Publications

4719

ABSTRACT Ponkan mandarin ( Citrus reticulata cv. Ponkan) is an important fruit crop planted across different countries. One of its most devastating diseases is citrus huanglongbing (HLB), which is associated with infection by the bacterium Candidatus Liberibacter asiaticus ( C Las). Although studies on HLB in Ponkan mandarin have been conducted, symptom heterogeneity and its association with C Las titer under field conditions remain underexplored. We examined the association between symptom variation and C Las infection status in a Ponkan mandarin grove by categorizing symptoms at both the leaf and canopy (tree) level. Chlorotic leaves were sampled and classified into different categories, including leaves exhibiting interveinal chlorosis (Cat. A), those showing vein yellowing (Cat. B), and blotchy mottled leaves (Cat. C). Polymerase chain reaction (PCR) and quantitative PCR (qPCR) assays targeting C Las revealed that Cat. A leaves, which resembled nutrient-deficient leaves, had higher C Las detection rates and a larger proportion of samples with high C Las titers. Canopy-level assessment of symptom diversity within each tree also revealed that plants bearing Cat. A leaves were more likely to carry C Las and have high pathogen loads. The likelihood was also greater in trees with higher leaf symptom diversity. Additional prophage typing of the detected C Las strains revealed that leaf symptom variations were independent of prophage type difference. Nutrient analyses revealed reduced magnesium, manganese, and zinc contents in Cat. A leaves, indicating that leaves prone to C Las infection also exhibited nutrient deficiencies. These findings highlight the complexity of potential cultivar-specific associations between C Las and citrus under field conditions. IMPORTANCE Although it is well-established that HLB in Ponkan mandarin is associated with C Las infection, the relationship between leaf symptom variation in this cultivar and the presence and titer of C Las under complex field conditions remains unclear. By examining symptom heterogeneity in natural field conditions at both the leaf and canopy level, this study demonstrated that HLB leaf symptom variation in naturally infected Ponkan mandarin corresponds to differences in C Las presence and titer at both levels. Notably, although blotchy mottling is considered the typical leaf symptom of HLB, we found that C Las detection rates and titers tended to be greater in leaves showing nutrient deficiency-like symptoms, which contrasts with patterns in other citrus cultivars. Recognizing inconsistencies among cultivar-specific patterns is important for interpreting HLB symptoms, devising sampling strategies, and exploring uncharacterized factors affecting symptom expression under field conditions.

Abstract Witches’ broom disease of lime (WBDL) associated with ‘ Candidatus Phytoplasma aurantifolia’ is a destructive disease affecting Mexican lime in southern Iran. Typical symptoms of the disease including internode shortening, little leaf and witches'-broom. This study was conducted to investigate changes in the concentrations of nutrient elements in the symptomatic branches, asymptomatic branches, and roots of Mexican lime trees infected with ‘ Ca. P. aurantifolia’, compared to healthy trees in an orchard in Roudan, Hormozgan Province, Iran. Leaf samples were collected from symptomatic branches, asymptomatic branches of infected trees, and healthy trees. In addition, root samples were separately collected from symptomatic and healthy trees. The concentrations of twelve nutrient elements were determined in leaves and roots. The results showed that in symptomatic branches, phosphorus (P), potassium (K), copper (Cu), iron (Fe), chlorine (Cl) and sodium (Na) elements were significantly higher than asymptomatic ones. Conversely, magnesium (Mg), calcium (Ca), manganese (Mn), and boron (B) levels were significantly lower in symptomatic branches. No significant differences were observed in nitrogen (N) and zinc (Zn) levels between symptomatic and asymptomatic leaves. The Na and Cu concentrations showed the most significant increases in symptomatic versus asymptomatic leaves. In the roots, symptomatic lime trees exhibited significantly lower concentrations of N, P, K, Na, Cu, Mn, Fe, and Mg compared to healthy trees; however, no significant differences were found for Cl, B, Zn, and Ca. Overall, these findings suggest that nutritional management, particularly targeting symptomatic trees, could potentially play a role in the management of WBDL in infected orchards.

