera showed 124 core genes that reveal a novel genus-level clade including the strain SL12-8T. Analysis of the SL12-8T genome revealed the presence of the beta-lactone and terpene biosynthetic gene clusters. The phylogenomic, phylogenetic, phenotypic and chemotaxonomic data showed that strain SL12-8T (=UCCCB 131T=CECT 30762T) represents the type of a novel species and genus, for which we propose the name Amphibiibacter pelophylacis gen. nov., sp. nov.

The highly invasive field bindweed (Convolvulus arvensis), native to Europe and Asia, competes with crops, reduces yields, and acts as a reservoir for plant pathogens, increasing the potential for disease spread in agricultural systems (Sosnoskie et al. 2020; Wenninger et al. 2019). In July 2023, bindweed plants exhibiting little leaf, leaf discoloration, thickening, and overall stunted growth were observed in a 0.3 ha green cherry orchard in Kharja, Bani Kenanah, Irbid Governorate, Jordan (32°37'37.883"N, 35°52'49.185"E) (Fig. 1). Twenty out of 35 plants displayed symptoms, resulting in a 57% incidence rate. Since the symptoms resembled those associated with phytoplasma infections and bindweed's known role as a phytoplasma host (Jovic et al. 2021), DNA was extracted from six symptomatic plants and two healthy controls using the Qiagen DNeasy Plant Mini Kit, and PCR assay was conducted with the primer pair P1A/16S-SR to amplify near full-length phytoplasma 16S rDNA (about 1.5 kb) as described by Lee et al. (2004). All healthy controls tested negative, while all symptomatic plants tested positive. Amplicons were cloned into the pCRII-TOPO vector, sequenced, and a representative sequence was deposited in GenBank (Accession number PQ497569). Using iPhyClassifier (Zhao et al. 2009), the strain was identified as a 'Candidatus Phytoplasma omanense' (GenBank accession: EF666051, 16SrXXIX-A)-related strain, with 98.95% sequence identity, belonging to the 16SrXXIX group. The similarity coefficients were 0.93 and 0.97 compared to reference strains 16SrXXIX-A (EF666051) and 16SrXXIX-B (OL873126, Abu Alloush et al. 2023a), respectively. The phylogenetic analysis indicated that the newly detected field bindweed little leaf phytoplasma strain (designated FBLL1) is genetically distinct from these known subgroups and represents a novel subgroup, 16SrXXIX-C (Fig. 2). Further characterization of the FBLL1 strains was performed by PCR amplification of the rp (1.9 kb) and secY (1.3 kb) gene segments using specially designed primers: FBRP-F2/FBRP-R4 (5’-TTGGCCGCCTTCAAATCCTA-3’/5’-TCGGAGGAGAAGTTTTGGCT-3') for rp, and FBSecYF1/FBSecR1 (5’-CTTCTTTTGGTGATATCCCA-3’/5’-TGGCGGAAGTATTGAGATAAGAA-3') for secY. The resulting rp and secY gene sequences were deposited in GenBank (Accession numbers PQ505136 and PQ505137). BLASTN analysis revealed that the rp gene's top hit was 'Ca. P. pruni' (CP119306) with 75.60% identity, while the secY gene's top hit was 'Ca. P. phoenicium' (MN076652), with 73.47% identity. Compared to the 16S rRNA gene, the lower percent identity is due to the limited availability of 16SrXXIX rp and secY gene sequences for comparison. Field bindweed hosts several phytoplasmas, such as ‘Ca. P. solani’ (16SrXII-A), which is linked to Bois noir (BN) in grapevines, and ‘Ca. P. convolvuli’ (16SrXII-H), associated with bindweed yellows (Jovic et al. 2021; Abu Alloush et al. 2023b). ‘Ca. P. omanense’ has also been documented in bindweed in Lebanon (Fossaic et al. 2019) and Iran (Esmailzadeh Hosseini et al. 2016). As a confirmed reservoir of the BN phytoplasma, bindweed supports the pathogen’s persistence and vector-mediated transmission to grapevines (Jovic et al. 2021). This report marks the first identification of ‘Ca. P. omanense’-related strains (16SrXXIX-C, novel subgroup) in bindweed in Jordan. These findings suggest that bindweed may serve as a potential reservoir for a broader range of phytoplasmas than previously recognized, with a wider geographical distribution. Its invasive growth near crops, such as grapevines, raises concerns about the potential phytoplasma spillover. This highlights the need for improved monitoring and management, which includes targeted herbicide application, mechanical removal, and biological control to protect agricultural biosecurity.

AbstractCandidatusLiberibacter asiaticus (CLas) is a host-dependent obligate parasite that causes citrus greening disease incitrus spp. The effective measures to control the bacteria are hindered due to the lack of an established culturing method for the characterization of CLas.Liberibacter crescens(Lcr) is the only culturable model organism to study possible strategies to cultureCandidatusLiberibacter species. Thein-silicocharacterization of CLas was conducted through functional and comparative annotation of sequence data using sequence similarity search, metabolic pathway analysis, and prophage detection tools. The comparative genome analysis of CLas strain gxpsy and Lcr strain BT-1 showed a significant genome size reduction in the CLas genome and compensating for the lost essential genes with the help of transport proteins. The study focuses on the missing genes and the unique genes in CLas, decoding the genomic and metabolic complexities. The study revealed the alternative pentose phosphate pathway utilized by the bacteria with the help of pyrophosphate fructose 6-phosphate 1-phosphotransferase (PPi-PFK) to bypass the loss of the transaldolase gene and to complete the glycolytic pathway. Also, the prophage genes are detected in the genome of CLas strains, possibly contributing to the genetic drift. This is the first report on thein-silicocharacterization of CLas exploring the survival strategies adopted by CLas in the metabolism to adapt to the host ecological conditions.