Non-destructive methods of detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in mungbean seeds

An indirect immunofluorescent assay (IF) with specific monoclonal antibodies (MAbs) and a semi-selective agar medium for Curtobacterium flaccumfaciens pv. flaccumfaciens , developed in this study, were compared with foliar symptoms and microscopic bacterial ooze for detection of this pathogen in mungbean seed 6 d and 28 d after germination. The IF method detected more infected seedlings than the other three methods at both samplings. Symptomless carriers of C. flaccumfaciens pv. flaccumfaciens in mungbean were detectable only by the IF method and, less frequently, by plating out on media. Poor agreement between the IF and other methods was found. The IF method gave the best agreement in the detection of the pathogen between early and late samplings of individual germinated seed. Currently, the IF technique with a specific MAb is being used for selecting clean seedlings for production of disease-free seed.     

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.     

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.     

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.     

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Cowpea Bacterial Wilt in Iran

During surveys in cowpea fields of Marand County, East Azerbaijan province, Iran, in the summer of 2013, a suspected bacterial disease was observed on cowpea leaves as tan spots and interveinal necrotic lesions surrounded by chlorotic margins. The disease was of high incidence where some fields had been fully destroyed and severity of the disease in some fields had reached up to 70%. Gram‐positive, yellow‐pigmented, coryneform bacteria were isolated from infected leaves. Pathogenicity of isolates was confirmed on 20‐day‐old cowpea (cv. Khoy) plants, and they were identified as Curtobacterium flaccumfaciens pv. flaccumfaciens based on biochemical test results confirmed using specific PCR primers. This is the first report of C. flaccumfaciens pv. flaccumfaciens, the causal agent of cowpea bacterial wilt in Iran.     

Curtobacterium flaccumfaciens pv. flaccumfaciens on Soybean in Germany – A Threat for Farming

Plant diseases caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are distributed in North and South America as well as in South and East Europe and occur mostly on beans (Phaseolus vulgaris). This is the first report of Cff on soybean in Germany. Cff was detected in complex with Pseudomonas syringae pv. glycinea on field‐grown soybeans that were not treated with pesticides. Cff, the causal agent of bacterial tan spot disease, was identified by 16S rDNA sequencing and by artificial infection and re‐isolation from the host plants soybean (Glycine max) and bush bean (Phaseolus vulgaris).     

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.     

Curtobacterium spp. and Curtobacterium flaccumfaciens: Phylogeny,Genomics-Based Taxonomy,Pathogenicity, and Diagnostics

The genus of Curtobacterium, belonging to the Microbacteriaceae family of the Actinomycetales order, includes economically significant pathogenic bacteria of soybeans and other agricultural crops. Thorough phylogenetic and full-genome analysis using the latest genomic data has demonstrated a complex and contradictory taxonomic picture within the group of organisms classified as the Curtobacterium species. Based on these data, it is possible to delineate about 50 new species and to reclassify a substantial part of the Curtobacterium strains. It is suggested that 53 strains, including most of the Curtobacterium flaccumfaciens pathovars, can compose a monophyletic group classified as C. flaccumfaciens. A genomic analysis using the most recent inventory of bacterial chromosomal and plasmid genomes deposited to GenBank confirmed the possible role of Microbacteriaceae plasmids in pathogenicity and demonstrated the existence of a group of related plasmids carrying virulence factors and possessing a gene distantly related to DNA polymerase found in bacteriophages and archaeal and eukaryotic viruses. A PCR diagnostic assay specific to the genus Curtobacterium was developed and tested. The presented results assist in the understanding of the evolutionary relations within the genus and can lay the foundation for further taxonomic updates.     

A simple and efficient PCR method for the specific detection of Pseudomonas syringae pv phaseolicola in bean seeds

Using Southern blot hybridizations, it was found that the gene encoding the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal agent of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolicola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thus avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi-selective media for 18h prior to PCR amplification. The detection threshold by visual detection using ethidium bromide staining was one naturally infected seed in lots of 400 to 600 seeds.     

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.     

甜菜银叶病菌的PCR检测

本研究用16S23S rDNA间的ITS 序列通用引物L1（5′AGTCGTAACAAGGTAGCCGT3′）和L2 （5′ GTGCCAAGGCATCCACC3）扩增甜菜银叶病菌（Curtobacterium flaccumfaciens pv. betae,Cfb）和其它相近细菌的基因组DNA;并对其PCR产物进行回收、克隆和测序,将所获序列和其它已报道的细菌内源转录间隔区(Internally Transcribed Spacer,ITS)序列进行多重比较后设计出Cfb的特异性引物B1（5′GGCCTCGTGTTGTCCCTTATC3′）和B2 （5′GTCACCAATCAACAACCCGAG3′）。此引物可以从Cfb中扩增出387bp 的特异性片段,而其余参试的21个细菌PCR反应结果均为阴性。该方法可以应用于病害防治工作中的Cfb快速、可靠的检测。     