Abstract Hemotropic mycoplasmas (hemoplasmas) infecting cattle were first detected in France in 2019 during a clinical outbreak, but their prevalence both at the species and strain levels remains unknown. This study therefore aimed to determine the prevalence of hemoplasmas and their coinfections, and to assess the within and between herd diversity in five fully sampled dairy herds. Detection was performed using 16S rRNA quantitative polymerase chain reaction (qPCR), as well as three strain-specific rnpB qPCR assays targeting Candidatus Mycoplasma haematobovis (CMh, formerly Ca . M. haemobos), Mycoplasma wenyonii strain Mexico (Mex), and Mycoplasma wenyonii strain Massachusetts (Mass). A total of 1011 cattle were sampled, of which 88.7% (897/1011) tested positive by 16S rRNA qPCR. Detection rates for CMh, Mex, and Mass were 67.7% (684/1011), 65.9% (666/1011), and 19.8% (200/1011), respectively. Concordance between 16S rRNA qPCR and strain-specific rnpB qPCR was 92% (931/1011). Coinfections were detected in 67.8% (565/833) of 16S PCR-positive animals, with the CMh + Mex combination being the most frequent. The Mass strain was predominantly found in association with the two other hemoplasmas. For all four PCR assays, hemoplasma positivity was significantly associated with both herd and animal age in the five fully sampled dairy herds. Further studies are needed to elucidate transmission routes and pathogenicity mechanisms of hemoplasmas.

Abstract Bovine hemoplasmas, including Mycoplasma wenyonii (M. wenyonii) and Candidatus Mycoplasma haemobos (C. M. haemobos) , are vector-borne pathogens that can cause significant clinical diseases in cattle. This case report from Türkiye describes the clinical findings, hematological analysis, and serum biochemistry results of a cow presenting with hemoglobinuria caused by C. M. haemobos . Blood samples from the infected animal were analyzed using hematological, biochemical, microscopic, and molecular methods. Multiplex polymerase chain reaction assay confirmed the infection, and microscopic analysis revealed a parasitemia rate of 6.99%. Clinical signs included hemoglobinuria, icterus, and anemia. The treatment included fluid therapy, antibiotic (oxytetracycline), and antianemic drugs. The cattle died 1 month post-treatment, reportedly due to a lower respiratory tract infection. This case report describes the diagnostic methods, clinical findings, and laboratory results. A widescale molecular survey of C. M. haemobos in cattle is warranted, with investigation of its pathogenicity and pathogenies.

Background: The Asian Haemaphysalis longicornis is capable of transmitting a wide range of zoonotic pathogens. This study aimed to investigate the prevalence and characteristics of major pathogens in questing H. longicornis ticks collected from two geographically and ecologically distinct regions in China. Methods: A total of 1004 questing H. longicornis ticks were collected from Liaoning Province (Northeast China; n = 882) and Anhui Province (Central and Eastern China; n = 122) and pooled into 670 mixed samples. Tick species were identified by mitochondrial cytochrome oxidase I gene sequencing. Nucleic acid samples from all pools were first screened for Dabie bandavirus using RT-PCR and for bacteria using universal 16S rRNA primers. Bacteria-positive samples were further tested using polygenic seminested PCR for Rickettsia spp. and Coxiella -like endosymbionts (CLEs). Phylogenetic analyses, including polygene sequence analysis, were performed to ensure the accuracy of species identification and to explore genetic relationships. Results: No Dabie bandavirus was detected in any samples. Among the 670 tick pools, 14 were positive for spotted fever group rickettsiae (SFGR), with an overall minimum infection rate (MIR) of 1.4% (14/1004). Notably, the detected SFGR species showed clear geographic segregation: Candidatus Rickettsia jingxinensis was detected only in Liaoning Province (MIR of 1.4%), while Rickettsia heilongjiangensis was found exclusively in Anhui Province (MIR of 1.6%). Additionally, CLEs (overall MIR of 2.0%) were identified in 20 tick pools, with a significantly higher prevalence in Anhui Province (10.7%) than in Liaoning Province (0.8%; p < 0.001). Conclusions: This surveillance revealed a distinct geographical distribution pattern of SFGR species in H. longicornis populations in China and confirmed the presence of Ca . R. jingxinensis in the northeast and R. heilongjiangensis in the central and eastern regions. These findings, supported by polygenic evidence, provide essential epidemiological data for understanding regional tick-borne disease risks and highlight the need for continued region-specific surveillance.

