Genetic diversity among Curtobacterium flaccumfaciens pv. flaccumfaciens populations in the American high plains

Curtobacterium flaccumfaciens pv. flaccumfaciens is a Gram-positive bacterium and has reemerged as an incitant of bacterial wilt in common (dry, edible) beans in western Nebraska, eastern Colorado, and southeastern Wyoming. Curtobacterium flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically and is represented by several different pathogen color variants. The population structure of 67 strains collected between 1957 and 2009, including some isolated from alternate hosts, was determined with 3 molecular typing techniques: amplified fragment length polymorphism (AFLP), repetitive extragenic palindromic polymerase chain reaction (rep-PCR), and pulsed-field gel electrophoresis (PFGE). All 3 typing techniques showed a great degree of population heterogeneity, but they were not congruent in cluster analysis of the C.?flaccumfaciens pv. flaccumfaciens populations. Cluster analysis of a composite data set (AFLP, PFGE, and rep-PCR) using averages from all experiments yielded 2 distinct groups: cluster A included strains with colonies of yellow, orange, and pink pigments, and cluster B had strains of only yellow pigment. Strains producing purple extracellular pigment were assigned to both clusters. Thus, C.?flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically.     

PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds

AIMS: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds. METHODS AND RESULTS: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using Cff DNA as template. Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h. CONCLUSIONS: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY:Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques.     

Methods for recovery and immunodetection of Xanthomonas campestris pv. phaseoli in navy bean seed

Non-selective enrichment procedures were evaluated for recovery of Xanthomonas campestris pv. phaseoli (fuscans strain) from artificially inoculated navy bean seed. A marked increase in recovery of the pathogen was obtained when the mixtures (bacterium plus bean seed) were suspended in Pseudomonas Agar F medium at 28°C for 48 h. Detection of this pathogen by indirect immunofluorescence microscopy (IF) and indirect enzyme-linked immunosorbent assay (ELISA) with a specific monoclonal antibody was compared. The IF system was not only more sensitive but also more reliable than ELISA for detection of the pathogen. The method is particularly useful for evaluation of the common bacterial blight status of seedlots before planting out.     

Serological and conductimetric assays for the detection of Pseudomonas syringae pathovar pisi in pea seeds

Test protocols for detecting Pseudomonas syringae pv. pisi , the causal agent of bacterial blight, in pea seeds are generally based on dilution-plating assays. These assays are usually very specific and reliable, but are time-consuming and laborious. Tests suited for large scale screening of seed lots are therefore needed. Conductimetric assays, immunofluorescence microscopy (IF) and an enzyme-linked immunosorbent assay (ELISA) for detecting Ps. syr. pv. pisi in pea seed extracts were compared with dilution-plating by two extraction methods, viz. 6 h soaking of seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Ps. syr. pv. pisi with conductimetric, IF and dilution-plating assays in the suspension water of the ground and 2 h-soaked pea samples was less sensitive than detection in suspension water of the 6 h-soaked pea seeds. The detection threshold of these assays varied per seed lot between 0 and 4.08 log cfu ml^-1 for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 4.08 and 6.08 log cfu ml^-1. Detection times recorded in conductimetric assays correlated well (— 0.89 < r < —0.98) with the log colony-forming units of Ps. syr. pv. pisi added to seed extracts at 27 as well as 17°. However, confirmation of results by isolation on semi-selective media after conductimetry was more successful at 17° than at 27°, because of the relatively lower activity of saprophytic Pseudomonas spp. at this temperature.     

Occurrence of a New Orange Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens,Causing Common Bean Wilt in Iran

A new orange variant of Curtobacterium flaccumfaciens pv. flaccumfaciens was isolated from seeds of common bean cv. Daneshkadeh and Dehghan stored in the seed banks in Khomein Bean Research Station, and field plants (cv. Local Khomein) in Arak, Iran. The pathogenicity of the isolates was confirmed on 5‐ to 7‐day‐old seedlings of cv. Daneshkadeh. Marginal necrosis and interveinal chlorosis on first trifoliate leaves were observed 10–15 days after inoculation. Amplification of 306 bp fragment of orange‐pigmented strains using CffFOR2‐ and CffREV4‐specific prime pair characterized them as C. flaccumfaciens pv. flaccumfaciens. Although the yellow‐pigmented variant of the causal agent was previously reported on cowpea, this is the first report of orange variant of C. flaccumfaciens pv. flaccumfaciens causing bacterial wilt on common bean in Iran.     

Resistance of Common Bean (Phaseolus vulgaris) to Bacterial Wilt Caused by Curtobacterium flaccumfaciens pv. flaccumfaciens

Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is an important new disease of common bean (Phaseolus vulgaris) in western Canada. Both yellow and orange variants of the pathogen were found in the region. A controlled environment study was conducted to assess 124 common bean cultivars and lines from eight market classes for resistance to the yellow and orange variants of the pathogen, using the hilum injury/seed inoculation method. Results of the screening tests showed significant (P < 0.05) differences in resistance to bacterial wilt among the cultivars or lines. The great northern line L02E317, the great northern cultivar Resolute and pinto lines L02B662 and 999S‐2A, were highly resistant to both variants of the pathogen, with disease severity indices of 0 on a rating scale of 0 (no wilt symptoms) to 5 (dead seedling). Resistant cultivars or lines were found among black, great northern, pink, pinto, small red and Flor de Mayo bean market classes. The study concludes that new bacterial wilt‐resistant germplasm exists among Canadian bean cultivars and lines, and constitutes a valuable resource for breeding common beans for resistance to both yellow and orange variants of C. flaccumfaciens pv. flaccumfaciens.     

