Pathotypes Research Articles

Screening for biofilm formation of E. coli pathotypes and determination of the effect of antimicrobial peptides on biofilm formation by automated fluorescence microscopy

Screening for biofilm formation of E. coli pathotypes and determination of the effect of antimicrobial peptides on biofilm formation by automated fluorescence microscopy

Genome-based Definition of an Inflammatory Bowel Disease-associated Adherent-Invasive Escherichia coli Pathovar

Genome-based Definition of an Inflammatory Bowel Disease-associated Adherent-Invasive Escherichia coli Pathovar

CHARACTERIZATION OF PSEUDOMONAS SYRINGAE pv. SYRINGAE ISOLATED FROM MANGO IN SICILY AND OCCURRENCE OF COPPER-RESISTANT STRAINS

Mango (Mangifera indica) is grown throughout a wide range of frost-free climates and is one of the world’s most important fruit crops. During 2010-2014, severe symptoms of bacterial apical necrosis (BAN) caused by Pseudomonas syringae pv. syringae (Pss) were observed on mango plants throughout all mango-growing areas in Sicily (southern Italy). The causal agent was identified based on phenotypic, genotypic and pathogenicity tests. The genetic variability of strains obtained from different areas and cultivars were assessed by rep- PCR and allowed clear differentiation of the Pss strains isolated from mango from other representative strains of the pathovar. In addition, the respective production of syringomycin, syringopeptin and mangotoxin was checked by in vitro tests and PCR detection. All tested strains showed the presence of mgoA and mgoB of the mangotoxin operon. Some copper- resistant Pss strains showing cusCBA genes were also found in some orchards. This feature could explain the failure of the disease control using copper compounds. Among 71 strains tested using in vitro assay, no copper-sensitive strains were detected, whereas 44 strains (62%) had high resistance, 16 strains (22.5%) were resistant and 11 strains (15.5%) had low resistance to copper sulfate. To our knowledge, this is the first report of copper resistance among Pss strains causing BAN on mango in Sicily.

Clinical and etiologic features of diarrheagenic Escherichia coli-associated diarrhea in children

Objective To investigate the pathotypes, epidemiological characteristics and antimicrobial resistance of diarrheagenic Escherichia coli (DEC) in children with acute bacterial diarrhea in Shanghai. Methods A total of 2 071 outpatient children with probable acute bacterial diarrhea referred to the enteric clinic of Children′s Hospital of Fudan University during June 2012 to June 2014 were enrolled in our study. The stool samples were processed for routine microbiologic and biochemistry tests to identify enteric bacteria, including enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli(EIEC) and enterohemorrhagic Escherichia coli (EHEC). Kirby-Bauer method was used to identify the antibiotic sensitivity. Difference of means between groups was compared by chi-square test. Results Of 2 071 enrolled children, DEC were identified in 145 (7.0%) cases. 148 strains were isolated with three of mix infection strains. All DEC isolates in this study included 106 (71.6%) EPEC, 24 (16.2%) ETEC, 16(10.8%) EIEC and 2(1.4%) EHEC. The median ages of diarrheal children with DEC infections were 14 months (range: 3 months to 13 years) and 62.8% of them were <2 years. Among 125 DEC isolates tested for antimicrobial susceptibility, the rates of resistance to ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, cefepime, gentamicin, ceftazidime, amoxicillin-clavulanate, ciprofloxacin, and ofloxacin in a descending order were 55.2%, 35.2%, 28.0%, 27.2%, 23.2%, 8.8%, 5.6%, 4.0% and 4.0%, respectively. Resistance rates of EIEC to cefotaxime, cefepime and ceftazidime were 50.0%, 43.8% and 25.0%, respectively, which were higher than those of EPEC, ETEC and EHEC. Conclusion DEC is the important enteric bacteria that causes bacterial diarrhea in children in this study. Key words: Diarrhea; Diarrheagenic Escherichia coli; Epidemiology; Child; Drug resistance, microbial

