Bacterial Pesticidal Protein Mpp51Aa1 Delivered via Transgenic Citrus Severely Impacts the Fecundity of Asian Citrus Psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri vectors the causative agent of citrus greening disease that has the capacity to decimate citrus production. As an alternative and more sustainable approach to manage D. citri than repeated application of chemical insecticides, we investigated the potential use of the bacteria-derived pesticidal protein, Mpp51Aa1, when delivered by transgenic Citrus sinensis cv. Valencia sweet orange or Citrus paradisi cv. Duncan grapefruit. Following confirmation of transcription and translation of mpp51aa1 by transgenic plants, no impact of Mpp51Aa1 expression was seen on D. citri host plant choice between transgenic and control Duncan grapefruit plants. A slight but significant drop in survival of adult psyllids fed on these transgenic plants was noted relative to those fed on control plants. In line with this result, damage to the gut epithelium consistent with that caused by pore-forming proteins was only observed in a minority of adult D. citri fed on the transgenic Duncan grapefruit. However, greater impacts were observed on nymphs than on adults, with a 40% drop in the survival of nymphs fed on transgenic Duncan grapefruit relative to those fed on control plants. For Valencia sweet orange, a 70% decrease in the number of eggs laid by adult D. citri on transgenic plants was noted relative to those on control plants, with a 90% drop in emergence of progeny. These impacts that contrast with those associated with other bacterial pesticidal proteins and the potential for use of Mpp51Aa1-expressing transgenic plants for suppression of D. citri populations are discussed. IMPORTANCE Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri. This insect is particularly important as the vector of the causative agent of citrus greening, or huanglongbing disease, which severely impacts global citrus production. In this study, we investigated the potential of transgenic citrus plants that produce the pesticidal protein Mpp51Aa1. While adult psyllid mortality on transgenic plants was modest, the reduced number of eggs laid by exposed adults and the decreased survival of progeny was such that psyllid populations dropped by more than 90%. These results provide valuable insight for potential deployment of Mpp51Aa1 in combination with other control agents for the management of D. citri.

Microscopic and Transcriptomic Analyses of Early Events Triggered by 'Candidatus Liberibacter asiaticus' in Young Flushes of Huanglongbing-Positive Citrus Trees.

'Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant-pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant-pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes.

Intracellular Life Cycle of 'Candidatus Liberibacter asiaticus' Inside Psyllid Gut Cells.

'Candidatus Liberibacter asiaticus' (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Cellular injury to 1- to 3+-year-old stems of Camellia sinensis by Tuckerella japonica.

Tuckerella japonica Ehara (Acari: Tuckerellidae) feeds on predigested plant cells beneath exposed periderm tissue of 1- to 3+-year-old stems of Camellia sinensis (L.) O. Kuntze (Theaceae) where longitudinal bark splitting occurs. Control samples from these tissues were compared with areas fed upon by T. japonica adults and immatures to characterize types of cellular injury. Stylet diameters ranged from 1.6 to 2.3 µm and were consistent with observed stylet punctures in the stems. Mite saliva was injected along tracts within the cortical tissue and resulted in cell wall disruption, collapsed cells and, in older tissue, hyperplasia. The range of potential stylet penetration into plant tissues was from 92 to 150 µm. Tuckerella japonica injects saliva in the cortical tissues. The paired stylet lengths would allow for possible injection of saliva into the upper areas of phloem tissue but not in the cambium area of wood exposed by splitting of the outer epidermis.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

Visualization of 'Candidatus Liberibacter asiaticus' cells in the vascular bundle of citrus seed coats with fluorescence in situ hybridization and transmission electron microscopy.

'Candidatus Liberibacter asiaticus' is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for 'Ca. L. asiaticus' were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the 'Ca. L. asiaticus' 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to 'Ca. L. asiaticus'. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no 'Ca. L. asiaticus' sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as 'Ca. L. asiaticus', providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were 'Ca. L. asiaticus'.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Canopy Density, but Not Bacterial Titers, Predicts Fruit Yield in Huanglongbing-Affected Sweet Orange Trees.

