Plasmodium falciparum and Plasmodium vivax co-exist and malaria rapid diagnostic test (RDTs) is vital in rendering parasite-confirmed treatment especially in areas where microscopy from 2008 to 2010 is not available. CareStartTM Malaria Pf/Pv combo test was evaluated compared to microscopy in Butajira area, south-central Ethiopia. This RDT detects histidine-rich protein-2 (HRP2) found in P. falciparum, and Plasmodium enzyme lactate dehydrogenase (pLDH) for diagnosis of P. vivax. The standard for the reporting of diagnostic accuracy studies was complied. Among 2,394 participants enrolled, 10.9% (n=87) were Plasmodium infected (household survey) and 24.5% (n=392) health facility-based using microscopy. In the household surveys, the highest positivity was caused by P. vivax , P. falciparum , and the rest due to mixed infections of both . In health facility, P. vivax caused 78.6% (n=308), P. falciparum caused 20.4% (n=80), and the rest caused by mixed infections 1.0% (n=4). RDT missed 9.1% (n=8) in household and 4.3% (n=17) in health facility-based surveys among Plasmodium positive confirmed by microscopy while 3.3% (n=24) in household and 17.2% (n=208) in health facility-based surveys were detected false positive. RDT showed agreement with microscopy in detecting 79 positives in household surveys (n=796) and 375 positives in health centre survey .Malaria is a major public health problem in Ethiopia. Plasmodium and P. falciparum (77.2%) in health centres; and Plasmodium (76.7%) and P. falciparum (87.5%) in household surveys. NPV was low in P. vivax in health centres (77.2%) and household (87.5%) surveys. Seasonally varying RDT precision of as low as 14.3% PPV (Dec. 2009), and 38.5% NPV (Nov. 2008) in health centre surveys; and 40-63.6% PPV was observed in household surveys. But the influence of age and parasite density on RDT performance was not ascertained. Establishing quality control of malaria RDT in the health system in areas with low endemic and where P. falciparum and P. vivax co-exist is recommendable. CareStartTM RDT might be employed for epidemiological studies that require interpreting the results cautiously. Future RDT field evaluation against microscopy should be PCR corrected.RDT performance varied in both survey settings, lowest PPV (64.3%) for  Plasmodium falciparum and Plasmodium vivax are epidemiologically the most important malaria parasites in the country [Plasmodium falciparum has been the major causes of high case fatality. Since 2005, the National Malaria Control Programme intensified the deployment of key malaria interventions including artemisinin-combination therapy (ACT), malaria rapid diagnostic tests (RDTs), and vector control measures [In Ethiopia, approximately 68% of the recently estimated 92 million total population, about 63 million people, is at risk of malaria infection ,2. Typic country . Plasmodmeasures ,8, and lP. falciparum, those which target the parasite enzyme lactate dehydrogenase (LDH) and aldolase that can detect non- falciparum from mixed infection. LDH test uses either monoclonal antibodies which react with LDH of all species including P. falciparum (or known as Pan-LDH), or antibodies specific for P. falciparum LDH [P.vivax-specific LDH.Malaria RDTs were introduced as one of the diagnostic methods following the revision of malaria diagnosis and treatment guideline in 2004 . The priarum LDH -13. RDTsP. falciparum were recommended [P. vivax was documented in some highland areas [P. falciparum, and consequently P. vivax may be misdiagnosed as malaria negative and treated based on clinical grounds. Moreover, World Health Organization (WHO) recommends that malaria case management be based on parasite-based diagnosis [P. falciparum and P. vivax is helpful in improving the early diagnosis and treatment of malaria at peripheral areas. CareStart™ Malaria Pf/Pv combo test was among multi-species detecting RDT according to WHO product testing [RDTs for detection of only ommended , and havommended ,15. Howeommended ,16. Follnd areas ,18. At ciagnosis . Thus, t testing . Various testing -25. Howe testing -28.There is paucity of information regarding the precision of malaria RDTs in the low-endemic highland zones of Ethiopia. Therefore, the present study was aimed at evaluating the performance of CareStart™ at health centres and concurrent surveys in highland areas of south-central Ethiopia.kebeles  part of the Demographic Surveillance site (DSS) of Butajira Rural Health Programme (BRHP) [This study was conducted in Butajira area, situated 130 km south of Addis Ababa, in six of ten e (BRHP) , and adme (BRHP) .kebele has a health post. Drug venders are also available in Butajira Town.There were 58,335 people living in the BRHP-DSS in 2008 and half  of the population were female. Of those 26,834 (46%) people lived in the study area. Malaria is endemic in the area with frequent outbreak that caused significant cases and deaths as documented previously . The locP. falciparum. Thus, CareStartTM Malaria Pf/Pv combo test was among the options for the field evaluation before introducing to malaria control program of the country.This study was conducted while the National Malaria Control Program of Ethiopia has initiated various local academic and research institutions for field evaluation of RDTs that could detect multi-species and replace RDTs that only detect A prospective household and health facility-based surveys were conducted to assess the performance of CareStart™ Malaria Pf/Pv combo test in reportedly febrile cases between 2008 and 2010. Six rural kebeles such as Hobe, Bati Lejano, Dirama, Shershera Bido, Yeteker and Wurib were randomly selected. A total of 750 households were selected randomly from those kebeles, using probability proportion to size (PPS) sampling. Individuals with the history of fever in the previous 48 hours of the survey period were included in both household and health facility-based surveys. Febrile cases visiting outpatient department (OPD) suspected of malaria cases at Butajira and Enseno health centres were recruited. In general, this study complied with Standards for Reporting of Diagnostic Accuracy guidelines (STARD) to improve the quality of reporting .Six cross-sectional surveys were performed on the same households for two consecutive years. The