Publications

4364

‘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.

Candidatus Liberibacter asiaticus (CLas) is a host-dependent obligate bacteria that causes citrus greening disease in citrus plants. The prolonged association of CLas with the host led to the loss of essential metabolic genes, including transaldolase in the pentose phosphate pathway, hindering the development of axenic culture in the host-free environment. In this study, in silico functional annotation of pyrophosphate-fructose 6-phosphate 1-phosphotransferase (PFP) protein was performed by utilizing sequence analysis and molecular modeling, revealing its potential role in bypassing the loss of transaldolase. The findings suggest how PFP and fructose bisphosphate aldolase (FBPA) can reconstruct the disrupted central carbon metabolism and help CLas in ATP production. The molecular modeling of PFP showed binding affinity towards fructose 6-phosphate and sedoheptulose 7-phosphate, showing its dual function. The sequence-based analysis and metabolic modeling study also highlight the presence of other alternate enzymes or transport proteins in CLas, elucidating the broader metabolic resilience. This is the first report to provide the structural insights of PFP in compensating for the lost metabolic function and survival strategies adopted by CLas despite the restrained metabolic capability.

Lethal wilt (LW), also known as “Marchitez Letal (ML)” in Colombia, is an endemic disease affecting oil palms (Elaeis guineensis Jacq.) and is a leading cause of crop loss. The disease is characterized by the drying of leaflets from the tip to the base, primarily impacting the lower third of the plant and progressively moving upward. This progression leads to physiological disturbances, including necrosis at the tips of immature inflorescence bracts (spines) and the detachment of bunch fruits, ultimately causing wilting. As a phytosanitary measure, infected palms are eradicated to prevent further spread of the disease. The primary goal of this research was to identify the bacteria associated with LW and to validate a molecular detection method. A 16S amplicon-based analysis was employed to identify and compare the microbial diversity in LW-affected palm tissues with those of healthy plants. Among the 16 OTUs corresponding to different bacterial genera found in all LW samples, taxonomic classification and symptomatology suggested that the bacteria closely associated with LW belong to the genus Candidatus Liberibacter. Further phylogenetic analysis indicated that these bacteria are part of the Rhizobiaceae family, grouping closely with other species of the genus Candidatus. Liberibacter. The concentration of the pathogen in different oil palm tissues was determined using droplet digital PCR (ddPCR) and quantitative PCR (qPCR), expressed in copies/µL in the LW samples. This study represents the first report of ‘Candidatus Liberibacter sp’. being associated with lethal wilt in oil palms of the Arecaceae family in Colombia. The findings from this research have the potential to contribute significantly to the development of effective management strategies to prevent crop losses.