In Florida, almost all citrus trees are affected with Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). We characterized various parameters of HLB-affected sweet orange trees in response to yield-improving nutritional treatment, including canopy volume, canopy density and CLas Ct values, and found that the treatment improved yield and maintained canopy density for over three years, whereas untreated HLB-affected trees declined in canopy density. The nutritional treatment did not affect CLas titer or the tree canopy volume suggesting that canopy density is a better indicator of fruit yield. To further validate the importance of canopy density, we evaluated three independent orchards (different in tree age or variety) to identify the specific traits that are correlated with fruit yields. We found that canopy density and fruit detachment force (FDF), were positively correlated with fruit yields in independent trials. Canopy density accurately distinguished between mild and severe trees in three field trials. High and low producing HLB trees had the same Ct values. Ct values did not always agree with CLas number in the phloem, as visualized by transmission electron microscopy. Our work identifies canopy density as an efficient trait to predict yields of HLB-affected trees and suggests canopy health is more relevant for yields than the CLas population.

Cry1Ba1-mediated toxicity of transgenic Bergera koenigii and Citrus sinensis to the Asian citrus psyllid Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan and Valencia expressing Cry1Ba1 exhibited a "delayed senescence" phenotype, similar to observations made for transgenic B. koenigii. In bioassays, significant reductions in the survival of adult psyllids were noted on transgenic B. koenigii and Valencia sweet orange plants expressing Cry1Ba1, but not on transgenic Duncan grapefruit or Carrizo citrange. In contrast to psyllids fed on wild type plants, the gut epithelium of psyllids fed on transgenic plants was damaged, consistent with the mode of action of Cry1Ba1. These results indicate that the transgenic expression of a bacterial pesticidal protein in B. koenigii and Valencia sweet orange offers a viable option for management of D. citri, that may contribute to solutions that counter citrus greening disease.

Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to 'Candidatus Liberibacter asiaticus' infection.

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by 'Candidatus Liberibacter asiaticus'. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to 'Ca. L. asiaticus' infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to 'Ca. L. asiaticus'. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.

Diaphorina citri flavi-like virus localization, transmission, and association with Candidatus Liberibacter asiaticus in its psyllid host.

Huanglongbing is caused by Candidatus Liberibacter asiaticus (CLas) and transmitted by Diaphorina citri. D. citri harbors various insect-specific viruses, including the Diaphorina citri flavi-like virus (DcFLV). The distribution and biological role of DcFLV in its host and the relationship with CLas are unknown. DcFLV was found in various organs of D. citri, including the midgut and salivary glands, where it co-localized with CLas. CLas-infected nymphs had the highest DcFLV titers compared to the infected adults and CLas-free adults and nymphs. DcFLV was vertically transmitted to offspring from female D. citri and was temporarily detected in Citrus macrophylla and grapefruit leaves from greenhouse and field. The incidences of DcFLV and CLas were positively correlated in field-collected D. citri samples, suggesting that DcFLV might be associated with CLas in the vector. These results provide new insights on the interactions between DcFLV, the D. citri, and CLas.

Genetic Modification of Bergera koenigii for Expression of the Bacterial Pesticidal Protein Cry1Ba1.

The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphorina citri, which vectors the bacterial causative agent of citrus greening or huanglongbing disease. This disease has decimated citrus production in Florida and in other citrus-producing countries. As D. citri exhibits high affinity for feeding on young leaves of B. koenigii, transgenic B. koenigii expressing bacteria-derived pesticidal proteins such as Cry1Ba1 have potential for D. citri management when planted in or adjacent to citrus groves. Importantly, the plant pathogenic bacterium that causes citrus greening does not replicate in B. koenigii. Transgenic plants of B. koenigii were produced by insertion of the gene encoding the active core of the pesticidal protein Cry1Ba1 derived from Bacillus thuringiensis. The transformation success rate was low relative to that of other citrus, at 0.89%. T-DNA integration into the genome and cry1ba1 transcription in transgenic plants were confirmed. Transgenic plants expressing Cry1Ba1 differed from wild-type plants, differed in photosynthesis parameters and hormone levels in some instances, and a marked delay in wilting of detached leaves. The gut epithelium of D. citri fed on transgenic plants was severely damaged, consistent with Cry1Ba1-mediated pore formation, confirming expression of the pesticidal protein by transgenic B. koenigii. These results demonstrate that transgenic B. koenigii expressing bacteria-derived pesticidal proteins can be produced for potential use as trap plants for suppression of D. citri populations toward protection of citrus groves from citrus greening.

