Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen ('Candidatus Liberibacter asiaticus') Using Cas12a-Based Methods.

Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, 'Candidatus Liberibacter asiaticus' (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids.

Huanglongbing (HLB) is the most destructive bacterial disease of citrus worldwide. While most citrus varieties are susceptible to HLB, Poncirus trifoliata, a close relative of Citrus, and some of its hybrids with Citrus are tolerant to HLB. No specific HLB tolerance genes have been identified in P. trifoliata but recent studies have shown that constitutive disease resistance (CDR) genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus (CLas), the pathogen of HLB. The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus. We identified 17 CDR genes in two citrus genomes, deduced their structures, and investigated their phylogenetic relationships. We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events. Through genome resequencing and transcriptome sequencing, we identified eight CDR genes in the Poncirus genome (PtCDR1-PtCDR8). The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7. Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes. PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant, Poncirus hybrids as revealed by real-time PCR analysis. These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Proteomics analysis reveals novel host molecular mechanisms associated with thermotherapy of 'Ca. Liberibacter asiaticus'-infected citrus plants.

BACKGROUND: Citrus Huanglongbing (HLB), which is linked to the bacterial pathogen 'Ca. Liberibacter asiaticus' (Las), is the most devastating disease of citrus plants, and longer-term control measures via breeding or genetic engineering have been unwieldy because all cultivated citrus species are susceptible to the disease. However, the degree of susceptibility varies among citrus species, which has prompted efforts to identify potential Las resistance/tolerance-related genes in citrus plants for application in breeding or genetic engineering programs. Plant exposure to one form of stress has been shown to serendipitously induce innate resistance to other forms of stress and a recent study showed that continuous heat treatment (40 to 42 °C) reduced Las titer and HLB-associated symptoms in citrus seedlings. The goal of the present study was to apply comparative proteomics analysis via 2-DE and mass spectrometry to elucidate the molecular processes associated with heat-induced mitigation of HLB in citrus plants. Healthy or Las-infected citrus grapefruit plants were exposed to room temperature or to continuous heat treatment of 40 °C for 6 days. RESULTS: An exhaustive total protein extraction process facilitated the identification of 107 differentially-expressed proteins in response to Las and/or heat treatment, which included a strong up-regulation of chaperones including small (23.6, 18.5 and 17.9 kDa) heat shock proteins, a HSP70-like protein and a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO)-binding 60 kDa chaperonin, particularly in response to heat treatment. Other proteins that were generally down-regulated due to Las infection but up-regulated in response to heat treatment include RuBisCO activase, chlorophyll a/b binding protein, glucosidase II beta subunit-like protein, a putative lipoxygenase protein, a ferritin-like protein, and a glutathione S-transferase. CONCLUSIONS: The differentially-expressed proteins identified in this study highlights a premier characterization of the molecular mechanisms potentially involved in the reversal of Las-induced pathogenicity processes in citrus plants and are hence proposed targets for application towards the development of cisgenic Las-resistant/tolerant citrus plants.

Long-Term Field Evaluation Reveals Huanglongbing Resistance in Citrus Relatives.

Citrus huanglongbing (HLB) is a destructive disease with no known cure. To identify sources of HLB resistance in the subfamily Aurantioideae to which citrus belongs, we conducted a six-year field trial under natural disease challenge conditions in an HLB endemic region. The study included 65 Citrus accessions and 33 accessions belonging to 20 other closely related genera. For each accession, eight seedling trees were evaluated. Based on quantitative polymerase chain reaction analysis of the pathogen titers and disease symptoms, eight disease-response categories were identified. We report two immune, six resistant, and 14 tolerant accessions. Resistance and tolerance observed in different accessions may be attributed to a multitude of factors, including psyllid colonization ability, absence of pathogen multiplication, transient replication of the bacterium, lack of pathogen establishment in the plant, delayed infection, or recovery from infection. Most citrus cultivars were considered susceptible: 15 citrons, lemons, and limes retained leaves in spite of the disease status. Resistance and high levels of field tolerance were observed in many noncitrus genera. Disease resistance/tolerance was observed in Australian citrus relative genera Eremocitrus and Microcitrus, which are sexually compatible with citrus and may be useful in future breeding trials to impart HLB resistance to cultivated citrus.

Heat treatment eliminates 'Candidatus Liberibacter asiaticus' from infected citrus trees under controlled conditions.

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus'. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by 'Ca. L. asiaticus', the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the 'Ca. L. asiaticus' bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the 'Ca. L. asiaticus' bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of 'Ca. L. asiaticus', while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate 'Ca. L. asiaticus' bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of 'Ca. Liberibacter'-infected plants in nursery and greenhouse settings.

EcpA, an extracellular protease, is a specific virulence factor required by Xanthomonas oryzae pv. oryzicola but not by X. oryzae pv. oryzae in rice.