Pruning date affects bacterial canker of sweet cherry

The effect of pruning date on the development of canker lesions caused by Pseudomonas syringae pv syringae (Pss) and Ps pv morsprunorum (Psm) was investigated in a field trial using three sweet cherry cultivars with or without Pss and Psm inoculation Four weeks after treatment the percentage of twigs showing bacterial canker lesions and the size of lesions were recorded Overall the percentage of twigs showing lesions did not vary between cultivars but did vary with the pruning date and between pathovars Uninoculated branches pruned in February 2014 did not develop canker lesions A moderate proportion (40) showed lesions when inoculated with Psm and 100 of twigs showed necrosis when inoculated with Pss However for later pruning dates (April and July 2014) the percentage of twigs showing lesions declined progressively for twigs inoculated with both pathovars up to the September pruning Thereafter a sharp increase was observed with nearly 100 of twigs showing necrosis after the final January 2015 pruning For branches that showed necrosis lesion size varied strongly with cultivar pruning time and pathovar Lesion size tended to be smaller with later pruning but the pattern varied considerably with pathovar and cultivar The significance of these findings in relation to bacterial canker management is discussed

RESISTANCE TO LEAF RUST IN A SET OF DURUM WHEAT CULTIVARS AND LANDRACES IN SPAIN

SUMMARY Durum wheat (Triticum turgidum var. durum) is an important crop in the Mediterranean basin, and in southern Spain. One of the most important biotic constraints to durum wheat production is leaf rust. Breeder deployed resistant cultivars, but mutations in the pathogen create new virulent pathotypes that erode resistance. In this work, two collections of genotypes have been characterized for their leaf rust resistance both in field trials (in Spain and Mexico) and in the greenhouse (with an array of Spanish pathotypes). The first collection included 14 cultivars and lines, mostly from CIMMYT origin, plus six genotypes with known resistance genes. The second collection comprised 29 cultivars commercially available in Spain, plus 11 landraces from the Spanish germplasm bank. Besides, six resistant cultivars of particular importance were crossed with two cultivars with known effective genes (Lr14a and Lr27 + Lr31), and a susceptible one to infer the genetic basis of their resistance. F2 populations were analyzed. At adult plant stage, 20 genotypes including four landraces were resistant in greenhouse to all Spanish pathotypes. In field trials, 24 genotypes were resistant in Spain, and 22 genotypes in Mexico. The presence of resistant genes Lr14a and the complementary genes Lr27+Lr31 has been deduced in some cultivars either by gene postulation or by genetic analysis. These results provide an opportunity for breeding programs in Spain and elsewhere to target their crossing and selection activities so they can yield lines with effective resistance in the Mediterranean region.

The Italian inter-laboratory study on the detection of Pseudomonas syringae pv. actinide

A severe form of bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has been detected in all the main areas of cultivation of kiwifruit (Actinidia deliciosa and A. chinensis). Since 2010 several research groups have been assessing methods and procedures to detect and identify Psa, both from symptomatic and symptomless host material. In 2011, a study to compare Psa diagnostic methods was performed with reference to Psa strains and related pathovars, and with plant extracts or DNA obtained from healthy and naturally infected leaves, pollen or wood. The study revealed the strengths and the weaknesses of the assessed methods. The procedure included screening tests for Psa detection and for identification of Psa colonies. The methods assessed were bacterial isolation on generic and semi-selective media, PCR analysis (single, duplex and rep-PCR assay, the latter for identification only). The results highlighted the best performance of semi-selective with respect the generic media; the usefulness of the direct-PCR as screening tests for Psa detection; and the greater specificity of duplex-PCR and sensitivity of simple-PCR. The use of semi-selective medium for isolation and of two PCR-based methods - in parallel - for Psa detection are suggested. Both rep-PCR and duplex-PCR, were found to be specific, and are recommended as an identification test for this pathogen.

