Characterization of isolates that cause black rot of crucifers in East Africa

A study was conducted in the East African countries of Kenya, Tanzania and Uganda in the months of July and August 2009 with the objectives of assessing the status of black rot and race structure of Xanthomonas campestris pv. campestris in the three countries. Samples infected with black rot were collected from farmers’ fields mainly from Brassica oleracea crops (broccoli, cabbage, cauliflower and kales). A total of 399 farms were surveyed of which 260 were from Kenya, 91 from Tanzania and 48 from Uganda. Following successful isolations, a total of 249 isolates of the causal agent, Xanthomonas campestris pv. campestris were recovered. Pathogenicity of all isolates was confirmed on B. oleracea susceptible cultivars Copenhagen Market F1 and Wirosa F1. Sixty of the 250 isolates were race-typed using a differential set Brassica spp. Only two races, 1 (Kenya and Tanzania) and 4 (Kenya, Tanzania and Uganda) were observed however, another race (5) was observed from one isolate recovered from a B. rapa sample obtained from Tanzania in 2003. Genomic fingerprinting with repetitive-PCR revealed clusters that did not depict significant correlations between isolates and geographical location, isolates and host adaptation or isolates and race. However, it did demonstrate existence of genetic differences within the East African X. campestris pv. campestris population indicating that it is not a similar clonal population of the same genetic background.     

Genetic fingerprinting of Iranian Xanthomonas campestris pv. campestris strains inducing black rot disease of crucifers

Northern Iran has one of the largest and most diverse populations of cultivated crucifers in Iran. Symptoms of black rot disease were observed in 40 % of fields. To assess the genetic diversity of Xanthomonas campestris pv. campestris (Xcc) strains, associated with black rot disease, 40 strains were isolated from infected samples of crucifers such as cabbage, radish, cauliflower, turnip and kohlrabi, and were collected from different geographic regions of northern Iran including West and East Azarbayjan and Ardabil provinces. Bacterial strains were characterized by their morphological, biochemical and physiological features and pathogenicity tests. Four races were found in northern Iran (1, 4, 5 and 6) and the majority of the tested strains belonged to either race 4 (45 %) or race 6 (20 %). To examine the distribution of dispersed repetitive DNA, Enterobacterial Repetitive Intergenic Consensus (ERIC), BOX, Repetitive Extragenic Palindromic (REP) and random amplified polymorphic DNA (RAPD) sequences in the genome of Xcc using conserved primers. The different markers produced characteristic banding patterns and the similarity matrices from binary banding data was derived with the similarity for qualitative data program (SIMQUAL). On the basis of the fingerprint patterns generated by the combination data set of both rep-PCR and RAPD, the Xcc strains were differentiated into seven clusters (A–G) at 76 % similarity level. The geographical origin of the Iranian strains does not seem to be correlated with the RAPD and rep-PCR clusters. The clusters seem to be more related to the race of the strains. This is the first study on genetic diversity of Xcc strains inducing black rot disease of crucifers in Iran.     

Copper resistance in Xanthomonas campestris pv. campestris affecting crucifers in Trinidad

Fifty-six native isolates collected in 12 farming districts of Trinidad and seven reference strains of Xanthomonas campestris pv. campestris were evaluated for resistance to copper in buffered (pH 7.0) and unbuffered (pH 5.6) nutrient agar media. All isolates and reference strains were pathogenic and elicited typical black rot symptoms on a susceptible variety of Brassica olearceae, ‘Copenhagen Market’. Thirty-four and thirty-three native isolates were highly resistant to copper (growth on?≥?200 ppm copper) in buffered and unbuffered media, respectively; however, all the reference strains were highly susceptible to copper. The mean minimum inhibition concentration for the 56 native isolates was 224.6 ppm copper indicating that high levels of copper resistance are present in X. campestris pv. campestris in Trinidad. The association between growth of the 56 isolates and seven reference stains on buffered and unbuffered media was strong (Pearson’s and Spearman’s r?=?0.93; P?<?0.01) suggesting that either medium can be used to evaluate resistance to copper in X. campestris pv. campestris. There was also a strong association between length of time of continuous applications of copper formulations to treat black rot disease and proportion of the native X. campestris pv. campestris with resistance to copper (Pearson’s r?=?0.96; Spearman’s r?=?0.93); however, there was no association between resistance to copper and aggressiveness at 10 days after inoculation.     

