Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate xanthomonas strains on citrus

ABSTRACT Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthomonas strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.     

壳聚糖和2株芽孢杆菌对柑橘溃疡病菌的杀菌效果评价

柑橘溃疡病是重要的柑橘细菌性病害,为探明壳聚糖和枯草芽孢杆菌对柑橘溃疡病的防治潜力,本文用平板菌落计数法评价了3种粘度壳聚糖对柑橘溃疡病菌的杀菌效果,以改良的纸碟法测定2株枯草芽孢杆菌(Bacillus subtilis)发酵液对柑橘溃疡病菌的抑菌效果。综合处理0.5 h、3 h和6 h的结果显示,3种粘度壳聚糖对柑橘溃疡病的杀菌强弱依次为:粘度12粘度100粘度20。枯草芽孢杆菌菌株BS 101和54-6在YPG培养基中有氧发酵3 d产生发酵液原液的抑菌圈直径分别为3.45 cm和3.21 cm,比0.1 g/mL氨苄青霉素的抑菌圈分别大45%和35%。可见,发酵液中存在对溃疡病菌高度有效的成分。因此,认为粘度12的壳聚糖和芽孢杆菌菌株BS 101和54-6具有防治柑橘溃疡病的潜力。     

柑橘溃疡病检疫与防治

细菌性溃疡病是严重危害世界柑橘产业的重大检疫性病害之一,柑橘溃疡病引起落叶、枯枝和落果,溃疡病斑导致果品质量降低,影响外贸出口。世界各国长期以来对病害采取严格苗木检疫、疫区病树铲除、零星病害药剂防控的综合治理措施;新近美国农业部推出"柑橘健康种植行动计划";2007年7月中国农业部正式启动"柑橘非疫区建设和维护"项目,总体目标在于防控柑橘溃疡病的发生和传播,确保柑橘产业的安全。     

柑桔溃疡病菌PCR快速检验检疫技术研究

 柑桔溃疡病是严重影响全世界柑桔生产的重大检疫性病害,根据柑桔溃疡病菌(Xanthomonas axonopodis pv. citri)新近公布的全基因组中独有的保守蛋白基因序列,设计筛选出一对种特异性引物(JYF5/JYR5),能专一地扩增检出柑桔组织表面所带溃疡病菌的DNA靶带(413 bp)。而柑桔叶面附生的非致病性黄单胞菌、野油菜黄单胞菌近缘种以及健康柑桔样品都不能扩增;靶细菌DNA检测下限1.56 pg/μL,靶细菌悬浮液检测下限10 cfu/μL;在不同PCR仪及各种控温方式下都能稳定地扩增出特征性靶带。这一特异、准确的柑桔溃疡病菌PCR检验技术和研制的预包被固相化PCR检测试剂盒已开始用于我国非疫生产区建设中柑桔苗木、果实的病害检疫检验。     

Detection of 'Candidatus Liberibacter asiaticus' in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of 'Ca. L. asiaticus' in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of 'Ca. L. asiaticus' in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.     

The Effect of Irrigation Practices on the Spatio-temporal Increase of Asiatic Citrus Canker in Simulated Nursery Plots in Reunion Island

Asiatic citrus canker is a potentially severe disease of several citrus species and cultivars in many tropical and subtropical areas. In such areas, infected nursery plants constitute an important source of primary inoculum for newly established citrus groves. The influence of overhead, drip, and mist irrigation systems on the development of Asiatic citrus canker was studied in simulated, Mexican-lime nurseries in Reunion Island. Overhead irrigation exacerbated the increase of disease incidence and severity caused by a streptomycin-resistant strain of Xanthomonas axonopodis pv. citri. The temporal development of Asiatic citrus canker for overhead irrigated nursery plots was best described by an exponential model, because disease incidence in these plots did not come close to an asymptote during the experimental period. This can be explained by the continuous production of new growth, susceptible to infection by Xanthomonas axonopodis pv. citri, and splash dispersal of Xanthomonas axonopodis pv. citri associated with overhead irrigation. Based on spatial correlation and spatio-temporal analyses, aggregated disease patterns were found irrespective of the irrigation system. In overhead-irrigated plots, the spread of Xanthomonas axonopodis pv. citri lacked directionnality. Rainstorms of short duration and high intensity were apparently associated with disease increase in drip-irrigated plots. There is a need to improve cultivation practices in Reunion Island citrus nurseries to minimize Asiatic citrus canker incidence in nurseries and to minimize the introduction of Xanthomonas axonopodis pv. citri to new groves.     