Phylogenetic group determination of faecal Escherichia coli and comparative analysis among different hosts

Summary Phylogenetic analysis has shown that Escherichia coli is composed of four main phylogenetic groups (A, B1, B2 and D). Characterization of phylogenetic groups is of clinical interest, as group A and B1 generally associated with commensals, whereas most enteropathogenic isolates are assigned to group D, and group B2 is associated with extra-intestinal pathotype. One hundred E. coli strains recovered from faecal samples of dog, chicken, ruminants (sheep, goat and cattle) and human were subjected to phylogenetic analysis based on triplex PCR method, according to a combination of three genetic markers chuA, yjaA and DNA fragment TspE4.C2. The majority of collected isolates belonged to group D (44%), followed by groups A (32%), and B2 (24%). By sample origin, groups D, A, and B2 were prevalent in 16.7, 50, and 33.3%, respectively for dog isolates; 52.8, 36.1, and 11.1% for chicken isolates; 41.2, 29.4, and 29.4% for ruminants isolates; and 60.9, 8.7, and 30.4% for human isolates, respectively and none of the strains among all the analysed hosts belong to group B1. This study suggests there was a significant difference in the E. coli phylogenetic groups, subgroups and genetic markers among the different hosts analysed.

Population changes and phenotypic diversity of Phytophthora infestans isolates from Estonia and Finland.

In Estonia and Finland, 386 and 671 isolates, respectively, of Phytophthora infestans were collected during the growing seasons of 2001-2007. Collection was from groups of potato cultivars of different origin from field plots at two research institutions in eastern and northern Estonia and from variety trials at two locations and commercial potato fields in Finland. Mating type, pathotype based on virulence on a differential set of 11 R genes of Solanum demissum and response to the fungicide metalaxyl were determined in both countries by comparable methods. The diversity of the P. infestans populations in both countries over the monitoring period was high. In Finland, the proportion of A1/A2 mating types was almost equal throughout the monitoring period, while in Estonia the A1 population, dominating in 2001-2004, shifted towards an equal proportion of both mating types in 2005-2007. The diversity of the P. infestans population, as measured by virulence pathotypes and the number of avirulence factors per isolate, developed in opposite directions in Estonia and Finland from 2001-2004 to 2005-2007: the diversity decreased in Estonia and increased in Finland. Metalaxyl sensitivity in the Estonian P. infestans population increased while in Finland it decreased during the same period. Results clearly show differences between Estonian and Finnish populations of P. infestans in several aspects, which may derive from cropping practices. The high A2 mating type percentage during 2005-2007 in both countries points to soil contamination with oospores.

Prevalence and distribution in different agro-ecologies and identification of resistance source for wheat stripe rust

To assess the prevalence and distribution of wheat stripe rust (Puccinia striiformis), a survey of the randomly selected wheat fields located in sub-tropical to temperate agro-ecologies of Jammu province of Jammu and Kashmir was conducted consecutively for three years during rabi seasons of 2009-2010, 2010-2011 and 2011-2012. The results revealed that, by and large, all the wheat growing areas under study were found infested by this disease with maximum and minimum Area Under Rust Progress Curve (AURPC) values of 2422.21 and 351.70 reported from Jammu sub-tropics and inter-mediate Doda districts, respectively. The cropping season of rabi 2010-2011 recorded highest disease prevalence, probably due to conducive environmental conditions coupled with virulent pathotypes out-break and monoculture of wheat varieties. Among twenty one germplasms of wheat screened for source of disease resistance against stripe rust under epiphytotic conditions, Agra local and PBW-343 were found susceptible, RSP-561 showed moderate resistance, whereas, all the other cultivars were moderately susceptible to Puccinia striiformis. Key words: Puccinia striiformis, stripe rust, wheat, distribution, resistance.

Diversity of Citrus tristeza virus in Jamaica.

