Phytopathology®

Candidatus Liberibacter solanacearum (Lso), haplotype D, is an insect-transmitted, phloem-limited bacterium that induces developmental abnormalities in carrots, including witches’ broom and hairy root symptoms. We hypothesize that these symptoms result from Lso-induced hormonal imbalances. To investigate this, we analyzed the spatial and temporal distribution of Lso in carrot plants and assessed its effects on hormone-related gene expression and phytohormone levels. Our findings revealed that Lso first accumulates in the shoot apical meristem before spreading to root tissues, aligning with phloem flow dynamics. Transcriptomic and biochemical analyses indicated that cytokinin (CK) biosynthesis and response genes were upregulated, while gibberellin biosynthesis genes were downregulated. In contrast, no significant changes were observed in auxin biosynthesis or signaling. Hormone quantification further demonstrated increased CK levels in lateral roots and decreased CK levels in the root meristem of infected plants, with no detectable changes in auxin levels. Additionally, salicylic acid (SA) and jasmonic acid (JA) were significantly elevated following Lso infection, suggesting a persistent plant defense response. To validate the role of CK and auxin in symptom development, we applied synthetic growth regulators to infected and uninfected plants. CK treatment exacerbated witches’ broom symptoms, while auxin application mitigated this phenotype but enhanced lateral root formation. These results suggest that Lso manipulates phytohormone homeostasis to induce disease symptoms, offering a potential avenue for symptom mitigation through targeted hormone applications. Our findings provide novel insights into the role of plant hormones in Lso pathogenesis and highlight new strategies for managing carrot yellows disease.

A Microviridae phage CLasMV1, was recently identified in “Candidatus Liberibacter asiaticus” (CLas), the bacterium associated with citrus Huanglongbing (HLB). The CLasMV1 strain GDHZ11D (CP045566) exhibited a circular genome of 8,869 bp containing eight open reading frames (ORFs). Phages of CLas play crucial roles in regulating bacterial population diversity that have potential use for HLB control. However, knowledge of CLasMV1 population diversity is currently limited. In this study, 1,520 CLas samples were collected from 10 citrus-growing provinces in China. PCR screening revealed that 1,042 samples (69%) harbored CLasMV1. Complete genome sequences of 43 CLasMV1 strains were obtained through Illumina HiSeq sequencing with genome size ranging from 8,696 bp to 8,881 bp. Variations were mostly related to single-nucleotide polymorphisms (SNPs) and sequence insertion/deletions (In/Dels). Notably, two hypervariable regions (HVRs) were identified. Two new ORFs were detected, ORFA (144 bp) in HRV-1 and ORFB (342 bp) in HRV-2. Whole genome phylogenetic analysis using 43 strains from this study, nine previously reported Chinese strains, and two Florida strains (CP040636, MZ382797) delineated five genomic groups. Chinese strains dominated Groups 1 to 4, while Group 5 contained the Florida strains. Further analyses of the HVR-1 across all 1,042 CLasMV1 samples detected a 173-bp sequence with suspected mobile activity. Analysis of PacBio long read assembly published previously detected a concatemer of CLasMV1 sequence, suggesting that the phage used the rolling-cycle mechanism for its replication. These findings significantly enhance our understanding of CLasMV1 genomic diversity and will facilitate future research in CLasMV1 classification and HLB biology.

Citrus Huanglongbing, caused by ‘ Candidatus Liberibacter asiaticus’ (CLas), is the most devastating citrus disease worldwide. The CLas genome is much smaller than those of its relatives, such as Sinorhizobium meliloti, due to its reductive evolution. Because CLas has not been cultured in artificial media, despite some progress in co-cultivating, and because genetic manipulation of CLas remains impossible, the understanding of CLas biology is very limited. Usually, 10% of total genes in bacteria are regulatory genes, but only 2% of CLas genes encode transcriptional factors. Here, 20 transcriptional regulators were predicted, including nine genes ( lsrB, ldtR, rem, visR, visN, ctrA, mucR, pelD, and atoC) directly or indirectly involved in regulating motility, and five genes ( rpoH, prbP, phrR, rirA, and lsrB) involved in oxidative stress response. We demonstrated that rem, lsrB, and visNR of CLas can complement the corresponding mutants of S. meliloti in their reduced motility. We further investigated traits controlled by Rem in S. meliloti and CLas using RNA sequencing analyses of rem mutant versus complementation strains with remSmc or remLas. Transcriptomic analysis showed that RemLas significantly regulates the expression of genes in S. meliloti, which was used to infer its regulation of CLas genes by identification of homologous genes. We found that Rem is involved in regulating motility, chemotaxis, transporters, and oxidative phosphorylation in S. meliloti and regulating flagellar and transporter genes in CLas. Among the 39 putative RemLas-regulated genes in CLas, 16 contain the Rem-binding motif, including 10 genes involved in flagellar assembly. Taken together, this study offers valuable insights regarding CLas regulatory genes, with many of them involved in regulating motility and oxidative stress response. The regulation of flagellar genes by Rem in CLas unravels critical information regarding motility in CLas infection of hosts.

Diaphorina citri (Hemiptera: Liviidae) is the main vector for the bacterium ‘ Candidatus Liberibacter asiaticus’, which is associated with citrus greening, also known as Huanglongbing. D. citri transmits ‘ Ca. L. asiaticus’ during its feeding on citrus phloem sap. Transmission occurs in a circulative, propagative, and persistent manner. ‘ Ca. L. asiaticus’ has a small genome (1.2 Mb). Therefore, it acquires most of its nutrients and energetic nucleotides from its hosts. The objective of this study was to assess the effect of ‘ Ca. L. asiaticus’ infection on the level of the free fatty acids in its vector. The fatty acids were extracted from adult D. citri using ethyl acetate, derivatized with boron trifluoride-methanol, and analyzed using gas chromatography-mass spectrometry. Nine fatty acids were identified in the D. citri extracts. Oleic acid was the most predominant fatty acid, followed by stearic and palmitic acid, whereas the rest of the fatty acids were present in low amounts. In general, the levels of the detected fatty acids in ‘ Ca. L. asiaticus’-infected D. citri were lower than those found in healthy psyllids. Our findings showed that the reduction of fatty acids in ‘ Ca. L. asiaticus’-infected psyllids resulted from the higher activity of β-oxidation to generate acetyl-coenzyme A, which causes more production of ATP. Our results indicated that ‘ Ca. L. asiaticus’ may enhance the β-oxidation of fatty acids in its vector insect to fulfill its nutrient and energetic nucleotide requirements.

'Candidatus Phytoplasma pyri', the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of 'Ca. P. pyri' using a strain from Chile, with its genomic features analyzed in comparison to the closely related 'Ca. Phytoplasma' species, 'Ca. P. mali' and 'Ca. P. prunorum'. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of 'Ca. P. mali' was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis (MLSA) of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from ten Chilean and ten Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of 'Ca. P. pyri'.

‘ Candidatus Liberibacter asiaticus’, the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, ‘ Ca. L. asiaticus’ is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of ‘ Ca. L. asiaticus’ becomes available.

The unculturable bacterium ‘ Candidatus Liberibacter solanacearum’ (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid ( Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by ‘ Candidatus Liberibacter asiaticus’ ( CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

Citrus Huanglongbing (HLB) caused by ‘ Candidatus Liberibacter asiaticus’ (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves—late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.

‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.