Inference of the phylogenetic diversity and population structure of Xanthomonas campestris affecting Brassicaceae using a multilocus sequence typing‐based approach

Xanthomonas campestris pathovars are widely distributed throughout the globe and have a broad host range, causing severe economic losses in the food and ornamental crucifers markets. Using an approach based on multilocus sequence typing, phylogenetic diversity and population structure of a set of 75 Portuguese and other Xanthomonas campestris isolates from several cruciferous hosts were assessed. Although this population displayed a major clonal structure, neighbour‐net phylogenetic analysis highlighted the presence of recombinational events that may have driven the ecological specialization of X. campestris with different host ranges within the Brassicaceae family. A high level of genetic diversity within and among X. campestris pathovars was also revealed, through the establishment of 46 sequence types (STs). This approach provided a snapshot of the global X. campestris population structure in cruciferous host plants, correlating the existing pathovars with three distinct genetic lineages. Phylogenetic relationships between the founder genotype and remaining isolates that constitute the X. campestris pv. campestris population were further clarified using goeBURST algorithm. Identification of an intermediate link between X. campestris pv. campestris and X. campestris pv. raphani provided new insights into the mechanisms driving the differentiation of both pathovars. Wide geographic distribution of allelic variants suggests that evolution of X. campestris as a seedborne pathogen was not shaped by natural barriers. However, as Portuguese isolates encompass 26 unique STs and this country is an important centre of domestication of Brassica oleracea crops, a strong case is made for its role as a diversification reservoir, most probably through host–pathogen coevolution.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

Characterization,genetic diversity and distribution of Xanthomonas campestris pv. campestris races causing black rot disease in cruciferous crops of India

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.     

Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Phenotypic and Genotypic Characterization of Xanthomonas campestris pv. zinniae Strains

During 1997 and 1998, serious outbreaks of bacterial leaf spot disease were observed on zinnia plants grown in home and commercial gardens in Ohio, USA. Twenty-two strains of Xanthomonas campestris pv. zinniae, isolated from diseased zinnia plants and contaminated seeds, were identified based on morphological, physiological and biochemical tests, fatty acid methyl ester analyses and pathogenicity tests on zinnia cv. Scarlet. Host range studies indicated that all of the X. campestris pv. zinniae strains were pathogenic on zinnia and tomato, but not on cabbage, lettuce, pepper and radish. The phenotypic and genotypic relationships among the strains determined based on serological reaction pattern, fatty acid profiles, repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) fingerprints and sequence analysis of the 16S–23S rDNA spacer region suggested that X. campestris pv. zinniae strains were closely related to each other, but clearly distinct from other Xanthomonas species including X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. vesicatoria and X. hortorum pv. vitians tested in this study. The results also demonstrated that rep-PCR fingerprinting is rapid, reliable and the most practical method for routine detection and identification of X. campestris pv. zinniae strains.     