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus

Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be studied.     

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003.Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.     

Bactericidal Efficacy of Oxidized Silver against Biofilms Formed by Curtobacterium flaccumfaciens pv. flaccumfaciens

Bacterial wilt is a re-emerging disease on dry bean and can affect many other crop species within the Fabaceae. The causal agent, Curtobacterium flaccumfaciens pv. flaccumfaciens (CFF), is a small, Gram-positive, rod-shaped bacterium that is seed-transmitted. Infections in the host become systemic, leading to wilting and economic loss. Clean seed programs and bactericidal seed treatments are two critical management tools. This study characterizes the efficacies of five bactericidal chemicals against CFF. It was hypothesized that this bacterium was capable of forming biofilms, and that the cells within biofilms would be more tolerant to bactericidal treatments. The minimum biocide eradication concentration assay protocol was used to grow CFF biofilms, expose the biofilms to bactericides, and enumerate survivors compared to a non-treated control (water). Streptomycin and oxysilver bisulfate had EC₉₅ values at the lowest concentrations and are likely the best candidates for seed treatment products for controlling seed-borne bacterial wilt of bean. The results showed that CFF formed biofilms during at least two phases of the bacterial wilt disease cycle, and the biofilms were much more difficult to eradicate than their planktonic counterparts. Overall, biofilm formation by CFF is an important part of the bacterial wilt disease cycle in dry edible bean and antibiofilm bactericides such as streptomycin and oxysilver bisulfate may be best suited for use in disease management.     

Biological control of bacterial spot of tomato caused by Xanthomonas campestris pv. vesicatoria by Rahnella aquatilis

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight ere obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.     

Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants

Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 10³ CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation.     

Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques

A polymerase chain reaction (PCR) technique was developed for detecting the presence of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight (BLB) pathogen in rice seed and for studying the transmission of this bacterium from seed to plant. Primers TXT and TXT4R from an insertion sequence (IS1113) of the pathogen were used to amplify a 964-bp DNA fragment. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed. The level of detection of TXT and TXT4R primers was 55 fg DNA of X. o. pv. oryzae, which is roughly the equivalent of seven cells (and four cells in pure culture suspension) of X. o. pv. oryzae. Hybridization of IS1113 with the amplified DNA fragment in Southern blot analysis confirmed that the 964-bp DNA fragment was amplified from X. o. pv. oryzae. The presence of the IS1113 element in strains of X. o. pv. oryzae from 16 rice-growing countries was confirmed by DNA dot blot analysis. X. o. pv. oryzae was detected from the seed washes and DNA extracted from the seed washes of naturally infected seeds of cvs Jaya and TN1. When stored at 4 degrees C, the pathogen was recovered up to 4 months and 9 months from naturally infected seeds of cvs Jaya and TN1, respectively. The BLB bacterium was also detected in seedlings, mature plants and seeds collected from plants raised from naturally infected seeds.     

Inhibition of Seed Germination and Root Development Caused by Xanthomonas campestris pv. vesicatoria in Pepper and Tomato

Inoculation of pepper seeds with the leaf pathogen Xanthomonas campestris pv. vesicatoria inhibited pepper germination. The inhibitory effect, which was stronger in non-sterilized light textured soils, decreased with time, and after 20, days or more, there was no difference between inoculated and non-inoculated seeds. Inhibitory substance(s) within the cytoplasmatic fraction of pathogen cells inhibited the germination of non-host tomato seeds. No relationship between pathogenicity to pepper leaves and inhibition of pepper seed germination was detected. The inhibitory substance(s) was found in two out of four X. campestris pv. vesicatoria strains. Heat-killed bacteria suppressed growth of pepper but not tomato seedlings. It is, therefore, suggested that the inhibition of seed germination and the decrease in root development are different modes of X. campestris pv. vesicatoria pathogenesis toward pepper plants.     

Bacterial blight pathogen concentration in cotton seeds; its role in seed quality parameters of fuzzy and acid delinted seeds

Abstract

The adverse effects of increasing concentration of Xanthomonas axonopodis pv. malvacearum on cotton seed quality parameters, seedling dry weight and on defense-related enzyme, Phenylalanine Ammonia Lyase (PAL) were studied. Different concentrations of pathogen (1×10² to 1×10⁸ CFU/ml) were treated on both fuzzy and acid delinted seeds and subjected to the standard blotter method to study the effect of the pathogen on seed quality parameters. The seedling symptom test following the roll towel method, the modified germination test to study the blight incidence and the field emergence test were carried out to discover the field planting value and disease incidence under laboratory and field conditions. From these experiments, it was found that the decrease in the seed quality parameters like seed germination, seedling vigour, and the dry weight of seedlings when X. axonopodis pv. malvacearum concentration was increased. Whereas the bacterial blight incidence increased with an increase in pathogen concentration, in both greenhouse and field conditions. Phenylalanine ammonia lyase activity was found to increase along with the pathogen concentrations, but total phenol content decreased as pathogen concentration increased. The effect of X. axonopodis pv. malvacearum load on seed quality parameters, PAL enzyme activity of fuzzy and acid delinted cotton seeds are discussed in the present study.     

