Seed-borne Bacterium Research Articles

Acidovorax citrulli type III effector AopU interferes with plant immune responses and interacts with a watermelon E3 ubiquitin ligase.

Acidovorax citrulli is a seed-borne bacterium that causes bacterial fruit blotch of watermelon and other cucurbit plants worldwide. It uses a type III secretion system to inject type III effectors (T3Es) into plant cells, which affect the host immune responses and facilitate pathogen colonization. However, the current understanding of the specific molecular mechanisms and targets of these effectors in A. citrulli is limited. In this study, we characterized a novel T3E called AopU in A. citrulli group II strain Aac5, which shares homology with XopU in Xanthomonas oryzae. The Agrobacterium-mediated gene transient expression system was used to study the effect of AopU on host immunity. The results showed that AopU localized on the cell membrane and nucleus of Nicotiana benthamiana, inhibited reactive oxygen species burst induced by flg22 and the expression of marker genes associated with pathogen-associated molecular pattern-triggered immunity, but activated salicylic acid and jasmonic acid signal pathways. Further investigations revealed that AopU interacts with E3 ubiquitin ligase ClE3R in watermelon, both in vitro and in vivo. Interestingly, the deletion of aopU did not affect the virulence of A. citrulli, suggesting that AopU may have functional redundancy with other effectors in terms of its role in virulence. Collectively, these findings provide new insights into the mechanism of plant immune responses regulated by A. citrulli T3Es.

Combined effect of thyme and clove phenolic compounds on Xanthomonas campestris pv. campestris and biocontrol of black rot disease on cabbage seeds.

The seed-borne bacterium Xanthomonas campestris pv. campestris (Xcc) as a causal organism of black rot disease remains the most serious bacterial problem of agricultural production of cruciferous plants worldwide. The eradication of a primary inoculum originating in seeds is available, but no treatment is totally effective. With the threat of developing chemical resistance and increasing pressure for sustainable disease management, biocontrol methods represent one of the main strategies currently applied in agriculture. Natural antimicrobials, including essential oils, are promising tools in disease management with low risks of environmental pollution and impact on human health. Thyme and clove essential oils were demonstrated to be highly effective in Xanthomonas studies in vitro; therefore, their application in black rot control was evaluated in this study. From five phenolic substances originating from thyme and clove essential oils (carvacrol, eugenol, linalool, p-cymene and thymol), the most promising in vitro results were observed with carvacrol, for which 0.0195% led to the death of all Xcc cells in 30 min. Moreover, a synergistic antibacterial effect of carvacrol and thymol solutions decreased the minimal inhibition concentration to 0.0049% and 0.0195% for carvacrol and thymol, respectively. Using the quadruple bactericidal values, the complete elimination of Xcc from the surface of infested cabbage seeds was obtained for both carvacrol and thymol solutions and their combined mixture at 2 MIC value. The elimination of bacterial infection from germinated cabbage plants was observed for both plate counting and quantitative real-time PCR methods. We also evaluated the effect of the application of phenolic treatment on the seed germination and germinated plants. Our results suggest a high potential of the application of carvacrol and thymol in vegetable seed production, specifically for cabbage, thus representing a suitable alternative to cupric derivatives.

A Seed-Borne Bacterium of Rice, Pantoea dispersa BB1, Protects Rice from the Seedling Rot Caused by the Bacterial Pathogen Burkholderia glumae.

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot.

Performance of Solanum phureja-derived bacterial-wilt resistant potato clones in a field naturally infested with Ralstonia solanacearum in Central Brazil

ABSTRACT Bacterial wilt (BW), or brown rot, caused by the soil and seed borne bacterium Ralstonia solanacearum, is one of the most devastating diseases of potatoes cultivated in warmer regions of the world. There are no potato cultivars with a desirable level of BW resistance, although it has been recognized that resistance can be an outstanding component for disease management. However, the sources of resistance available lack agronomic traits required by potato growers, therefore being of little interest to breeders. The objective of this work was to evaluate the performance of 11 clones selected for BW resistance and improved for tuber traits upon selection in the last two decades. The clones under test were compared with susceptible and resistant clones and cultivars, in a completely randomized blocks design with three replications of single lines of 10 plants, in a field naturally infested with race 1, biovar 1, phylotype II of R. solanacearum. BW incidence was assessed 60-70 days after planting and total tuber yield in each plot was recorded 110 days after planting. All the evaluated clones presented higher levels of resistance to BW compared with the commercial varieties, not differing from the resistant, not commercial, controls. In a next step, these clones will be characterized for other desirable traits and those which combine high level of resistance and commercial characteristics will be recommended for breeders for enriching the genotypic background in the search for commercial varieties. We also confirmed that the cultivar BRSIPR Bel displays an intermediate level of resistance, what makes it an interesting genitor for its good agronomic characteristics. The findings of this work demonstrate that the improved potato clones selected under tropical conditions in the Embrapa’s pre-breeding project possess high and stable levels of resistance to bacterial wilt, being a valuable resource for breeders.