Identification and Characterisation of Xanthomonas campestris pv. campestris Strains from Tanzania by Pathogenicity Tests, Biolog, rep-PCR and Fatty Acid Methyl Ester Analysis

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a major disease constraint to cabbage production by smallholder farmers in Africa. Variability exists within the pathogen, and yet differentiation of Xcc strains from other closely-related xanthomonads attacking crucifers is often difficult. The Biolog system, fatty acid methyl ester analysis using microbial identification system (MIS), rep-PCR and pathogenicity tests were used to identify and characterise Xcc strains from Tanzania. Great diversity was observed among Xcc strains in their Biolog and rep-PCR profiles. Specific rep-PCR genomic fingerprints were linked to some geographical areas in the country. Most of the Xcc strains were clustered in two groups based on their fatty acid profiles and symptom expression in cabbage although some deviant strains were found. Each of the methods allowed a degree of identification from species, pathovar to the strain level. Biolog and MIS identified all Xcc strains at least to the genus level. Additionally, Biolog identified 47% of Xcc strains to the pathovar and 43% to strain level, whereas MIS identified 43% of the strains to pathovar level. In the absence of a database, the utility of rep-PCR for routine diagnosis of strains was limited, although the procedure was good for delineation of Xcc to the strain level. These findings indicate the existence of Xcc strains in Tanzania that are distinct from those included in Biolog and MIS databases. The limitations noticed warrant continued improvement of databases and inclusion of pathogenicity testing, using universally susceptible cultivars, as an integral part of strain identification.     

Growth of Xanthomonas campestris pv. campestris populations at constant and variable temperatures

Quantitative data were collected to describe the relation between temperature and growth of the cabbage black rot pathogen,Xanthomonas campestris pv.campestris (Xcc). Relative growth rates derived from experiments at constant temperatures were used in dynamic simulation of bacterial population development. The relative growth rates were adequate to simulate growth ofXcc populations at constant temperatures but overestimated growth of populations at variable temperatures. This finding gives rise to the hypothesis, that under field conditions, disease development is slower than is expected on the basis of growth parameters obtained from studies with constant temperatures.     

Identification of Isolates that Cause a Leaf Spot Disease of Brassicas as Xanthomonas campestris pv. raphani and Pathogenic and Genetic Comparison with Related Pathovars

ABSTRACT Twenty-five Xanthomonas isolates, including some isolates received as either X. campestris pv. armoraciae or pv. raphani, caused discrete leaf spot symptoms when spray-inoculated onto at least one Brassica oleracea cultivar. Twelve of these isolates and four other Xanthomonas isolates were spray- and pin-inoculated onto 21 different plant species/cultivars including horseradish (Armoracia rusticana), radish (Raphanus sativus), and tomato (Lycopersicon esculentum). The remaining 13 leaf spot isolates were spray-inoculated onto a subset of 10 plant species/cultivars. The leaf spot isolates were very aggressive on several Brassica spp., radish, and tomato causing leaf spots and dark sunken lesions on the middle vein, petiole, and stem. Based on the differential reactions of several Brassica spp. and radish cultivars, the leaf spot isolates were divided into three races, with races 1 and 3 predominating. A differential series was established to determine the race-type of isolates and a gene-for-gene model based on the interaction of two avirulence genes in the pathogen races and two matching resistance genes in the differential hosts is proposed. Repetitive-DNA polymerase chain reaction-based fingerprinting was used to assess the genetic diversity of the leaf spot isolates and isolates of closely related Xanthomonas pathovars. Although there was variability within each race, the leaf spot isolates were clustered separately from the X. campestris pv. campestris isolates. We propose that X. campestris isolates that cause a nonvascular leaf spot disease on Brassica spp. should be identified as pv. raphani and not pv. armoraciae. Race-type strains and a neopathotype strain for X. campestris pv. raphani are proposed.     