Development of a PCR test for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens

A chromosomal DNA library of the bacterial pathogen of bean, Curtobacterium flaccumfaciens pv. flaccumfaciens NCPPB 559 was constructed in the plasmid pGEM-7Zf(+). Several clones were identified that hybridised to all Curtobacterium flaccumfaciens pathovars including: C. f. betae, C. f. flaccumfaciens, C. f. oortii, C. f. poinsettiae and, in addition, to some strains of Clavibacter michiganensis subsp. insidiosus and Clavibacter michiganensis subsp. One of these clones (pPMP-26), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 0.2 Kb (pPMP-26D) that hybridised specifically to C. f. flaccumfaciens and not to any of the other plant pathogenic members of the order Actinomycetales or any of the other prokaryotic bean pathogens tested. This fragment was subcloned and sequenced, analysis of the resultant 198 bp sequence showed that no significant homology existed with any other sequence currently deposited in public databases. Further analysis of these data facilitated the design of PCR primers which were subsequently tested against a wide range of plant pathogenic actinomycetes and other prokaryotic bean pathogens. Results show that these primers are highly specific for all strains of C. f. flaccumfaciens with no cross-reaction to strains from any other bacterial taxa tested.     

Detection of Xanthomonas arboricola pv. pruni by PCR using primers based on DNA sequences related to the hrp genes

Efficient control of Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot on stone fruit, requires a sensitive and reliable diagnostic tool. A PCR detection method that utilizes primers to target the hrp gene cluster region was developed in this study. The nucleotide sequence of the PCR product amplified with primers specific for the hrp region of the xanthomonads and genomic DNA of X. arboricola pv. pruni was determined, and the sequence was aligned with that of X. campestris pv. campestris, which was obtained from the GenBank database. On the basis of the sequence of the amplified hrp region, a PCR primer set of XapF/R specific to X. arboricola pv. pruni was designed. This primer set yielded a 243-bp product from the genomic DNA of X. aboricola pv. pruni strains, but no products from other 21 strains of Xanthomonas or from two epiphytic bacterial species. Southern blot hybridization with the probe derived from the PCR product with the primer set and X. aboricola pv. pruni DNA confirmed the PCR results. The Xap primer system was successfully applied to detect the pathogen from infected peach fruits. When it was applied in field samples, the primer set was proved as a reliable diagnostic tool for specific detection of X. aboricola pv. pruni from peach orchards.     

Detached leaf enrichment: a method for detecting small numbers of Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria in seed and symptomless leaves of tomato and pepper

A method for detecting 10¹-10² cells of phytopathogenic bacteria ( Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria ) in either tomato or pepper seed was developed. The method is based on the enrichment of the compatible pathogen inside a detached leaf of its host when placed on a water agar medium. It was found to be superior to the diagnostic growth media method commonly used and to permit the detection of the pathogens in symptomless plants.     

植物病原棒形细菌的RAPD分析

采用RAPD分析技术对萎蔫短小杆菌落葵致病变种（Curtobacterium flaccumfaciens pv. basellae pv. Nov.）和萎蔫短小杆菌糖甜菜致病变种（Curtobacterium flaccumfaciens pv. beticola pv. Nov.）及植物病原棒形细菌4个属的15个菌株进行分类研究：从50条随机引物中筛选出20条,共产生225条带,多态性条带占804%。遗传相似矩阵及UPGMA聚类分析,表明这两个新致病变种与萎蔫短小杆菌属亲缘关系近,最小相似系数为0.6511;与其他属细菌亲缘关系较远;结合前人研究结果对植物病原棒形细菌新近提出的分类地位的改变进行了探讨。     

Detection of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in seeds using a specific TaqMan probe

Xanthomonas oryzae pv. oryzae is the pathogen that causes bacterial leaf blight in rice. Bacterial leaf blight is the main cause for severe rice underproduction in many countries. However, with conventional methods it is difficult to quickly and reliably distinguish this pathogen from other closely related pathogenic bacteria, especially X. oryzae pv. oryzicola, the causal organism of bacterial leaf streak in rice. We have developed a novel and highly sensitive real-time method for the identification of this specific bacteria based on a TaqMan probe. This probe is designed to recognize the sequence of a putative siderophore receptor gene cds specific to X. oryzae pv. oryzae, and can be identified from either a bacterial culture or naturally infected rice seeds and leaves in only 2 h. The sensitivity of the method is 100 times higher than that of the current polymerase chain reaction (PCR) gel electrophoresis method for diagnosis.     

Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method.

pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.