Xanthomonas citri pv. citri Pathotypes: LPS Structure and Function as Microbe‐Associated Molecular Patterns

Xanthomonas citri pv. citri is the pathogen responsible for Asiatic citrus canker, one of the most serious citrus diseases worldwide. The lipopolysaccharide (LPS) molecule has been demonstrated to be involved in X. citri pv. citri virulence. Despite enormous progress in investigations of the molecular mechanisms for bacterial pathogenicity, determination of the detailed LPS structure–activity relationship is limited, as the current knowledge is mainly based on structural determination of one X. citri pv. citri strain. As X. citri pv. citri strains are distinguished into three main pathogenicity groups, we characterized the full structure of the LPS from two pathotypes that differ in their host‐range specificity. This revealed an intriguing difference in LPS O‐chain structure. We also tested the LPSs and isolated lipid A moieties for their ability to act as microbe‐associated molecular patterns in Arabidopsis thaliana. Both LPS/lipid As induced ROS accumulation, but no difference was observed between the two pathotypes.     

Attractiveness of Host Plant Volatile Extracts to the Asian Citrus Psyllid, <Emphasis Type="Italic">Diaphorina citri,</Emphasis> is Reduced by Terpenoids from the Non-Host Cashew

Diaphorina citri is a vector of the bacterial causative agent of Huanglongbing (HLB?=?Citrus greening), a severe disease affecting citrus crops. As there is no known control for HLB, manipulating insect behaviour through deployment of semiochemicals offers a promising opportunity for protecting citrus crops. The behavioural responses of D. citri to plant volatiles, and the identity of these plant volatiles were investigated. Volatiles were collected from host plants Murraya paniculata, Citrus sinensis, C. reshni, C. limettioides, Poncirus trifoliata, and from non-host plants Psidium guajava, Mangifera indica, Anacardium occidentale. In behavioural assays, female D. citri spent more time in the arms containing volatiles from either M. paniculata or C. sinensis compared to the control arms. When D. citri was exposed to volatiles collected from A. occidentale, they preferred the control arm. Volatiles emitted from the other studied plants did not influence the foraging behaviour of D. citri. Chemical analyses of volatile extracts from C. sinensis, M. paniculata, and A. occidentale revealed the presence of the terpenoids (E)-4,8-dimethylnona-1,3,7–triene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) in higher amounts in A. occidentale. In further behavioural bioassays, female D. citri spent less time in arms containing a synthetic blend of DMNT and TMTT compared to the control arms. Female D. citri also spent less time in arms containing the synthetic blend in combination with volatile extracts from either M. paniculata or C. sinensis compared to the control arms. Results suggest that higher release of the two terpenoids by A. occidentale make this species unattractive to D. citri, and that the terpenoids could be used in reducing colonisation of citrus plants and therefore HLB infection.     

Exposure to Diflubenzuron Results in an Up-Regulation of a Chitin Synthase 1 Gene in Citrus Red Mite,Panonychus citri (Acari: Tetranychidae)

Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB.     

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis “Xuegan” plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.     

3′ Tth Endonuclease Cleavage Polymerase Chain Reaction (3TEC-PCR) Technology for Single-Base-Specific Multiplex Pathogen Detection using a Two-Oligonucleotide System

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3′ Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.     

Development of Real-Time and Conventional PCR Assays for Identifying a Newly Named Species of Root-Lesion Nematode (Pratylenchus dakotaensis) on Soybean

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (Pratylenchus dakotaensis) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of P. dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P. dakotaensis DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R² value of 0.95 between quantification cycle number and log number of P. dakotaensis. To validate the assays to distinguish P. dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P. dakotaensis and discriminated it from other Pratylenchus spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of P. dakotaensis.     

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the Xfm rpoD gene from both pure bacterial cultures and infected plant material using the Genie^® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.     

Upscaled CTAB-Based DNA Extraction and Real-Time PCR Assays for Fusarium culmorum and F. graminearum DNA in Plant Material with Reduced Sampling Error

Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5–1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5–1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed.     