Pantoea agglomerans, a seed-borne plant pathogenic bacterium, decreased seed germination, seedling growth and seed quality of oat

Pantoea agglomerans, a seed-borne plant pathogenic bacterium, decreased seed germination, seedling growth and seed quality of oat

QTLs for potato tuber resistance to Dickeya solani are located on chromosomes II and IV

AbstractThe goal of this research was to identify quantitative trait loci (QTLs) for potato tuber resistance to the soil‐ and seedborne bacterium Dickeya solani and for tuber starch content, to study the relationship between these traits. A resistant diploid hybrid of potato, DG 00‐270, was crossed with a susceptible hybrid, DG 08‐305, to generate the F1 mapping population. Tubers that were wound‐inoculated with bacteria were evaluated for disease severity, expressed as the mean weight of rotted tubers, and disease incidence, measured as the proportion of rotten tubers. Diversity array technology (DArTseq) was used for genetic map construction and QTL analysis. The most prominent QTLs for disease severity and incidence were identified in overlapping regions on potato chromosome IV and explained 22.4% and 22.9% of the phenotypic variance, respectively. The second QTL for disease severity was mapped to chromosome II and explained 16.5% of the variance. QTLs for starch content were detected on chromosomes III, V, VI, VII, VIII, IX, XI, and XII in regions different from the QTLs for soft rot resistance. Two strong and reproducible QTLs for resistance to D. solani on potato chromosomes IV and II might be useful for further study of candidate genes and marker development in potato breeding programmes. The relationship between tuber resistance to bacteria and the starch content in potato tubers was not confirmed by QTL mapping, which makes the selection of genotypes highly resistant to soft rot with a desirable starch content feasible.

Characterization of Xanthomonas campestris pv. campestris in Algeria

Xanthomonas campestris pv. campestris (Xcc) causes the black rot of cruciferous plants. This seed-borne bacterium is considered as the most destructive disease to cruciferous crops. Although sources of contamination are various, seeds are the main source of transmission. Typical symptoms of black rot were first observed in 2011 on cabbage and cauliflower fields in the main production areas of Algeria. Leaf samples displaying typical symptoms were collected during 2011 to 2014, and 170 strains were isolated from 45 commercial fields. Xcc isolates were very homogeneous in morphological, physiological and biochemical characteristics similar to reference strains, and gave positive pathogenicity and molecular test results (multiplex PCR with specific primers). This is the first record of Xcc in Algeria. Genetic diversity within the isolates was assessed in comparison with strains isolated elsewhere. A multilocus sequence analysis based on two housekeeping genes (gyrB and rpoD) was carried out on 77 strains representative isolates. The isolates grouped into 20 haplotypes defined with 68 polymorphic sites. The phylogenetic tree obtained showed that Xcc is in two groups, and all Algerian strains clustered in group 1 in three subgroups. No relationships were detected between haplotypes and the origins of the seed lots, the varieties of host cabbage, the years of isolation and agroclimatic regions.

Double antibody pairs sandwich-ELISA (DAPS-ELISA) detects Acidovorax citrulli serotypes with broad coverage.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called “double antibody pairs sandwich ELISA” (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.