Bacterial antagonists and their cell-free cultures efficiently suppress canker disease in citrus lime

Journal of Plant Diseases and Protection - Proliferation of citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) has increasingly become a serious threat and has resulted in a...     

Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia

Strains of Xanthomonas axonopodis pv. citri were isolated from Mexican lime (Citrus aurantifolia) trees in several countries in southwest Asia. These strains produced typical erumpent bacterial canker lesions on Mexican lime but not on grapefruit (C. paradisi). Lesions on grapefruit were watersoaked and blister-like in contrast to the typical erumpent lesions seen after artificial inoculation with all described pathotypes of X. axonopodis pv. citri. This group of strains hydrolysed gelatin and casein and grew in the presence of 3% NaCl as is typical of X. axonopodis pv. citri pathotype A. RFLP analyses and DNA probe hybridization assays also gave results consistent with X. axonopodis pv. citri pathotype A. Metabolic fingerprints prepared with the Biolog® system showed similarities as well as differences to X. axonopodis pv. citri pathotype A. In spite of the physiological and genetic similarities to pathotype A of X. axonopodis pv. citri, these strains had no or very little affinity for polyclonal antiserum prepared against any of the reference strains of X. axonopodis pv. citri and also did not react with monoclonal antibody A1, an antibody that detects all strains of pathotype A of X. axonopodis pv. citri. These strains were also insensitive to bacteriophage Cp3 like X. axonopodis pv. citri pathotype A and unlike X. axonopodis pv. citri pathotype B. We conclude that these strains, designated Xcc-A*, represent a variant of X. axonopodis pv. citri pathotype-A with pathogenicity limited to C. aurantifolia. The existence of extensive genotypic and phenotypic variation within pathotype A of X. axonopodis pv. citri was unexpected and further complicates the systematics of this species.     

Development of a real-time PCR-based method for detection of Xylophilus ampelinus

A real-time PCR MGB-probe-based detection method specific to Xylophilus ampelinus , the cause of grapevine bacterial blight, was developed. Used in combination with the DNeasy plant mini kit, the sensitivity of X. ampelinus detection was approximately 100 cells from tissue extracts, surpassing the sensitivity of an existing nested PCR method at least tenfold. In field samples a high correlation was observed between real-time PCR cycle threshold (Ct) values obtained and X. ampelinus isolation on artificial media. Isolation was successful from samples with Ct values below 25. Lower concentrations of X. ampelinus , with Ct values up to 36, could also be reliably detected in real-time PCR. The newly developed method offers a reliable and sensitive test for X. ampelinus, suitable as a screening test, complementary to isolation on media or other methods, and could also be used for fast and specific identification of isolated colonies and for relative quantification of X. ampelinus bacteria.     

Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR

A single‐tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0 × 10² Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10‐fold and 8500‐fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single‐tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker.     

Survival of Xanthomonas axonopodis pv. citri in Leaf Lesions Under Tropical Environmental Conditions and Simulated Splash Dispersal of Inoculum

