Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild‐type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.     

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

A Comparison of Pathogen Isolation in Culture and Injection–infiltration Bioassay of Citrus Leaves for Detecting Xanthomonas citri subsp. citri

Citrus canker [caused by Xanthomonas citri subsp. citri (Xcc)] can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. For both regulatory purposes and epidemiological studies, accurate detection and quantification of viable inoculum are critical. Two accepted methods used to detect and quantify Xcc are injection–infiltration bioassay and culture, but these two methods have not been directly compared using field‐obtained samples. The two methods were compared using washates of lesions taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009–2010 and 2010–2011, with bioassay being the assumed standard. Despite some misclassifications, true positives (sensitivity) and true negatives (specificity) were the dominant classes using culture. False positives for lesions from shoots ranged from 13.1 to 21.4% in 2009–2010 and 2010–2011, respectively, and false positives for lesions from fruit and leaves ranged from 4.3 to 15.7%, in the two seasons, respectively. The false positive rate for culture compared with injection–infiltration bioassay was highest (0.16–0.55), due to more frequent recovery of Xcc by culture at ≤10³ colony‐forming units (CFU) Xcc per ml. The false negative rate was consistently lower (0.02–0.21), confirming that in only a few cases did culture fail to detect Xcc when it was present. The area under the curve for receiver operator characteristic analysis ranged from 0.80 to 0.97, confirming that culture provided an accurate diagnosis in most cases. There was a higher frequency of lesions from shoots with a CFU ≤10³ Xcc compared with lesions from fruit or leaves, making culture more effective at detecting these. The data demonstrate that culture is a reliable way to detect and quantify Xcc compared with injection–infiltration bioassay, particularly when the CFU is ≤10³ Xcc per ml.     

Molecular Typing and Genetic Characterization of Pathogenic Variants of Xanthomonas axonopodis pv. citri in Taiwan

Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac‐A^f, Xac‐A^p and Xac‐A^r. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine‐responsive regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive‐sequence PCR (rep‐PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac‐A^f, Xac‐A^p and Xac‐A^r, and additional reference strains from pathotypes Xac‐A, Xac‐A*, Xac‐A^w, X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep‐PCR clearly grouped the atypical symptom–producing variants in types Xac‐ A^f, Xac‐A^r and Xac‐A^p into the same cluster with typical symptom‐producing strains in pathotype Xac‐A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac‐A* and Xac‐A^w in these genotypic analyses. Strains of Xac‐A* and Xac‐A^w were closely related to Xac‐A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac‐A.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

柑橘溃疡病及其微生物防治研究进展

柑橘是我国第一大水果,柑橘溃疡病是导致柑橘产量和品质下降的最具破坏性细菌性病害之一,给柑橘产业造成了巨大的经济损失,严重阻碍了柑橘产业的可持续发展。微生物防治柑橘溃疡病具有安全、环保、高效等优点,是当前研究的热点。本文主要概述了柑橘溃疡病特征及其病原菌分类、分布,全面分析了柑橘溃疡病病原菌主要致病机理和协助致病机理;系统梳理了柑橘溃疡病生防微生物的多样性;重点总结了微生物通过产生活性物质、诱导激活植物免疫防御系统等防治柑橘溃疡病的作用机制;最后,我们提出了柑橘溃疡病微生物防治面临的挑战以及未来可能的解决途径,以期为柑橘产业的健康发展提供参考。     

Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Asiatic citrus psylla, Diaphorina citri, Kuwayama (Hemiptera: Liviidae) is an economic pest of citrus groves and a vector of the bacterium, Candidatus Liberibacter spp., one of the causative agents of citrus greening. In order to estimate the infectivity of six different isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea, fungal bioassay was performed on the adults of D. citri. Adults of D. citri were treated individually with 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, 2 × 10⁸ spores/mL fungal concentrations by the immersion method. Subsequent to fungal bioassay, treated D. citri were used to determine the levels of esterase and glutathione S‐transferases (GST) enzymes over a period of 3–7 days. The mortality results suggest that I. fumosorosea isolates (If‐02) caused 82.2% mortality on the seventh day of treatment. However, B. bassiana isolate (Bb‐08) with lowest LC₅₀ (1.4 × 10⁷ spores/mL) proved to be highest potential isolate against D. citri. Biochemical determination of esterase and GST activity assay showed significant differences in activities after infection of fungi. Significantly high activity of esterase was observed by Bb‐01 (27.0 unit per mg protein) on the seventh day, while Ma‐11.1 and If‐2.3 (16.9 and 36.3 unit per mg protein) on the third day post treatment. However, maximum GST's activity was showen by isolates Bb‐08,Ma‐M₂ and If‐2.3 (37.6, 1.40 and 10.9 unit per mg protein) on the third day. The current investigation will help to explore the relations between the insect defense system and entomopathogenic fungi. Moreover, the determination of enzymatic activities will be useful for selecting the most pathogenic isolates.     

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g⁻¹, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g⁻¹ by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g⁻¹ to 4.70, 5.85 and 3.36 log cfu g⁻¹. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.     