Previously, 12 protease-deficient mutants of the Xanthomonas oryzae pv. oryzicola (Xoc) RS105 strain were recovered from a Tn5-tagged mutant library. In the current study, the Tn5 insertion site in each mutant was mapped. Mutations in genes encoding components of the type II secretion apparatus, cAMP regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme and extracellular protease (ecpA(Xoc)) either partially or completely abolished extracellular protease activity (ECPA) and reduced virulence in rice. Transcription of ecpA(Xoc) was induced in planta in all the mutants except RΔecpA. Complementation of RΔecpA with ecpA(Xoc) in trans restored ECPA, virulence and bacterial growth in planta. Purified EcpA(Xoc) induced chlorosis- and necrosis-like symptoms similar to those induced by the pathogen when injected into rice leaves. Heterologous expression of ecpA(Xoc) conferred ECPA upon the vascular bacterium X. oryzae pv. oryzae (Xoo) and upon non-pathogenic Escherichia coli. Genetic analysis demonstrated that the C-terminal residues of EcpA in Xoo PXO99(A) and Xoc RS105 are different, and a frame shift in ecpA(Xoo) may explain the absence of EcpA activity in Xoo. Collectively, these results suggest that EcpA(Xoc) is a tissue-specific virulence factor for Xoc but not Xoo, although the two pathovars are closely related bacterial pathogens of rice.

Multilocus microsatellite analysis of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing worldwide.

BACKGROUND: Huanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited α- proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed. RESULTS: A panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide. CONCLUSIONS: Our results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.

Improved real-time PCR detection of 'Candidatus Liberibacter asiaticus' from citrus and psyllid hosts by targeting the intragenic tandem-repeats of its prophage genes.

'Candidatus Liberibacter asiaticus' (CLas) is a Gram-negative α-proteobacterium, and the prominent species of Liberibacter associated with a devastating worldwide citrus disease known as huanglongbing (HLB). This fastidious bacterium resides in phloem sieve cells of host plants and is vectored by the Asian citrus psyllid (Diaphorina citri). Due to its uneven distribution in planta and highly variable bacterial titers, detection of HLB bacteria can be challenging. Here we demonstrated a new utility of nearly identical tandem-repeats of two CLas prophage genes for real-time PCR by SYBR Green 1 (LJ900fr) and TaqMan(®) (LJ900fpr). When compared with conventional 16S rDNA-based real-time PCR, targeting the repeat sequence reduced the relative detectable threshold by approximately 9 and 3 real-time PCR cycles for LJ900fr and LJ900fpr, respectively. Additionally, both LJ900 methods detected CLas from otherwise non-detectable samples by other methods. CLas was also detected from globally derived samples including psyllids, various citrus varieties, periwinkle, dodder, and orange jasmine, suggesting the new detection method can be applicable worldwide. Additionally, we demonstrated the presence of the hyv(I)/hyv(II) repeat sequence within the 'Ca. Liberibacter americanus' strain. The method thereby provides sensitive HLB detection with broad application for scientific, regulatory, and citrus grower communities.

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

Low Incidence of 'Candidatus Liberibacter asiaticus' in Murraya paniculata and Associated Diaphorina citri.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. 'Candidatus Liberibacter asiaticus' is the prevalent species of three HLB-associated Liberibacter spp., which is vectored by the psyllid Diaphorina citri. The vector and the bacteria have host plants outside the genus Citrus, and these plants have the potential to affect disease epidemiology within citrus groves. Murraya paniculata could be especially problematic because it is a popular ornamental plant and a host of both psyllid and bacteria. We conducted a year-long survey of eight urban plantings of M. paniculata in east-central Florida to characterize 'Ca. L. asiaticus' infection rates in plants and associated psyllids. Using sensitive quantitative polymerase chain reaction (qPCR) primers targeting two prophage genes of 'Ca. L. asiaticus', we found infection to be extremely low: less than 1% of psyllids and 1.8% of plants. With qPCR primers targeting 'Ca. L. asiaticus' 16S rDNA, none of the plants and only one psyllid were 'Ca. L. asiaticus'-positive. Therefore, the titer of 'Ca. L. asiaticus' is low in M. paniculata and associated psyllids. These results suggest that urban plantings of M. paniculata may serve as a minor source of 'Ca. L. asiaticus' inoculum.

'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections.

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.

Effects of cadmium stress on alternative oxidase and photosystem II in three wheat cultivars.

The effects of Cd stress (200 micromol/L, 8 days) on respiration and photosynthesis of three wheat cultivars were investigated: Chuanyu 12 (CY12), Chuanmai 42 (CM42), and Chuanmai 47 (CM47). Fifteen-day-old seedlings were exposed to 200 micromol/L CdCl2 for 4 days and 8 days, respectively. The results indicated that Cd was accumulated largely in roots, but little in leaves of all three cultivars. CY12 accumulated the highest level of Cd in roots and showed the weakest resistance. On the contrary, the other two cultivars, CM42 and CM47, adapted better to Cd stress, and their thiobarbituric acid-reactive substances (TBARS) contents were lower than in CY12, but the chlorophyll contents and water contents were higher than in CY12. Additionally, Cd stress prompted the alternative oxidase (AOX) activity and upregulated the cyanide-resistant respiration in CM42 and CM47 after 8 days; no such induction was observed for CY12. The CO2 assimilation rate, leaf stomatal conductance and chlorophyll fluorescence were inhibited by Cd stress in all cultivars, but more severe in the CY12 cultivar. Western blots indicated that the content of the photosystem II proteins LHCII and D1 decreased in CY12, but did not change in CM42 and CM47. While the content of the mitochondrial AOX protein increased markedly in CM42 and CM47, it did not in CY12. These results suggested that AOX and LHCII could be regarded as indicators of plant's resistance to heavy metals.

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.