ABSTRACT Asiatic citrus canker (ACC) is a severe disease of several citrus species and hybrids in many tropical and subtropical areas. Populations of Xanthomonas axonopodis pv. citri in leaf and twig lesions are the most important inoculum source for secondary infections. In areas with a marked winter season (e.g., Argentina and Japan), low temperatures induce a decrease of 10(2) to 10(4) in population sizes in lesions, thus creating a discontinuity in the X. axonopodis pv. citri life cycle. The purpose of this study was to evaluate the dynamics of X. axonopodis pv. citri populations in leaf lesions exposed to the mild winter temperatures prevailing in a tropical environment. Internal X. axonopodis pv. citri population levels in Mexican lime leaf lesions reached 10(6) to 10(7) CFU lesion(-1) whatever the lesion size. These densities, however, were not strongly negatively affected by winter temperatures prevailing under experimental conditions. The estimated decrease in internal X. axonopodis pv. citri population sizes was approximately 10-fold. When exposed to 35 mm h(-1) of simulated rainfall, internal population sizes decreased over time by approximately 1 log unit for lesions 1 and 2 months old, but did not for older lesions. A microscopic examination indicated that lignin-like compounds are present in lesions more than 6 months old. The slow decrease over time of X. axonopodis pv. citri population sizes in leaf lesions may be the balanced result of defense reactions by the host at late stages of disease development, and the concomitant multiplication of the pathogen at the margin of old lesions. We conclude that the epidemiological significance of overwintered leaf lesions in the tropics is higher than that reported in other areas.     

三种PCR方法检测柑橘黄龙病菌的效果比较

为了比较常规PCR、巢式PCR和实时荧光定量PCR方法在大田检测中对柑橘黄龙病(Huanglongbing, HLB)的检测效果, 首先比较了3种检测方法对柑橘黄龙病菌检测的灵敏度, 结果发现：3种检测方法的灵敏度依次为常规PCR<巢式PCR<实时荧光定量PCR。运用3种检测方法对广东5个柑橘品种上的189个黄龙病疑似病样进行检测, 结果发现：黄龙病检出率依次为常规PCR<巢式PCR<实时荧光定量PCR。研究表明：常规PCR适合以较低成本大规模检测黄龙病; 实时荧光定量PCR具有最大的检测灵敏度; 巢式PCR检测技术同时具有前两者的一些优点, 但操作较复杂, 适合技术熟练的研究者使用。     

Narrow host range phages associated with citrus canker lesions in Florida and Argentina

Bacteriophages were isolated from naturally infected citrus canker lesions from diverse locations in Florida and Argentina and characterized for host range using a world-wide collection of Xanthomonas citri subsp. citri (Xcc) strains. Sixty-seven bacteriophages isolated from citrus canker lesions in Florida (37 bacteriophages) and Argentina (30 bacteriophages) revealed little diversity. All 30 phages isolated from four locations in Argentina had identical host ranges (group ARG), while 37 phages from Florida made up two groups (FLA and FLB). ARG and the 31 FLA phages produced clear plaques and had nearly identical host ranges as phage CP2 of Japan in that they only reacted with typical A strains and none of the atypical A strains (A^* and A^W) or other Xanthomonas spp. FLB phages had a different host range from the other strains and produced turbid plaques. We used phage typing, fatty acid analysis and riboprinter analysis to classify citrus-associated xanthomonads. Phage typing using 12 phages isolated from Xcc, X. fuscans subsp. aurantifolii (Xfa), X. alfalfae subsp. citrumelonis (Xacm), and other sources proved useful for classifying all major Xcc pathotypes and/or strains (A, A*, Miami (MI), Manatee (MA) and Wellington (A^W)), as well as B and C types of Xfa. X. citri subsp. citri strains from a worldwide collection were diverse in phage susceptibility. The majority of Xcc strains, which originated from different regions of the world and which were typical “A” pathotype strains based on pathogenicity characteristics, was sensitive to most phages (including CP2, FLA and ARG), and had nearly identical phage sensitivity profiles. MA strains were quite unique in that they reacted with none of the phages; furthermore, they were different from the putative progenitor MA strain, ATCC 49118, which reacted with a group of phages. Fatty acid analysis revealed considerable variation in Xcc-A, Xfa-B, Xfa-C and Xacm strains. Using riboprinter analysis, we identified a unique riboprinter pattern for strains isolated from an etrog tree (Citrus medica) in Florida that were “A” pathotype strains based on pathogenicity characteristics. Phage typing and fatty acid analysis were useful in corroborating that the etrog strains represent a unique new Xcc strain in Florida.     