Dual specificity phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases that regulate diverse cellular processes through dephosphorylation of phosphorylated substrates. DUSPs are commonly found in eukaryotes, bacteria, archaea, and viruses. However, structural and biochemical characterization of bacterial DUSP remains limited, as only one bacterial DUSP has been identified thus far. In this study, we investigated a novel putative bacterial DUSP from Candidatus Chlorohelix allophototropha, referred to as CCaDUSP. The crystal structure of CCaDUSP showed the presence of a well-conserved catalytic motif with a characteristic phosphate-binding loop. Biochemical analyses further confirmed that CCaDUSP exhibits phosphatase activity and contains dual general acid/base residues, both of which contribute to its enzymatic activity. These findings not only represent the first characterization of a novel bacterial DUSP with dual general acid/base residues but also provide a foundation for understanding the diversity of DUSP proteins in bacteria.

In July 2025, two loblolly pines (Pinus taeda L.) growing within the 761-acre coastal Carolina Beach State Park (New Hanover County, NC) were observed with conspicuous growth abnormalities. One tree displayed a large witches’ broom arising from the main stem approximately 7 meters above the ground, immediately below the primary canopy, whereas the second showed extensive proliferation of epicormic shoots bearing needles along much of its 12-meter trunk, accompanied by reduced crown development. Needle tissues were collected from both trees, along with samples from four asymptomatic loblolly pines in the same area for the investigation of the possible phytoplasma presence. Total DNA was extracted using a Qiagen DNeasy Plant Pro Kit according to the manufacturer’s instructions. DNA extracts were screened by phytoplasma-specific real-time PCR (Hodgetts et al. 2009), which resulted in detection of phytoplasmas only in samples from the symptomatic trees. The full-length 16S rRNA gene (1,531 bp) was amplified with PCR primers flanking the gene: Tyr-F2 (5’-CAGTCTGTAAAACTGYTCCG-3’) and Ile-R2 (5’-TATCAGGCGTGTGCTCTAAC-3’). Amplicons of the expected size (~1.9 kb) were purified and sequenced using Oxford Nanopore sequencing. Identical consensus sequences were obtained and deposited in GenBank as strains PES-NC3 and PWB-NC4 (accession numbers PZ173280–PZ173281). BLASTn analysis of the 16S rRNA gene sequence showed the highest identity (1,530/1,531 bp; 99.93%) with a ‘Candidatus Phytoplasma pini’-related strain MDPP4 from Maryland (GenBank acc. no. OR147379), and 98.43% identity with the reference strain of ‘Ca. P. pini’ Pin127S from Spain (subgroup 16SrXXI-A; GenBank acc. no. AJ632155). Virtual RFLP analysis of the F2n/R2 region using iPhyClassifier (Zhao et al. 2009) produced a restriction pattern identical to that of the designated reference strain of subgroup 16SrXXI-B (GenBank acc. no. KU242428), supporting assignment to subgroup 16SrXXI-B. To further characterize the phytoplasma strains, a 1960 bp fragment of the secA gene was amplified from each symptomatic tree as previously described by Valiunas et al. (2015). The secA sequences differed by 10 nucleotides and were deposited in GenBank under acc. nos. PZ184669 (PES-NC3) and PZ184670 (PWB-NC4). BLASTn analysis showed 99.64% identity (1,932/1,939 bp) for PES-NC3 and 99.74% identity (1,934/1,939 bp) for PWB-NC4 to the corresponding locus of ‘Ca. P. pini’ strain MDPP (GenBank acc. no. KU242429). Phylogenetic analyses based on both the 16S rRNA and secA gene sequences indicated that the two strains detected in North Carolina cluster with previously reported strains from Maryland, forming a distinct, well-supported clade that is clearly separated from European ‘Ca. P. pini’ strains. This is the first report of ‘Ca. P. pini’-related strains infecting loblolly pine in North Carolina and only the second documented occurrence of this phytoplasma in the United States of America, following its detection in mountain pine (P. pungens Lamb.) in Maryland (Costanzo et al. 2016). The 16S rRNA gene sequences of the Maryland and North Carolina strains differ by a single nucleotide, whereas both U.S. strains differed from the European reference strain of ‘Ca. P. pini’ by 24 nucleotides across the 1,531-bp 16S rRNA gene sequence. These findings expand the known host range and geographic distribution of phytoplasmas associated with pines and suggest that they may be more widespread in the U.S.A. than previously recognized.