Application of polyclonal and monoclonal antibodies for the detection of Xanthomonas campestris pv. campestris in crucifer seeds using immunofluorescence microscopy

Polyclonal and monoclonal antibodies (PCAs and MCAs) were tested for the detection ofXanthomonas campestris pv.campestris (Xcc) in cabbage seeds using immunofluorescence microscopy (IF). It was concluded that PCA 94, MCAs 20H6, 2F4, 18G12 and a mixture of MCAs 20H6, 18G12, 2F4 and 16B5 could be used to detect Xcc in seed extracts when 5 min and 2.5 h shaking of seeds are used as extraction methods. The reliability of confirming suspect colonies with MCAs and PCA 94 in IF depended in part on the seed lot tested and the antibody used. Some virulent Xcc strains derived from seed lots, did not react with MCAs 10C5, 2F4, 18G12, 17C12 and 16B5. On the other hand, saprophytic isolates obtained from one seed lot cross-reacted with MCA 17C12 and to a lesser extent with MCAs 2F4, 18G12 and PCA 94. No relationship was found between IF-reactions of Xcc strains using MCAs and reactions of Xcc strains in pathogenicity testing. Xcc andX. c. pv.amoraciae (Xca) could in general not be distinguished on the basis of reactions with MCAs and PCAs. Also in pathogenicity tests Xcc and Xca were hard to distinguish.     

Bacterial leaf spot of hemp caused by Xanthomonas campestris pv. cannabis in Japan

Bacterial leaf spot disease of hemp was observed in Tochigi Prefecture, Japan in 1982 and characterized by necrotic lesions ca. 1–2 mm diameter on leaves with a yellow halo 2–3 mm wide. In this report, we describe the pathological, physiological and genetic properties of the causal bacterium. Our results indicated that this bacterium is identical with Xanthomonas campestris pv. cannabis reported in Romania.     

Control by metham-sodium ofxanthomonas campestris pv.Campestris and the pathogen’s survival in soil

Detection and quantitative estimation ofXanthomonas campestris pv.campestris (XCC) from soils were done on Kritzman’s selective medium (KSM). The composition of KSM, its selectivity and the recovery of 64 isolates ofXCC obtained from Israel, Holland, Taiwan, the USA, and Germany are given in detail.XCC was isolated from soil in Israel 22 months after infected cauliflower residues were incorporated into the soil. Metham-sodium (MES) at 16 ml/kg (32.7% active compound) of air-dried soil killed more than 99.9% and 99% ofXCC in soil in laboratory and field tests, respectively. MES applied at this ratevia the irrigation system in a field infested withXCC, significantly reduced counts of the pathogen in stems, siliques and seeds, and the incidence of plants with black rot symptoms. The treatment was more successful in reducing disease incidence when applied to the soil at a depth of 30 or 40 cm rather than 20 cm. It is recommended that a seed treatment be combined with fumigation of soil with MES for the production of seeds free ofXCC.     

Genetic diversity and virulence of Xanthomonas campestris pv. campestris isolates from Brassica napus and six Brassica oleracea crops in Serbia

The present study provides insight into the diversity of 147 Xanthomonas campestris pv. campestris (Xcc) isolates obtained from six Brassica oleracea vegetable crops (broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi) and the winter oilseed rape crop Brassica napus, collected from different regions in Serbia in 2014. The XCF/XCR pathovar-specific primer set was used for fast preliminary identification. In repetitive sequence-based PCR (BOX, ERIC and REP) of all isolates, a higher level of genetic diversity was found in winter oilseed rape isolates compared to isolates from the other hosts. ERIC and REP-PCR showed the highest heterogeneity, with 10 and nine banding patterns, respectively. The REP-PCR results showed the highest correlation (70%) with those obtained with multilocus sequence analysis (MLSA), performed with 10 housekeeping genes (fusA, gap-1, gltA, gyrB1, lacF, lepA, rpoD, dnaK, fyuA and gyrB2). Three distinct phylogenetic groups of winter oilseed rape isolates were detected using MLSA. Two genes, gltA and rpoD, showed the greatest ability to identify and discriminate winter oilseed rape Xcc isolates from isolates of the other six hosts. The lepA gene exhibited specific three-nucleotide changes in sequences of some of the isolates. Results of virulence testing of 18 representative isolates showed statistically significant host–pathogen specialization for Xcc isolates from winter oilseed rape, cauliflower, kale and kohlrabi. In conclusion, oilseed rape isolates are more genetically diverse and show greater specialization to their host in comparison to the rest of the tested isolates from other brassica hosts.     