PCR-based detection of Phomopsis azadirachtae in die-back affected neem seeds

Abstract

Phomopsis azadirachtae Sateesh, Bhat & Devaki is the incitant of die-back disease of neem trees. Delayed appearance of conidia and presence of other microorganisms in the neem tissues are the obstacles in the rapid and accurate identification of P. azadirachtae. This work was carried out to develop a methodology for rapid detection of the pathogen in diseased tissues especially in the neem seeds. rDNA sequences of many Phomopsis spp. were retrieved from the database and were subjected for multiple alignment to select a 179 bp conserved sequence. This was used to design Phomopsis specific primer pair (Forward and Reverse) having the potential to produce a 154 bp product in PCR. The primer pair was utilised to detect the presence of P. azadirachtae in diseased neem seeds and other tissues. This is the first report on the PCR-based detection of P. azadirachtae directly in die-back diseased neem tissues. This method can be employed for rapid and reliable detection of P. azadirachtae in die-back affected neem seeds. Hence it will have very good application in seed health testing laboratories.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef.

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0. The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.     

PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli

A PCR-based method was developed for the specific detection of Xanthomonas campestris pv. phaseoli var. fuscans from plant material. Primers Xf1 and Xf2, based on a sequence conserved amplified region (SCAR) derived from RAPD PCR analysis of X. c. pv. phaseoli var. fuscans , amplified a DNA fragment of 450 bp from all such isolates. In contrast, no amplification product was obtained from any X. c. pv. phaseoli isolates, or from any other DNAs tested. As few as 10 cells of X. c . pv. phaseoli var. fuscans (equivalent to about 100 fg DNA) could be detected in vitro . In planta , following an initial inoculation of as little as one cell, an amplification product was generated after only 2 d of incubation, allowing highly sensitive detection 10 d before disease symptoms were observed. Moreover, the failure to amplify DNA from X. c . pv. phaseoli isolates shows that these primers provide a rapid, improved method to differentiate these two varieties using PCR.     

Introduction of a validation concept for a PCR-based Mycoplasma detection assay

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.     

PCR-based assay for the detection of Xanthomonas euvesicatoria causing pepper and tomato bacterial spot

Aims:  To develop a PCR-based assay for Xanthomonas euvesicatoria detection in culture and in planta .
Methods and Results:  A fragment of 1600 bp specific for X. euvesicatoria was found by repetitive extragenic palindromic sequence-PCR. Among the primers designed on the basis of the partially sequenced fragment, the primers Xeu2.4 and Xeu2.5 direct amplification of the expected product (208 bp) for all the X. euvesicatoria strains and not for other related and unrelated phytopathogenic bacteria or saprophytic bacteria isolated from pepper and tomato phyllosphere. The assay permits the detection of X. euvesicatoria in pure culture, with a limit of detection of two bacterial cells and 1 pg of DNA per PCR, and in extracts obtained from asymptomatic inoculated tomato and pepper plants.
Conclusions:  Primers Xeu2.4 and Xeu2.5 provide a specific, sensitive and rapid assay for the detection of X. euvesicatoria in culture and in pepper and tomato plants.
Significance and Impact of the Study:  Because X. euvesicatoria is a quarantine organism in the European Union, and it is subjected to stringent international phytosanitary measures, this highly sensitivity PCR-based assay is suitable for its detection in pepper and tomato plant materials to avoid the introduction and spread of the bacterium.     

Development of a PCR-based assay for rapid detection of class IIa bacteriocin genes

Aims: We have developed a PCR‐based assay using custom designed panel of primers which allows rapid detection of class IIa bacteriocin‐coding genes. To demonstrate the applicability of the developed assay, the method was applied on 40 metagenomic DNA preparations isolated from native microbiota of Polish artisanal cheeses produced in the Tatra Mountains. Methods and Results: The developed assay was designed on the basis of a large scale alignment of class IIa bacteriocin‐coding genes. A panel of seven primer pairs with confirmed ability to detect class IIa bacteriocin‐coding sequences was obtained. The following study has revealed a superb bacteriocinogenic potential of all forty analysed cheese samples. Conclusions: The majority of obtained sequences were lactic acid bacteria (LAB) related, although some sequences showed significant similarity to bacteriocin‐coding sequences present in non‐LAB bacteriocin producers. The results suggest that several potentially new bacteriocin‐coding sequences were found. Significance and Impact of the Study: The developed assay can be extremely helpful in establishing whether isolates from the environment of interest have a potential of synthesizing antilisterial class IIa bacteriocins. Application of the approach may represent a useful tool contributing to ecological studies looking for valuable probiotic, bacteriocinogenic microbiota developing in foods.