Two Volatile Organic Compounds Trigger Plant Self-Defense against a Bacterial Pathogen and a Sucking Insect in Cucumber under Open Field Conditions

Systemic acquired resistance (SAR) is a plant self-defense mechanism against a broad-range of pathogens and insect pests. Among chemical SAR triggers, plant and bacterial volatiles are promising candidates for use in pest management, as these volatiles are highly effective, inexpensive, and can be employed at relatively low concentrations compared with agrochemicals. However, such volatiles have some drawbacks, including the high evaporation rate of these compounds after application in the open field, their negative effects on plant growth, and their inconsistent levels of effectiveness. Here, we demonstrate the effectiveness of volatile organic compound (VOC)-mediated induced resistance against both the bacterial angular leaf spot pathogen, Pseudononas syringae pv. lachrymans, and the sucking insect aphid, Myzus persicae, in the open field. Using the VOCs 3-pentanol and 2-butanone where fruit yields increased gave unexpectedly, a significant increase in the number of ladybird beetles, Coccinella septempunctata, a natural enemy of aphids. The defense-related gene CsLOX was induced by VOC treatment, indicating that triggering the oxylipin pathway in response to the emission of green leaf volatiles can recruit the natural enemy of aphids. These results demonstrate that VOCs may help prevent plant disease and insect damage by eliciting induced resistance, even in open fields.     

Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

A Phagostimulant Blend for the Asian Citrus Psyllid

Chemical cues that elicit orientation by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), are of interest because it is the primary vector of the causal pathogen of citrus greening disease. Non-pesticidal control methods for D. citri remain a high priority for the citrus industry. While searching for semiochemicals that may be involved in orientation to host plants, we previously identified a blend of formic and acetic acids that stimulated substrate probing by D. citri. Here, we applied geometric mixture designs and response surface modeling to identify and optimize a 3-component blend that further increased the number of salivary sheaths produced by D. citri on a wax substrate containing a 3.5:1.6:1 blend of formic acid, acetic acid, and p-cymene, respectively. No evidence was found for remote orientation by D. citri adults through olfaction to the phagostimulant blends. Increased probing in response to the presence of phagostimulants in the wax matrix occurred after contact with the substrate. Yellow wax beads always attracted more D. citri adults and received more probes compared with white wax beads. Yellow beads containing the 3-component blend of phagostimulants were probed by D. citri 2 to 3 times more often compared with yellow beads alone. The phagostimulant effect also was tested by covering wax beads containing the 3-component blend with a plastic film to minimize olfaction or contact chemoreception by antennation. The plastic film did not affect the probing response, thus suggesting that chemosensation was associated with mouthparts and not olfactory receptors. Salivary sheaths produced in wax beads containing the phagostimulant blend were 4.5 times longer than sheaths produced in beads without tastants. This phenomenon might be used to improve a trap, design an attract-and-kill product, or enhance other means of managing D. citri and citrus greening disease.     

Synthesis and field bioassay of some analogs of sex pheromone of citrus mealybug,Planococcus citri (Risso)

A series of structural analogs of (s+)-cis-(1R)-3-isopropenyl-2,2-dimethylcyclobutanemethanol acetate, sex pheromone of the citrus mealybug,Planococcus citri (Risso), was synthesized. The analogs were tested in a field bioassay in order to determine the structure-activity relationships of the pheromone. All changes in structure reduced the activity of the test compounds, to various degrees. The most active analog tested was the homolog (+)-cis-(1R)-3-isopropenyl-2,2-dimethylcyclobutaneethanol acetate (IV), whose activity, at a higher dosage, was comparable to that of the pheromone. The alcohol (+)-cis-(1R)-3-isopropenyl-2,2-dimethylcyclobutanemethanol was tested in mixtures with the pheromone and found to be neither an inhibitor nor a Synergist. The results show that all functional groups of the pheromone molecule are essential for optimal biological activity.Homoptera: Coccoidea: Pseudococcidae.Contribution from the Agricultural Research Organization (ARO), The Volcani Center, Bet Dagan, Israel, No. 1667-E, 1986 series.Levi Eshkol Postdoctoral Fellow, 1984–1986.     

The acylation and phosphorylation pattern of lipid A from Xanthomonas campestris strongly influence its ability to trigger the innate immune response in Arabidopsis

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.