Characterization of plant growth-promoting traits and antagonistic potentials of endophytic bacteria from bean plants against Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola (Psp) is a seed-borne bacterium which causes halo blight disease in common bean. This study aimed the determination of plant growth-promoting traits (such as IAA, phosphate solubilization) and antagonistic potentials (such as siderophore and ammonia productions) of endophytic plant growth-promoting bacteria (PGPB) from healthy bean plants growing in different regions against Psp in vitro conditions. A total of 131 bacteria were primarily characterized as putative PGPB and tested for antagonist activity against Psp in dual culture tests. Seventy-one bacterial isolates demonstrated antagonistic activities against Psp isolate in varying ratios. Among these 71 isolates, 30 representative bacterial isolates from the different regions/fields were selected. On the basis of morphological, physiological, biochemical characteristics and confirmation by MALDI-TOF analyses, 30 endophytic antagonist isolates were identified as belonging to 10 genera, 24 different species. According to results obtained, 10 isolates belong to Bacillus spp., 6 isolates belong to Pseudomonas spp., 4 isolates belong to Rhizobium radiobacter, 2 isolates belong to Arthrobacter spp., 2 isolates belong to Achromobacter spanius, 2 isolates belong to Serratia liquefaciens, 1 isolate belongs to Acinetobacter calcoaceticus, Exiguobacterium sp., Microbacterium hydrocarbonoxydans, and Ochrobactrum anthropi. The largest and lowest inhibition zone was produced by endophytic bacterial isolates Pseudomonas gessardii (4.85) and Bacillus licheniformis (1.35). Among the tested antagonist bacterial isolates, 10 isolates were positive for the production of α-amylase, 7 isolates positive for phosphate solubilization, 29 isolates positive for siderophore production, 11 isolates positive for protease production. All selected bacterial isolates produced IAA and ammonia in relatively varying amounts. P. gessardii produced a relatively large amount of extracellular siderophore (5.83), Exiguobacterium sp. produced a relatively large amount of extracellular protease (5.25), P. gessardii and O. anthropi produced a relatively large amount of extracellular IAA (161.39 µg/ml) and Acinetobacter calcoaceticus produced a relatively large amount of phosphatase (2.63). This is the first study reporting bean plants harbor endophytes having plant growth promoting activities with antagonistic potential against Psp.

Detection of Xanthomonas campestris pv. campestris through a real-time PCR assay targeting the Zur gene and comparison with detection targeting the hrpF gene

Xanthomonas campestris pv. campestris (Xcc) is a seedborne bacterium that causes black rot of crucifers. A real-time PCR assay based on a dual-labeled hydrolysis TaqMan® probe has been developed for the rapid and sensitive detection of Xcc and related pathovars that affect mainly Brassicaceae crops and ornamentals. Primers were designed to specifically amplify a 152 bp fragment of the Zur gene from X. campestris. To confirm the specificity of the detection, primers targeting the Zur and hrpF genes were used for standard and real-time PCR with DNA samples from 13 Xcc strains, seven Xanthomonas species and pathovars and five different bacterial endophytes including Bacillus, Erwinia, Klebsiella, Pantoea and Pseudomonas, previously isolated from tissues of crucifers. PCR products amplified with Zur and hrpF primers were sequenced to assess the genetic diversity of these genes in the tested isolates. The real-time PCR protocol was optimized to allow the detection at the level of ten copies of Zur PCR fragment per one microliter of DNA. Although the real-time based on detection of Zur also detected X. campestris pvs raphani, armoraciae, incanae and a strain of X. hortorum pv. carotae, it improved the specificity in relation to the previously published hrpF based real-time method. A multiplex assay for Zur and hrpF genes further improved the specificity by excluding X. hortorum pv. carotae. Tests of brassica tissues and seeds artificially inoculated with Xcc showed that the real-time PCR based on detection of Zur is an efficient and robust assay.

Pest categorisation of Curtobacterium flaccumfaciens pv. flaccumfaciens.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of the seed‐borne bacterium Curtobacterium flaccumfaciens pv. flaccumfaciens. The pest is regulated in Council Directive 2000/29/EC (Annex IIB) as a harmful organism whose introduction into, and spread within, the protected zones (PZ) of Greece, Portugal and Spain shall be banned if present on seeds of Phaseolus vulgaris and of Dolichos. The bacterium is widely distributed outside the EU and causes a systemic vascular disease (bacterial wilt of bean) as well as bacterial tan spot disease on soybean. The pest was sporadically recorded in several EU Member States in the past, but is currently not known to occur in the EU. The identity of the bacterium is well established and identification methods are available. The major host is common bean (Phaseolus vulgaris), but other crops and weeds are, or may be, hosts or play a role as reservoirs, with uncertainties. Seed transmission remains uncertain for minor and alternative host species. The main pathway for entry is seed. The role of other pathways (e.g. irrigation water and infected residues) is uncertain. Should the bacterium enter the EU (including the PZ), it may establish, spread and have an impact on its host crops. The use of healthy seeds is the most effective control measure. Curtobacterium flaccumfaciens pv. flaccumfaciens fits all the criteria assessed by EFSA to be regarded as a Union quarantine pest.