Development of a lateral flow device for in‐field detection and evaluation of PCR‐based diagnostic methods for Xanthomonas campestris pv. musacearum,the causal agent of banana xanthomonas wilt

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in‐field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross‐reacted with non‐target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 10⁵ cells mL^?1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first‐line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non‐scientists and is cost‐effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.     

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The present study developed a pathovar‐specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265‐bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant‐associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm‐specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.     

Genetic analyses of Pseudomonas syringae isolates from Belgian fruit orchards reveal genetic variability and isolate-host relationships within the pathovar syringae, and help identify both races of the pathovar morsprunorum

A collection of Pseudomonas syringae and viridiflava isolates was established between 1993 and 2002 from diseased organs sampled from 36 pear, plum and cherry orchards in Belgium. Among the 356 isolates investigated in this study, phytotoxin, siderophore and classical microbiology tests, as well as the genetical methods REP-, ERIC- and BOX- (collectively, rep-) and IS50-PCR, enabled identification to be made of 280 isolates as P. syringae pv. syringae (Pss), 41 isolates as P. syringae pv. morsprunorum (Psm) race 1, 12 isolates as Psm race 2, three isolates as P. viridiflava and 20 isolates as unclassified P. syringae. The rep-PCR methods, particularly BOX-PCR, proved to be useful for identifying the Psm race 1 and Psm race 2 isolates. The latter race was frequent on sour cherry in Belgium. Combined genetic results confirmed homogeneities in the pvs avii, and morsprunorum race 1 and race 2 and high diversity in the pv. syringae. In the pv. syringae, homogeneous genetic groups consistently found on the same hosts (pear, cherry or plum) were observed. Pathogenicity on lilac was sometimes variable among Pss isolates from the same genetic group; also, some Psm race 2 and unclassified P. syringae isolates were pathogenic to lilac. In the BOX analyses, four patterns included 100% of the toxic lipodepsipeptide (TLP)-producing Pss isolates pathogenic to lilac. Many TLP-producing Pss isolates non-pathogenic to lilac and the TLP-non-producing Pss isolates were classified differently. Pseudomonas syringae isolates that differed from known fruit pathogens were observed in pear, sour cherry and plum orchards in Belgium.     

Xanthomonas campestris pv. translucens in North and South America and in the Middle East1

Bacterial leaf streak and black chaff is a seed-transmitted disease of cereals caused by Xanthomonas campestris pv. translucens. The bacterium can be identified by its slow growth, slime production, yellow pigment, beta-galactosidase production, and ice nucleation activity. Aspects of resistance, dissemination, symptoms, and distribution are discussed.     

Symptomless Spread of Blight-inducing Strains of Xanthomonas campestris pv. campestris on Cabbage Seedlings in Misted Seedbeds

Xanthomonas campestris pv. campestris induces two types of symptoms, namely, black rot and blight. Black rot symptoms are V-shaped lesions and black veins on the leaf, and blight symptoms are sudden collapse of interveinal tissues following the lack of veinal necrosis at early stages of infection. These two symptoms can occur simultaneously. However, the tendency to induce either symptom type is strain-dependent. Six strains were evaluated for their rate and pattern of spread in misted seedbeds by using strain-specific monoclonal antibodies and miniplate enrichment/ELISA. Data on pathogen incidence was defined as the presence of the pathogen in or on plants rather than visual symptoms. The results indicated that blight-inducing strains spread to more seedlings than black rot-inducing strains. The high incidences of blight-inducing strains in experimental plots were associated with non-randomness of spatial pattern of pathogen spread, indicating that high incidence is primarily due to the spread from adjacent plants by leaf contact and water splash. Most ELISA-positive seedlings were symptomless, indicating that the sensitivity of the system used in this study was adequate for detection of latent or epiphytic spread.