Publications

4537

Citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), is a destructive disease threatening global citrus industry. Although citrus cultivars differ in HLB sensitivity, how infection alters endophytic bacterial communities in cultivars with contrasting susceptibility remains unclear. Here, we compared endophytic microbiome shifts in leaf and root tissues of HLB-susceptible Shatangju mandarin (Citrus reticulata cv. Shatangju) and HLB-tolerant Shatian pomelo (C. maxima cv. Shatian) at 10 months postgrafting with CLas-infected buds. Infected Shatangju mandarin showed severe symptoms and higher CLas levels in leaves than in roots, while infected Shatian pomelo remained asymptomatic with higher CLas in roots than in leaves. High-throughput 16S rRNA gene sequencing revealed that CLas infection restructured the endophytic microbial community in a tissue-specific manner. CLas-infected Shatian pomelo leaves displayed increased microbial diversity and network complexity, while CLas-infected Shatangju mandarin roots harbored more disease-suppressive taxa like Streptomyces. Across both cultivars, network complexity correlated inversely with CLas abundance. Key genera (e.g., Pseudomonas, Streptomyces, and Prevotella) correlated positively with CLas, suggesting roles in mitigating pathogen effects. Predicted functional profiling indicated activated pathways (amino acid metabolism, terpenoid/polyketide biosynthesis, and xenobiotic biodegradation) potentially supporting host defense in infected tissues. The HLB tolerance of Shatian pomelo appeared linked to tissue-specific bacterial restructuring, particularly recruitment of antibiotic-producing bacteria and enhanced metabolic resilience. These findings elucidated cultivar-specific microbial strategies against CLas, offering insights for microbiome-mediated HLB management.

Ocean warming, disease, and pollution have contributed to global declines in coral abundances and diversity. In the Caribbean, corals previously dominated reefs, providing an architectural framework for diverse ecological habitats, but have significantly declined due to infectious disease and anthropogenic climate change. Key species like the coral Acropora cervicornis are critically endangered, prompting researchers to focus on scientific endeavors to identify factors influencing coral disease resistance and resilience. We previously showed that disease susceptibility, growth rates, and bleaching risk were all associated with the abundance of a single bacterial parasite, Candidatus Aquirickettsia rohweri which proliferates in vivo under nutrient enrichment. Yet how nutrients influence parasite physiology in vivo remains unknown. Here, we analyzed parasite gene expression from a disease-susceptible A. cervicornis genotype exposed to ambient or nutrient enrichment conditions. Electron microscopy showed that Ca. A. rohweri was abundant in coral tissue and densely packed in mucocytes prior to nutrient enrichment. Under ambient conditions, the parasite upregulated genes involved in translation, protein maintenance, and cell envelope integrity, consistent with a conserve-and-maintain strategy. Nutrient enrichment induced expression of genes associated with central metabolism, nutrient import, stress response, host interaction, and two-component systems. Together, these results indicate that nutrient enrichment activates a growth-and-exploitation strategy, likely exacerbating parasitic pressure on A. cervicornis .

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.

‘Candidatus Phytoplasma rubi’ (elm yellows group, 16SrV-E phytoplasma subgroup) is the causal agent of rubus stunt disease, a disorder affecting economically important plants—raspberries and blackberries. Although this phytoplasma has been extensively studied in cultivated Rubus crops, its occurrence and molecular identity in wild Rubus species populations in North-Eastern Europe remain poorly documented. In this study, phytoplasmas associated with symptomatic wild Rubus idaeus L. and Rubus nessensis Hall plants were investigated in natural forest ecosystems of the Curonian Spit, Lithuania. A total of 65 symptomatic plants were surveyed, and phytoplasma infection was detected in 30 samples by nested PCR targeting the 16S rRNA gene. Positive samples were characterized using a multilocus molecular approach based on sequence analysis of the additional cpn60 and secA genes. All strains showed high nucleotide sequence similarity across the analysed loci and consistently clustered with reference strains of ‘Candidatus Phytoplasma rubi’. Virtual RFLP profiles derived from the 16S rRNA and cpn60 genes were nearly identical to those of established 16SrV-E phytoplasma subgroup reference strains and clearly distinct from other 16SrV phytoplasma subgroups. These results provide not only the first detailed multilocus molecular characterization of ‘Candidatus Phytoplasma rubi’-related strains infecting wild Rubus species in Lithuania but also represent the first report of this phytoplasma in naturally occurring R. idaeus and R. nessensis plants in the country, thereby extending the known geographical occurrence of this pathogen and documenting its presence in wild Rubus hosts from unmanaged forest habitats in the Eastern Baltic region of Northern Europe.

Citrus huanglongbing (HLB), associated with the bacterium ‘Candidatus Liberibacter asiaticus’ and spread by the Asian citrus psyllid (Diaphorina citri; ACP), poses a significant threat to California’s citrus industry. First identified in Los Angeles in 2012, HLB has since spread through residential areas across Southern California. A risk-based survey (RBS) model has been developed to improve HLB surveillance and intervention. Within this framework, model components change as HLB dynamics shift, requiring regular updates to maintain data accuracy and model reliability. Disease spread is influenced by natural factors, such as ACP establishment and confirmed HLB locations, as well as human-mediated factors like global mobility (travel introduction from HLB-infected countries), transportation of citrus materials, nurseries, packinghouses, farmers’ markets, and proximity to private or otherwise inaccessible lands. Human-mediated risk factors account for approximately 26.3% (18.4 to 38.4%) of HLB incidence across different years, and natural causes predominantly explain the remaining 73.7% (61.6 to 81.7%). Notably, global mobility was crucial for early HLB detection in new areas, whereas ACP density strongly correlated with disease spread once established. A retrospective analysis from 2015 to 2022 evaluated the RBS model’s performance, showing a predictive power of 88 to 97%, which confirms its validity for developing targeted interventions and early detection strategies in California.

