Publications

3986

Abstract Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm), and Candidatus Mycoplasma turicensis (CMt) are the major feline hemotropic Mycoplasma (FHM) species identified in cats worldwide. Data concerning FHM in Egypt is limited; therefore, the current study aimed to gain further insights into disease epidemiology by investigating FHM molecular prevalence, risk factors, and hemogram abnormalities in 246 Egyptian cats (shelter-housed and client-owned) during 2022–2024. Additionally, 16 S rRNA gene-based maximum-likelihood (ML) phylogenetic analysis was performed for all CMt-positive samples for the first time in Egypt. FHM was detected in 16.3% (n = 40) of cats and typed as CMhm, Mhf, and CMt in 15.4% (n = 38), 3.6% (n = 9), and 2.4% (n = 6) of cats, respectively. Single CMhm and Mhf infections were detected in 12.6% (n = 31) and 0.8% (n = 2) of cats, respectively. Dual (Mhf and CMhm) and triple (Mhf, CMhm, and CMt) infections were found in 0.4% (n = 1) and 2.4% (n = 6) of cats, respectively. CMhm was significantly associated with single infections rather than coinfections compared to other FHM species. Male sex and anemia were identified as predictors of FHM PCR positivity. FHM-infected cats had significantly lower hematocrit %, RBC counts, and hemoglobin concentrations than non-infected ones. Egyptian CMt sequences showed 97.6–100% nucleotide identity with each other. Egyptian and reference CMt strains represented nine nucleotide sequence types clustered into three well-supported clades on the ML tree without clear geographic distinction. The data generated in this study, conducted in Egypt, is crucial for enhancing our understanding of disease epidemiology and implementing effective preventive measures.

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.

Candidatus Liberibacter asiaticus (CLas) is a host-dependent obligate parasite that causes citrus greening disease in citrus 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 culture Candidatus Liberibacter species. The in-silico characterization 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 the in-silico characterization of CLas exploring the survival strategies adopted by CLas in the metabolism to adapt to the host ecological conditions.

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.

ABSTRACT Although the high-altitude limit for microbial survival in the Earth–atmosphere system has remained a scientific curiosity and topic of study, the ecological significance of long-distance microbial dispersal in the atmosphere has been perceived to have marginal relevance. Here, we report the characterization of novel plant pathogenic species of Curtobacterium that were isolated from samples collected at altitudes ranging from 1.5 to 29 km above sea level. Whole genome-based phylogenies of three strains, paired with plant challenge assays, indicate that each is a previously unrecognized species and causes disease on beans comparable to Curtobacterium flaccumfaciens . Isolates from the stratosphere (strain L6-1) and agricultural millet (G77) were identified to be the same species and designated as Curtobacterium aetherium sp. nov. C. aetherium displays high levels of tolerance to desiccation and UV radiation, which are stresses that increase in intensity with altitude. Back trajectory air mass analysis implied that the phytopathogens may have had an intercontinental source, but regional origins in the continental US cannot be excluded. The environmentally robust phytopathogens we have documented in the upper atmosphere provide new perspective on the role that high-altitude transport may play in microbial dispersal, gene flow, and the epidemiology of aerially dispersed plant disease. IMPORTANCE Enormous quantities and varieties of microorganisms are continually aerosolized and transported in the atmosphere, yet there is a limited understanding of the consequences to downwind ecosystems. While studying bacteria that survive extreme conditions at altitudes up to 29 km in the atmosphere, we discovered new species that have the capacity to cause disease in agriculturally relevant bean varieties. The hardiest isolate we characterized from the stratosphere is a member of the same species as an isolate from an agricultural source, which we have designated Curtobacterium aetherium . C. aetherium is a phytopathogen capable of enduring high-altitude and long-distance atmospheric transport while also possessing the potential to opportunistically infect crops in deposition locations.