Analysis of Citrus Tristeza Virus Incidences within Asian Citrus Psyllid (Diaphorina citri) Populations in Florida via High-Throughput Sequencing.

The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida's citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.

Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin.

Huanglongbing (HLB) is a devastating disease of citrus, caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas). Here, we present evidence that HLB is an immune-mediated disease. We show that CLas infection of Citrus sinensis stimulates systemic and chronic immune responses in phloem tissue, including callose deposition, production of reactive oxygen species (ROS) such as H2O2, and induction of immunity-related genes. The infection also upregulates genes encoding ROS-producing NADPH oxidases, and downregulates antioxidant enzyme genes, supporting that CLas causes oxidative stress. CLas-triggered ROS production localizes in phloem-enriched bark tissue and is followed by systemic cell death of companion and sieve element cells. Inhibition of ROS levels in CLas-positive stems by NADPH oxidase inhibitor diphenyleneiodonium (DPI) indicates that NADPH oxidases contribute to CLas-triggered ROS production. To investigate potential treatments, we show that addition of the growth hormone gibberellin (known to have immunoregulatory activities) upregulates genes encoding H2O2-scavenging enzymes and downregulates NADPH oxidases. Furthermore, foliar spray of HLB-affected citrus with gibberellin or antioxidants (uric acid, rutin) reduces H2O2 concentrations and cell death in phloem tissues and reduces HLB symptoms. Thus, our results indicate that HLB is an immune-mediated disease that can be mitigated with antioxidants and gibberellin.

Widespread Occurrence and Low Genetic Diversity of Colombian datura virus in Brugmansia Suggest an Anthropogenic Role in Virus Selection and Spread.

Brugmansia (Brugmansia spp.) is a perennial shrub in the Solanaceae, originating from South America, that is a popular landscape plant in the tropics and subtropics and container plant in temperate regions. Virus-like symptoms including mosaic, rugosity, and faint chlorotic spots were first observed on leaves of Brugmansia plants in a south Florida nursery in November 2003. Colombian datura virus (CDV) was identified in these initial plants and subsequent Brugmansia and Datura metel (a Brugmansia relative also grown as an ornamental) plants obtained from Florida, Connecticut, Wisconsin, and California. Overall, 77.5% of Brugmansia and two of four D. metel plants tested were infected with CDV. Partial NIb/CP sequences of 28 Brugmansia CDV isolates from this study were compared with all 16 CDV isolates in GenBank and found to share high levels of nucleotide and amino acid identity, with negative selection estimated to be occurring. A single Brugmansia plant was also infected with a recently described tobamovirus. The low genetic diversity of CDV observed, along with negative selection pressure on NIb/CP, suggests a recent ancestry (<400 years) of the worldwide population of CDV, coinciding with anthropogenic collection and dissemination of Brugmansia plants from their center of origin.

Early events of citrus greening (Huanglongbing) disease development at the ultrastructural level.