应用Nested PCR技术检测柑桔木虱及其寄主九里香的柑桔黄龙病带菌率

应用PCR及Nested PCR技术检测柑桔木虱及其寄主九里香的结果表明:PCR只可检测最低2头带菌木虱,Nested PCR可检测到单个带菌木虱。100头带菌木虱中,单虫检出率为96％。检测田间重、中等、轻病的柑桔园内的木虱,其带菌率依次为87％、53％和21％。在病芦柑上饲菌不同天数的木虱均能检测出带菌,其饲菌时间最短为1d。城市九里香叶片及在其叶上取食的木虱单虫,均能用Nested PCR检测出病原。饲菌木虱接种九里香及芦柑健苗,在植株尚未表现症状时,常规PCR难检测出病原,但用Nested PCR则能检测到病原,说明九里香不仅是木虱的寄主,而且是黄龙病病原的隐症寄主。     

Over-expression of the Arabidopsis <Emphasis Type="BoldItalic">NPR1</Emphasis> gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.     

柑桔溃疡病内生拮抗细菌Bc51的研究

 从中国南宁柑桔叶片中分离到对柑桔溃疡病菌有拮抗作用的细菌菌株Bc51。根据16S rDNA序列同源性、形态学特征和生理生化反应,将该菌株鉴定为洋葱伯克霍尔德氏菌(Burkholderia cepacia)。温度、pH值和培养基对洋葱伯克霍尔德氏菌抑制柑桔溃疡病菌生长的能力有显著影响。在温室测试中,观察到60.3%柑桔溃疡病斑的形成受到洋葱伯克霍尔德氏菌抑制。连续8次在人工培养基上转代培养,洋葱伯克霍尔德氏菌对柑桔溃疡病菌生长的抑制力没有发生明显改变。洋葱伯克霍尔德氏菌对植物病原菌有较宽的抑菌谱,表明除柑桔溃疡病外,该菌对其它作物病害的防治也具有潜在的应用价值。     

Pathogenic Interactions Between Xanthomonas axonopodis pv. citri and Cultivars of Pummelo (Citrus grandis)

ABSTRACT The aggressiveness of strains of Xanthomonas axonopodis pv. citri on seven Citrus species, including Citrus sinensis (navel orange), C. paradisi (grapefruit), C. unshiu (Satsuma mandarin), C. junos (Yuzu), C. aurantifolia ('Mexican' lime), C. tachibana (Tachibana), and C. grandis (pummelo: 'Otachibana', 'Banpeiyu', and 'Anseikan'), were assessed by comparing lesion expansion and growth in planta, using a prick inoculation method. The existence of two groups distinct in aggressiveness was demonstrated on the pummelo cultivars, whereas the remaining species tested were uniformly susceptible. The two groups of strains were distinct in lesion expansion and growth in planta; however, both caused canker lesions on the 'Otachibana' pummelo. The sensitivity of the bacterial strains to phages Cp1 and Cp2 was associated with differences in aggressiveness. Namely, all the strains sensitive to Cp2 but resistant to Cp1 were aggressive to 'Otachibana', whereas all the strains sensitive to Cp1 but resistant to Cp2 were weakly aggressive. When a repetitive sequence-based polymerase chain reaction amplification was carried out by enterobacterial repetitive intergeneric consensus (ERIC) sequences (ERIC1R and ERIC2) as the primers, these two groups were also distinguishable by the presence or absence of a 1.8-kb DNA fragment among otherwise identical fragments. The 1.8-kb fragment was amplified only from the strains aggressive to C. grandis.     

Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay

Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real-time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S-23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real-time PCR was 10³ colony forming units per mL (cfu mL⁻¹) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.     

柑桔溃疡病菌存活期的研究

本文对鄂东南地区生态条件下柑桔溃疡病菌在不同场所的存活期进行了研究 ,并对其能否作为侵染源进行了评价。证实在病株病斑内的病菌存活时间可达一年以上 ,是此病发生最主要的侵染源。病菌在田间条件下的土壤、落叶、落果、果皮及自然水中的存活期均相当有限 ;其中以冬季病落叶中的病菌存活期最长 ,也不超过 3个月 ;故年前存在于这些场所的病菌均不能成为第 2年的初侵染源