Seed-borne bacterium interacts with air-borne fungus in rice fields

Seed-borne bacterium interacts with air-borne fungus in rice fields

Strains of the Group I Lineage of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops, are Predominant in Brazil.

Bacterial fruit blotch (BFB), caused by the seedborne bacterium Acidovorax citrulli, is an economically important threat to cucurbitaceous crops worldwide. Since the first report of BFB in Brazil in 1990, outbreaks have occurred sporadically on watermelon and, more frequently, on melon, resulting in significant yield losses. At present, the genetic diversity and the population structure of A. citrulli strains in Brazil remain unclear. A collection of 74 A. citrulli strains isolated from naturally infected tissues of different cucurbit hosts in Brazil between 2000 and 2014 and 18 A. citrulli reference strains from other countries were compared by pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes, and pathogenicity tests on seedlings of different cucurbit species. The Brazilian population comprised predominantly group I strains (98%), regardless of the year of isolation, geographical region, or host. Whole-genome restriction digestion and PFGE analysis revealed that three unique and previously unreported A. citrulli haplotypes (assigned as haplotypes B22, B23, and B24) occurred in Brazil. The greatest diversity of A. citrulli (four haplotypes) was found among strains collected from the northeastern region of Brazil, which accounts for more than 90% of the country's melon production. MLSA clearly distinguished A. citrulli strains into two well-supported clades, in agreement with observations based on PFGE analysis. Five Brazilian A. citrulli strains, representing different group I haplotypes, were moderately aggressive on watermelon seedlings compared with four group II strains that were highly aggressive. In contrast, no significant differences in BFB severity were observed between group I and II A. citrulli strains on melon and squash seedlings. Finally, we observed a differential effect of temperature on in vitro growth of representative group I and II A. citrulli haplotypes. Specifically, of 18 group II strains tested, all grew at 40 and 41°C, whereas only 3 of 15 group I strains (haplotypes B8[P], B3[K], and B15) grew at 40°C. Three strains representing haplotype B8(P) were the only group I strains that grew at 41°C. These results contribute to a better understanding of the genetic diversity of A. citrulli associated with BFB outbreaks in Brazil, and reinforce the efficiency of MLSA and PFGE analysis for assessing population structure. This study also provides the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations.

Insights from the Genome Sequence of Acidovorax citrulli M6, a Group I Strain of the Causal Agent of Bacterial Fruit Blotch of Cucurbits.

Acidovorax citrulli is a seedborne bacterium that causes bacterial fruit blotch of cucurbit plants including watermelon and melon. A. citrulli strains can be divided into two major groups based on DNA fingerprint analyses and biochemical properties. Group I strains have been generally isolated from non-watermelon cucurbits, while group II strains are closely associated with watermelon. In the present study, we report the genome sequence of M6, a group I model A. citrulli strain, isolated from melon. We used comparative genome analysis to investigate differences between the genome of strain M6 and the genome of the group II model strain AAC00-1. The draft genome sequence of A. citrulli M6 harbors 139 contigs, with an overall approximate size of 4.85 Mb. The genome of M6 is ∼500 Kb shorter than that of strain AAC00-1. Comparative analysis revealed that this size difference is mainly explained by eight fragments, ranging from ∼35–120 Kb and distributed throughout the AAC00-1 genome, which are absent in the M6 genome. In agreement with this finding, while AAC00-1 was found to possess 532 open reading frames (ORFs) that are absent in strain M6, only 123 ORFs in M6 were absent in AAC00-1. Most of these M6 ORFs are hypothetical proteins and most of them were also detected in two group I strains that were recently sequenced, tw6 and pslb65. Further analyses by PCR assays and coverage analyses with other A. citrulli strains support the notion that some of these fragments or significant portions of them are discriminative between groups I and II strains of A. citrulli. Moreover, GC content, effective number of codon values and cluster of orthologs’ analyses indicate that these fragments were introduced into group II strains by horizontal gene transfer events. Our study reports the genome sequence of a model group I strain of A. citrulli, one of the most important pathogens of cucurbits. It also provides the first comprehensive comparison at the genomic level between the two major groups of strains of this pathogen.