Citrus greening (Huanglongbing [HLB]) is one of the most destructive diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be 'Candidatus Liberibacter asiaticus'. Understanding of the early events in HLB infection is critical for the development of effective measures to control the disease. In this work, we conducted cytopathological studies by following the development of the disease in citrus trees graft inoculated with 'Ca. L. asiaticus'-containing material under greenhouse conditions to examine the correlation between ultrastructural changes and symptom production, with the main objective of characterizing the early events of infection. Based on our observations, one of the first degenerative changes induced upon invasion of the pathogen appears to be swelling of middle lamella between cell walls surrounding sieve elements. This anatomical aberration was often observed in samples from newly growing flushes in inoculated sweet orange and grapefruit trees at the early "presymptomatic" stage of HLB infection. Development of symptoms and their progression correlated with an increasing degree of microscopic aberrations. Remarkably, the ability to observe the bacterium in the infected tissue also correlated with the degree of the disease progression. Large numbers of bacterial cells were found in phloem sieve tubes in tissue samples from presymptomatic young flushes. In contrast, we did not observe the bacteria in highly symptomatic leaf samples, suggesting a possibility that, at more advanced stages of the disease, a major proportion of 'Ca. L. asiaticus' is present in a nonviable state. We trust that observations reported here advance our understanding of how 'Ca. L. asiaticus' causes disease. Furthermore, they may be an important aid in answering a question: when and where within an infected tree the tissue serves as a better inoculum source for acquisition and transmission of the bacterium by its psyllid vector.

Colonization of dodder, Cuscuta indecora, by 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus'.

Huanglongbing, or citrus greening, threatens the global citrus industry. The presumptive pathogens, 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' can be transferred from citrus to more easily studied experimental hosts by using holoparasitic dodder plants. However, the interaction between 'Candidatus Liberibacter' spp. and the dodder has not been studied. We combined quantitative polymerase chain reaction with electron microscopy to show that only 65% of tendrils of Cuscuta indecora grown on 'Ca. Liberibacter' spp.-infected host plants had detectable levels of the pathogen. Among tendrils that were colonized by Liberibacter in at least one 2 cm segment, most were not colonized in all segments. Furthermore, the estimated population levels of the pathogen present in serial 2 cm segments of dodder tendrils varied widely and without any consistent pattern. Thus, there was generally not a concentration gradient of the pathogen from the source plant towards the recipient and populations of the pathogen were sometimes found in the distal segments of the dodder plant but not in the proximal or middle segments. Populations of the pathogens ranged from 2 x 10(2) to 3.0 x 10(8) cells per 2 cm segment. On a fresh weight basis, populations as high as 1.4 x 10(10) cells per g of tissue were observed demonstrating that 'Ca. Liberibacter' spp. multiplies well in Cuscuta indecora. However, 55% of individual stem segments did not contain detectable levels of the pathogen, consistent with a pattern of nonuniform colonization similar to that observed in the much more anatomically complex citrus tree. Colonization of dodder by the pathogen is also nonuniform at the ultrastructural level, with adjacent phloem vessel elements being completely full of the pathogen or free of the pathogen. We also observed bacteria in the phloem vessels that belonged to two distinct size classes based on the diameters of cross sections of cells. In other sections from the same tendrils we observed single bacterial cells that were apparently in the process of differentiating between the large and round forms to the long and thin forms (or vice versa). The process controlling this morphological differentiation of the pathogen is not known. The highly reduced and simplified anatomy of the dodder plant as well as its rapid growth rate compared with citrus, and the ability of the plant to support multiplication of the pathogen to high levels, makes it an interesting host plant for further studies of host-pathogen interactions.

Walking Together: Cross-Protection, Genome Conservation, and the Replication Machinery of Citrus tristeza virus.

"Cross-protection", a nearly 100 years-old virological term, is suggested to be changed to "close protection". Evidence for the need of such change has accumulated over the past six decades from the laboratory experiments and field tests conducted by plant pathologists and plant virologists working with different plant viruses, and, in particular, from research on Citrus tristeza virus (CTV). A direct confirmation of such close protection came with the finding that "pre-immunization" of citrus plants with the variants of the T36 strain of CTV but not with variants of other virus strains was providing protection against a fluorescent protein-tagged T36-based recombinant virus variant. Under natural conditions close protection is functional and is closely associated both with the conservation of the CTV genome sequence and prevention of superinfection by closely similar isolates. It is suggested that the mechanism is primarily directed to prevent the danger of virus population collapse that could be expected to result through quasispecies divergence of large RNA genomes of the CTV variants continuously replicating within long-living and highly voluminous fruit trees. This review article provides an overview of the CTV cross-protection research, along with a discussion of the phenomenon in the context of the CTV biology and genetics.