Citrus tristeza virus (CTV), one of the most serious pathogens affecting Rutaceae species, causes considerable economic losses to citrus production worldwide. While mild pathotypes have been known for many years in Jamaica, decline outbreaks were only recently recognized after the introduction of the vector Toxoptera citricidus in 1993. In this study, coat protein (CP) gene sequences of isolates from four major citrus-growing regions in Jamaica were obtained and their molecular diversity compared with reference CTV genotypes from other regions. CP sequences from Jamaican isolates showed identities between 90 to 100% at the nucleotide level and shared similar identities with comparable sequences reported for other Jamaican isolates in 2009 and 2010. Phylogenetic analysis revealed segregation into five of seven lineages. One third of the Jamaican isolates clustered with Florida-T30 and Spain-T385 genotypes, with 99 to 100% identity within the lineage. The remaining isolates grouped closely with Florida T36-like genotypes or with the grapefruit and orange stem pitting B249 from Venezuela and the New Zealand CTV-resistance breaking genotypes. Identities among these groups ranged between 91 and 99%. The detection of genetic variants and potential recombination events involving parents from different lineages provide further evidence of the diversity among Jamaican CTV isolates.

Genetic diversity of microsatellite alleles located at quantitative resistance loci for Ascochyta blight resistance in a global collection of chickpea germplasm

A global collection of 43 chickpea (Cicer arietinum L.) genotypes, resistant and susceptible to Ascochyta blight caused by Ascochyta rabiei was evaluated for the disease under controlled conditions. In this study three known pathotypes (P-I, P-II, and P-III) were used to evaluate the reactions of this collection. the chickpea genotypes were also characterized using 14 microsatellite markers flanking the genomic regions associated with Ascochyta blight resistance quantitative trait loci (QTLs). Phenotyping results indicated that 27 genotypes were re- sistant to P-I, 14 to P-II, and five to P-III, revealing the possible erosion of resistance through the evolution of virulent pathogen pathotypes. the genetic diversity analysis revealed 67 alleles at 14 microsatellite loci with an average of 4.8 alleles per locus among the genotypes tested. Genetic similarity estimates differentiated four subclusters (A, B, C, and D) of the genotypes. However, none of sub-clusters were separated into resistant genotypes for a specific pathotype. The genetic diversity ranged from 0.48 to 0.80 which indicated that there is considerable variation in QTL regions associated with Ascochyta blight resistance among the collections of chickpea genotypes studied, as assessed using the hyper-variable microsatellite markers.

Pathotype characterization of the cereal cyst nematode Heterodera filipjevi (Madzhidov, 1981) Stelter in Turkey

Turkey is ranked among top ten wheat producing countries in the world. Cereal cyst nematodes are considered one of the most damaging plant parasitic nematodes on wheat worldwide. Up to date, the most effective method of controlling this group of nematodes is the use of crop rotation and resistant varieties. The successful development of disease-resistant plants is contingent on knowledge of the pathotypes of the causal agent. In this study, three populations of Heterodera filipjevi (Madzhidov, 1981) Stelter were collected from different locations (Afsin, Elbistan and Yozgat) in Turkey in order to identify their pathotype using “The International Test Assortment of Cereal Cultivars”. The results showed that two populations gave a similar reaction to the resistant and susceptible varieties. These populations were determined as Ha 33 pathotype which is a new report for Turkey.

Recent progress on detecting understanding and controlling &lt;i&gt;Pseudomonas syringae&lt;/i&gt; pv &lt;i&gt;actinidiae&lt;/i&gt; a short review

In the last few years the causal agent of bacterial canker of kiwifruit Pseudomonas syringae pv actinidiae (Psa) has become a global pathogen of economic importance Since the beginning of this global outbreak many laboratories in the world have been working on Psa Today it is known that Psa is not a homogeneous pathovar and tools that allow the distinction between biovars (subpathovar classification) have been developed The whole genome sequence of several strains of Psa has now been published Some of the assumptions on the life cycle (ports of entry epiphytic survival etc) made in the early days of the outbreak have now been confirmed Although few new methods have been found to control Psa there is now a better understanding of how to reduce the incidence of this disease This paper reviews the progress made in understanding the pathogen the disease and how to control it