A field investigation was conducted during the winter seasons of 2021–22 and 2022–23 at the Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, Central India, to identify and characterize the phytoplasma strains associated with niger (var. JNC6) and sesame (var. RT315) exhibiting severe phyllody symptoms. Disease incidence ranged from 0-3%, and 3 to 15% were recorded in niger and sesame crops during the study period. Nested PCR assays using universal phytoplasma primer pairs P1/P7 followed by R16F2n/R16R2 consistently amplified ~1.25 kb fragments of the 16S rRNA gene from both the symptomatic plants, confirming phytoplasma association. Additional amplification of phytoplasma-specific sec A (~600 bp) and sec Y (~1.7 kb) gene fragments further validated the phytoplasma infection in both crops. Two predominant leafhopper species, Amrasca biguttula and Orosius albicinctus , colonizing and feeding niger and sesame fields were identified. Notably, only Amrasca biguttula was tested positive for phytoplasma using the similar PCR assays mentioned above, implicating it as a putative vector. Sequence identity, phylogenetic analysis, and in silico RFLP profiling of 16S rRNA, sec A, and sec Y gene sequences from both the symptomatic plants and leafhoppers confirmed the presence of a ‘ Ca. P. asiaticum’ related strain (16SrII-C subgroup). This study represents the first global report of 16SrII-C subgroup phytoplasma infection in niger, highlighting a new host record. The detection of an identical phytoplasma strain in niger, sesame, and Amrasca biguttula suggests a possible epidemiological link and underscores the role of these natural hosts and the leafhopper in the dissemination of ‘ Ca. P. asiaticum’-related phytoplasma strains in Central India.

Areca palm yellow leaf phytoplasma (AYLP) and Areca palm velarivirus 1 (APV1) are two critical pathogens associated with yellow leaf in areca palm, resulting in devastating damage to areca production. However, evidence of their coinfection remains unclear. Areca palms showing yellow leaf symptoms (suspected to be caused by phytoplasma and velarivirus 1) were surveyed and sampled around Hainan Island of China, and the pathogens were identified and analyzed in the study. The results showed that infectious leaf yellowing symptoms of areca palms primarily occurred in eastern and southern cities and counties of Hainan Island. Molecular identification revealed coinfection of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) and APV1 in individual areca palm samples. Among 520 samples tested by qPCR, AYLP was detected in 23.46% (n = 122), APV1 in 53.46% (n = 278), and a coinfection with both AYLP and APV1 in 10.96% (n = 57). A statistically significant difference in APV1 detection rate was observed between asymptomatic and symptomatic plantations (P < 0.0001), suggesting that APV1 infection likely represents an important risk factor for yellowing development in cultivated areca palm populations. These results confirm the coinfection of AYLP and APV1 on Hainan Island, offering epidemiological evidence for targeted disease management.

Candidatus Liberibacter asiaticus (CLas) is an unculturable, phloem-limited gram-negative bacterium associated with citrus Huanglongbing (HLB) disease in Florida and most other citrus-producing countries around the world. HLB is considered the most destructive disease of citrus and is severely affecting the fruit production worldwide. Natural transmission of CLas occurs by the Asian citrus psyllid (ACP) vector Diaphorina citri. Under greenhouse conditions, the experimental transmission of CLas to non-infected trees usually is accomplished using infected ACP or by grafting infected citrus tissue. CLas was confirmed infecting citrus in Florida in 2005 and many characteristics of the disease have been described. However, the transmission of CLas for controlled greenhouse studies is often prob lematic and poorly understood. Due the complexity in maintenance of ACP colonies for controlled vector transmission, the experimental infection of new plants is accomplished primarily by grafting, but several factors may influence the reliability of this method. This study evaluates the propagation and transmission efficiency of CLas using graft inoculation methods under greenhouse conditions. Three experiments were conducted for with graft inoculation: 1) seedlings of 12 cultivars; 2) ‘Tango’ mandarin on 13 rootstock cultivars; and 3) ‘Navel’ orange on 14 rootstock cultivars. Plants were inoculated with buds from infected greenhouse-grown ‘Valencia’ orange plants that were poly merase chain reaction (PCR) positive for CLas and symptomatic for HLB. Three bud pieces were used to inoculate each plant using the inverted-T method.

Citrus huanglongbing (HLB), or citrus greening, is caused by a bacterial pathogen (Candidatus Liberibacter asiaticus, CLas) and is considered the most destructive citrus disease worldwide. The disease has affected millions of citrus trees both in Florida and around the world, resulting in billions of dollars in annual production losses. Infection by the CLas disrupts vascular movement, weakens the tree root system, and cause a rapid decline in tree health. Despite extensive research efforts, there are still no effective management tools to cure HLB-positive trees or prevent new infections. Current HLB management strategies include chemical application of insecticides to control the insect vector of the CLas bacterium (the Asian citrus psyllid) and traditional antibiotics that kill the CLas bacterium. Vector control has not been effective and antibiotic use has been complicated because the CLas bacterium resides inside the plant in the vascular system, and it is difficult to get enough antibiotics to penetrate at effective concentrations. We present a novel method of biological molecule delivery method referred to as Symbionts™. In Symbionts™, host cells are modified to divide autonomously using plant growth regulator genes from Agrobacterium tumefaciens and to produce biological molecules that can improve host-plant defenses against CLas. To produce the Symbiont, host cells expressing cargo of interest can either be induced on citrus trees by infection with A. tumefaciens, or cultured symbiont cells can be produced in the lab and transplanted (free of A. tumefaciens).