Effective molecular detection of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in bulk insect samples from sticky traps

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the principal vector of citrus greening (huanglongbing) disease. Invasion of new areas by the vector increases the risk of further spread of the disease and has economic impacts on the global citrus industry. Effective implementation of vector surveys is essential to contain disease outbreaks. This is especially true in countries such as Japan, where most of the major citrus‐producing areas are free from citrus greening. Recently, vector surveys have been routinely conducted to maintain ‘disease‐free’ and ‘disease‐ and vector‐free’ areas in Japan, and improvement of methods that can detect D. citri in native insect populations is imperative. Here, we developed a method of using conventional and real‐time PCR to detect D. citri among bulk insects captured in sticky traps without the need for preliminary differentiation steps based on morphology. DNA fragments of D. citri were specifically detected by both conventional and real‐time PCR in a mixture of a 10^?3 dilution (ca. 0.008–0.009 ng/μl) of D. citriDNA and 100 ng/μl of bulk insect DNA, indicating that small body parts such as pieces of leg or parts of wings of D. citri were detectable in the bulk insect samples. No misleading amplification of fragments from the other psyllid species and citrus pests we used occurred under our PCR conditions. Our results suggest that the technique is applicable to extensive surveys of D. citri in early warning programmes.     

Environmental conditions for Diaphorina citri Kuwayama (Hemiptera: Liviidae) take‐off

Environmental factors that influence flight activity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) may have implications for Huanglongbing spread and management. In this work, four studies were conducted to evaluate the effect of environmental conditions on D. citri take‐off. In the first, insects were transferred to sweet orange seedlings and confined inside an acrylic cage to verify the take‐off periodicity and the effect of environmental factors on this process. In the second, take‐off temperature threshold was estimated by recording the number of insects that initiated flight from a platform when subjected to gradual temperature increases from 15 to 39°C. In the other studies, we evaluated the effect of different photoperiods and temperature regimes (third study) and of constant temperatures (fourth study) on the propensity for D. citri flight. Insects were confined in clear plastic bottle cages with tubes of 50 ml placed on the cab, to collect emerged adults that initiated flights. Results showed that a small portion of the tested population (maximum 10%) tends to take off from plants and this behaviour is more prevalent in the afternoon (14:00–16:00 h), coinciding with daytimes of lower humidity and higher thermal amplitude. Adults that were submitted to lower temperatures (18°C) and short light periods (10 h) showed less propensity to flight. In contrast, at constant 27°C, the insects were more prone to flight, and this result was confirmed when individuals were submitted to increases in temperature, indicating that 27.14°C is the take‐off temperature threshold of D. citri. Results show that temperature plays an important role in the flight activity of D. citri and suggest that control measures of the insect may be more effective in the morning and in temperatures below 27°C, when the probability to take off from a host is lower.     

Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.     

Efficacy of different copper compounds in the control of Xanthomonas citri subsp. citri pathotypes A and A*

Citrus canker disease is one of the most devastating diseases that attacks citrus, especially limes in the Southern parts of Iran, and is caused by Xanthomonas citri subsp. citri (Xcc). The efficacy of several formulations of copper compounds including Bordeaux mixture, copper oxychloride and copper sulphate in controlling Xcc in Key lime was estimated in vitro and in planta using artificial inoculation. Specific primers were used to detect copper-resistant genes copA, copB and copL in 30 isolates of Xcc. The copA and copL genes were present in all isolates, and copB was detected only in 6 strains. In this study, we observed a very good in vitro growth inhibition activity of copper compounds against Xcc pathotype A. S14 strain (pathotype A^*) was the sole isolate that grew on media amended with 2/4 mM of Bordeaux mixture, copper oxychloride and copper sulphate. All other strains (pathotype A) failed to grow on media amended with this concentration. Bordeaux mixture exhibited high efficacy in controlling Xcc in both conditions. However, there were no significant differences in the efficacy of copper oxychloride and copper sulphate at 1.2 mM concentration in planta. A significantly minimum canker necrotic spot and highest disease control was achieved with Bordeaux mixture and copper oxychloride. There was a significant difference in disease severity of the type strain LMG9322 (pathotype A) and Xcc strain S14 (pathotype A^*). Our experiments showed that Bordeaux mixture exhibited satisfactory efficacy in controlling the causal agent of citrus canker.     

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris

Citrus is an economically important fruit crop that is severely afflicted by citrus canker, a disease caused by Xanthomonas citri ssp. citri (X. citri); thus, new sustainable strategies to manage this disease are needed. Although all Citrus spp. are susceptible to this pathogen, they are resistant to other Xanthomonas species, exhibiting non-host resistance (NHR), for example, to the brassica pathogen X. campestris pv. campestris (Xcc) and a gene-for-gene host defence response (HDR) to the canker-causing X. fuscans ssp. aurantifolii (Xfa) strain C. Here, we examine the plant factors associated with the NHR of C. limon to Xcc. We show that Xcc induced asymptomatic type I NHR, allowing the bacterium to survive in a stationary phase in the non-host tissue. In C. limon, this NHR shared some similarities with HDR; both defence responses interfered with biofilm formation, and were associated with callose deposition, induction of the salicylic acid (SA) signalling pathway and the repression of abscisic acid (ABA) signalling. However, greater stomatal closure was seen during NHR than during HDR, together with different patterns of accumulation of reactive oxygen species and phenolic compounds and the expression of secondary metabolites. Overall, these differences, independent of Xcc type III effector proteins, could contribute to the higher protection elicited against canker development. We propose that Xcc may have the potential to steadily activate inducible defence responses. An understanding of these plant responses (and their triggers) may allow the development of a sustained and sustainable resistance to citrus canker.     

Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.