FIRST REPORT OF BACTERIAL LEAF SPOT OF MANGO CAUSED BY A XANTHOMONAS ARBORICOLA PATHOVAR IN CHINA

A disease of mango (Mangifera indica) observed in 2009-2010 in Hainan and Guangxi provinces (China) is characterized by small water-soaked spots on the leaves turning with age into an- gular, sunken, deep-brown to black necrotic lesions with a yellow halo, whose necrotic tissue may fall, resulting in a “shot-hole” condition of the blades. These symptoms differ from those of mango bacterial black spot caused by X. campestris pv. mangiferindicae (Xcm) (Gagnevin and Pruvost, 2001). Purified bacterial colonies appeared yellow, slimy and convex on YDCA medium, and induced hypersensitive responses on tobacco (HC9511). The field syndrome was reproduced on inoculated mango plants and the same bacterium was reisolated from symp- tomatic but not from control leaves, which remained symptom- less The sequences of 16S rDNA gene fragment (ca.1.4 kb) (JF803830-JF803839) of ten isolates (GXMG2, GXMG5, GXMG8, GXMG9, GXMG10 and HNMG23-HNMG27) am- plified with the universal primer pair 27f/1492r (Weisburg et al., 1991), showed a complete identity with those of X. arboricola pv. juglandis (AB558555), X. arboricola (AB558553), X. gardneri (FR749911) and X. campestris (GU596424). Furthermore, the gyrB gene sequences (HQ538517-HQ538526) of the same iso- lates (Parkinson et al., 2007), showed 99-100% identity with those of X. arboricola strains from GenBank, but only 88% iden- tity with references strains of Xcm (HQ538527-HQ538538). This is the first report of a X. arboricola pathovar on mango in China inducing a disease for which we propose the name of “bacterial leaf spot” to distinguish it form the “bacterial black spot” caused by Xcm. Further studies are needed for determining pathovar sta- tus of the strain.

Pathotype diversity among Algerian isolates of Pyrenophora teres f. teres

Barley Net Blotch caused by Pyrenophora teres f. teres (Ptt) is one of the most important foliar diseases in north western Algeria. The disease could cause appreciable yield losses under favorable environmental conditions. Studies on pathogen variability and distribution of virulence in Ptt are essential to identify effective sources of resistance to net blotch of barley. In this study, pathogenic variability in 48 isolates of Ptt , collected from different barley-growing areas of north-western Algeria were evaluated using 22 barley genotypes as differential hosts. Trials carried out under controlled conditions confirmed the large variation in the virulence of this fungus . The genotypes exhibited variability in response ranging from complete resistance to high susceptibility. This is the first report of pathogenic variability in Ptt in north western Algeria. The 48 isolates tested were further differentiated into 12 pathotypes according to their reaction on the host. Eight differential cultivars were resistant to all isolates tested, but others were susceptible to one or more of the isolates. The variability in the pathogen and the resistance identified in some genotypes used in this study are being investigated further to develop superior, adapted germplasm for use in barley breeding programs in north western Algeria.

Screening of Brassica germplasm for resistance to Plasmodiophora brassicae pathotypes prevalent in Canada for broadening diversity in clubroot resistance

Hasan, M. J., Strelkov, S. E., Howard, R. J. and Rahman, H. 2012. Screening ofBrassicagermplasm for resistance toPlasmodiophora brassicae pathotypes prevalent in Canada for broadening diversity in clubroot resistance. Can. J. Plant Sci. 92: 501–515. Clubroot disease of crucifers, caused by Plasmodiophora brassicae, poses a threat to the Canadian canola industry, and the development of resistant cultivars is urgently needed. Germplasm resistant to local pathotype(s) is the prime requirement for breeding clubroot-resistant cultivars. The objective of this study was to identify Brassica germplasm possessing resistance to P. brassicae pathotypes prevalent in Alberta. Pathotype-specific resistance was identified in the diploid species Brassica rapa (AA) and B. oleracea (CC), and in the amphidiploid B. napus (AACC). Among B. rapa genotypes, turnip was the most resistant, followed by winter type and spring type oilseed rape. The rutabaga group of B. napus, on the other hand, was homogeneous for resistance to Can...