Effects of nitrogen nutrition on disease development caused by Acidovorax citrulli on melon foliage

Bacterial fruit blotch (BFB) of cucurbits, caused by the seed-borne bacterium Acidovorax citrulli, is a destructive disease that threatens the melon and watermelon industries worldwide. The available means to manage the disease are very limited and there are no reliable sources of BFB resistance. Mineral nutrition has marked effects on plant diseases. To the best of our knowledge, no studies reporting effects of mineral nutrition on BFB severity have been reported to date. In the present study we assessed the influence of nitrogen nutrition on BFB severity and A. citrulli establishment in the foliage of melon plants under greenhouse conditions. Our results show that nitrogen fertilization, based on nitrate only, led to reduced disease severity and bacterial numbers in melon leaves, as compared with two combinations of nitrate and ammonium. No consistent effect of nitrogen nutrition on expression of several plant defense-associated transcripts was found, except for hydroperoxide lyase (HPL), which upon inoculation was repressed to a greater extent under the “nitrate-only” nitrogen regime compared with combined nitrate and ammonium. Reducing BFB severity and A. citrulli establishment in the plant foliage are of particular importance since establishment of the pathogen during the growing season is assumed to increase the incidence of fruit infection, leading to serious yield losses. Further research is needed to elucidate the mechanisms by which nitrogen nutrition influences BFB development, and to assess the effects of nitrogen as well as other minerals on the disease under field conditions.

Development of a new technique to detect living cells of Xanthomonas campestris pv. campestris in crucifers seeds: the seed-qPCR

Xanthomonas campestris pv. campestris is a seed-borne bacterium that causes black rot on Brassicaceae. Ensuring seed lot sanitary quality is the most efficient control strategy against bacterial diseases. Currently, the procedures adopted in the control of seed lots are mainly based on microbiological techniques combined with PCR or plant inoculation which is time and money consuming. The aim of this study was to propose a reliable and rapid detection technique of living X. c. pv. campestris in cabbage seeds. We have shown that not all cells of X. campestris is able to grown on rich medium after washing and soaking seeds as no colony of X. campestris was detected on inoculated seeds, whereas plantlets develop symptoms 7–14 days after germination. The PCR technique used alone does not address the viability of bacteria in samples. We set up a technique named seed-qPCR for the detection of living X. c. pv. campestris bacterial cells in seed lots. This technique is based on an enrichment of bacterial population associated with infected seeds by seed germination coupled with real-time Taq-man PCR after extraction of the target DNA. It is an inexpensive technique that allow the detection of down to 1 contaminated seed among 10 000 healthy seeds. The seed-qPCR method combines an efficient extraction based on bacterial multiplication on seedlings with a sensitive technique qPCR for the detection of bacteria in seed lots.

Detection of Xanthomonas axonopodis pv. vignicola, causal agent of bacterial blight of cowpea in seeds by non-serological methods

Among the diseases infecting cowpea, bacterial blight caused by Xanthomonas axonopodis pv. vignicola (Burkholder,1944). Vauterin et al . (1995) is a major production constraint. First necrotic lesions are formed on leaves and later the stem is attacked and the pathogen reaches vascular bundles and the disease becomes systemic. In the present study, attempted have been made to develop the suitable methods for the detection of the pathogen in the seeds and to find out the nature of transmission using selective and semi-selective media and compare them for their efficacy. Results indicated NSCAA medium to be more efficient in recovering the colonies of seed borne bacterium Xanthomonas axonopodis pv. vignicola with 13 2 ×10 5 cfu/ml as compared to 75×10 5 cfu/ml of colonies on Nutrient agar. The next best medium was SIBU agar (126×10 5 cfu/ml) followed by XTS medium (118×10 5 cfu/ml). All the media for isolation of plant pathogenic bacterium from seeds revealed that the cowpea bacterium is seed borne in nature. Another method employed to detect pathogen in seeds is Van Vuurde et al.(1983) method and results revealed that unsterilized seeds of susceptible cultivar C-152 yielded more number of colonies 35×10 2 cfu/ml by direct plating the seed extract on NSCAA whereas the resistant germplasm, DCS 47-1 yielded very less number of colonies 2×10 2 from diseased unsterilized seeds confirming its seed borne nature.

Draft Genome Sequence of Pseudomonas putida Strain S610, a Seed-Borne Bacterium of Wheat

We report the genome sequence of a seed-borne bacterium, Pseudomonas putida strain S610. The size of the draft genome sequence is approximately 4.6 Mb, which is the smallest among all P. putida strains sequenced to date.

Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed.

A novel monoclonal antibody (MAb) specific to the seedborne bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comamonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 104 CFU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection of A. citrulli in cucurbits and can be applied for routine field inspection.

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli ( Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli ( Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10 6 cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5 × 10 5 cfu/ml. The LOD for the ELISA technique was 5 × 10 4 cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.