Bacterial canker of plum caused by Pseudomonas syringae pathovars, as a serious threat for plum production in the Netherlands

In the Netherlands, bacterial canker of plum trees (Prunus domestica) caused by Pseudomonas syringae pathovars syringae and morsprunorum is a recent and serious problem. The trunks of the affected plum trees are girdled by cankers resulting in relatively sudden death of the trees 1 to 4 years after planting. Disease incidence can be very high, and sometimes complete orchards have to be removed. Recently, plum cultivation in the Netherlands has changed from a relatively extensive into an intensive cultivation. However, due to the risks of losses of trees due to bacterial canker, growers are reluctant to plant new orchards. In general, nurseries and fruit growers are not familiar with bacterial diseases and lack knowledge to prevent infections. Therefore, control strategies to manage plum decline have to be developed.

DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS DEVELOPED AND USED IN THE FRAMEWORK OF COST 873 FOR BACTERIA PATHOGENIC TO STONE FRUITS AND NUTS

Xanthomonas arboricola pv. juglandis (Pierce) Vauterin et al. (1995) is the causal agent of walnut bacterial blight, the major nut and foliage disease of Persian (English) walnut (Juglans regia). The disease limits walnut production in most regions of the world. All new walnut tissues are susceptible to infection and necrosis can occur in catkins, female flowers, leaves, fruit and green shoots (Miller and Bollen, 1946). Over time, the bacterium has been classified into different genera under different names, i.e. Pseudomonas juglandis Pierce (1901); Bacterium juglandis (Pierce) Smith (1905); Phytomonas juglandis (Pierce) Bergey et al. (1930); Xanthomonas juglandis (Pierce) Dowson (1939) and Xanthomonas campestris pv. juglandis (Pierce) Dye (1978), which is officially accepted in the International standards for naming pathovars of phytopathogenic bacteria (Dye et al., 1980). Recently, however, it was re-classified by Vauterin et al. (1995) as Xanthomonas arboricola pv. juglandis (Xaj). Xaj is the causal agent of two new emerging diseases of Persian (English) walnut (J. regia) known as vertical oozing canker (VOC) (Hajri et al., 2010), and apical necrosis (Moragrega and Ozaktan, 2010; Moragrega et al., 2011). According to molecular studies based on fluorescent amplified fragment length polymorphism (FAFLP), in France VOC is caused by a distinct genetic lineage of the bacterium which is also able to induce blight symptoms on leaves and fruits (Hajri et al., 2010). Bacterial strains isolated from apical necrosis lesions in Spain are also able to infect walnut leaves, causing typical blight lesions (Moragrega et al., 2011).

Pathotype determination of the cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) in the Eastern Mediterranean Region in Turkey

Dogu Akdeniz Bolgesi'nde Tahil kist nematodu, Heterodera avenae patotiplerinin belirlenmesi amaciyla Karlik (Adana-Saricam), Imece (Hatay-Kirikhan) ve Besaslan (Hatay-Reyhanli) populasyonlari kullanilmistir. H. avenae’nin patotipleri “Uluslararasi konukcu tahil test ayirim materyalleri” kullanilarak arastirilmistir. Bu materyaller icerisinde bulunan, yirmi adet arpa, alti adet yulaf ve alti adet bugday cesit ve hatlari ile dort kontrol hatti (milan, seri, silverstar ve croc) denemeye alinmistir. Test materyalleri icerisinde dayaniklilik genleri (Rha”E”, Rha1, Rha2, Rha3, Cre1) tasiyan ve dayanikli geni tasimayan cesit ve hatlara karsi, denemeye alinan 3 populasyonun gosterdigi reaksiyon esas alinarak gruplandirilmistir. Calisma sonuclarina gore, Rha1 ve Rha3 genleri tasiyan materyaller dayaniklilik gostermesine karsin, Rha2 ve Cre1 genleri tasiyanlar ayni tepkiyi gostermemistir. Sonuc olarak konukcu test ayrim hatlari genellikle benzer reaksiyonlar gostermis ve denemeye alinan 3 populasyonun da ayni patotip, Ha1 grubundan Ha21 oldugu saptanmistir. Bu sonuclar Turkiye’de H. avanea patotipinin belirlenmesi acisindan ilk kayit niteligindedir.