surveys were conducted in October-November 2008 (a month after the main rainy season), January-February 2009 (dry season), June-July 2009 (main rainy season), October-November 2009, January-February 2010, and June 2010 in the household surveys. The health facility-based survey was done during October 2008, November 2008, August 2009, September 2009, October 2009, November 2009, and December 2009 .Data collection was done using pre-tested structured questionnaire and format prepared for this purpose. Pre-testing of the data collection tool was performed in an adjacent The health facility-based survey was organized as a research team of four health personnel assigned from the same health centre. Overall, the study was supervised by the principal investigator (AW), a laboratory technician, and two OPD staffs were the member of the team. A health centre head was responsible for coordination of the survey. Those OPD staffs each assigned to screen malaria suspected febrile cases at pediatric and adult OPD separately. During the data collection, participants were requested for blood specimen to be screened for malaria parasite using CareStart™ Malaria Pf/Pv combo test . In addition, thin and thick film was prepared immediately for microscopic examination. CareStart™ RDTs are individually packaged with alcohol swab, lancet, capillary tube, buffer, and test device.Both blood slides and the RDT devices were given identical code for later comparison of results. Specimen collection and reporting result for the malaria RDT was performed using the recommended procedures by the company . First, Plasmodium positive, asexual parasite count was performed using standard procedures of light microscopy as recommended by World Health Organization [Then, thin and thick films were prepared and Giemsa-stained for comparison using light microscopy. Blood specimen processing, examination, and reporting of results were performed using standard guidelines . In brienization . The numnization .P. vivax. Other lines (test line 1) is printed with a monoclonal antibody specific to HRP2 of P. falciparum, and the control area (line 3). So, the CareStart™ Malaria Pf/Pv combo test is designed for the differential diagnosis between P. vivax and P. falciparum[P. falciparum; ii) two colour bands (one band in the control area and another band in the “2” area) indicates a positive result for P. vivax; iii) three colour bands  indicates a positive result for mixed infection for P. falciparum and P. vivax; iv) only one band in the control area within the result window indicates a negative result; and v) the test is invalid if the line in the control area does not appear were used in the interpretation of RDT results [CareStart™ Malaria Pf/Pv combo test contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line 2) is specific to pLDH of alciparum. The fol results . In addiA malaria microscopist temporarily stationed at BRHP-DSS , did theIn order to assure quality of the microscopic examinations, in both surveys, the entire positive and 10% of the negative slides were sent to senior malaria microscopist and re-examined at Adama Malaria Control Laboratory, Adama Town, Ethiopia. In the household surveys, seven of 2,094 slides (0.3%) showed discordant results. A third reader, blinded to the previous results, re-examined the seven discrepant slides . Similar procedure was followed for the survey held at health centres. Each reader was blinded to the RDT results and others’ slide reading results. To ensure maximum participation, households with absentees were re-visited once.This study obtained ethical approval from Institutional Review Board of the College of Health Sciences of the Addis Ababa University, and from the Ministry of Science and Technology of Ethiopia. Local health authorities at all levels also gave permission to conduct the study. Individual informed consent was obtained from adults, and from the parents or guardians of children aged less than 18 years. In addition, minors gave verbal assent. All study participants including minors had the right to refuse. Blood specimens were collected aseptically using an alcohol swab and disposable blood lancets by trained staff. All people found to be malaria positive during the survey were treated directly according to the national guideline .Plasmodium vivax positives were treated with chloroquine, 25 mg/kg for three days . Artemether-lumefantrine (20 mg artemether plus 120 mg lumefantrine in a fixed dose combination) was administered, based on body weight, two times a day for three days to Plasmodium falciparum-positive patients [Febrile patients were screened for malaria parasites on the spot using the RDT, and blood slides were also collected for the survey to investigate the nature of the malaria parasite using light microscopy. Malaria positive cases detected using malaria RDTs were treated immediately. Moreover, those found to be malaria positive on microscopy were also treated. Data entry and cleaning was done using Epi Info version 6  and exported to SPSS. Data analysis was performed using IBM SPSS Statistics 20. Sensitivity, specificity, positive and negative predictive values of the test was computed with 95% confidence interval (CI) by taking malaria microscopy as reference.The kappa statistic or agreement between the test and microscopy was calculated using data observed and probability of expected as recommended . A kappaTM performance was based on self-reported 2,394 febrile cases from public health facilities  and household surveys . The participants’ mean (±SD) age was 13.9 ± 13.0 (from one month to 70 years), and median 10 years in health centre surveys, and 19.7 ± 18.2 (from one month to 99 years) with median 13 years in household surveys. More participants were recruited from Enseno Health Centre  than from Butajira Health Centre  in health facility-based surveys. A total of 31 infants  were suspected for malaria screened (Data not shown).The evaluation of CareStartSeasonal variation of malaria suspected cases observed in both survey settings, which was higher during October-November 2008 in household surveys, and October 2009 and November 2009 at health centres Figure .Plasmodium positive in the household surveys. Of those, 83.9% (n = 73) were caused by P. vivax, 15.0% (n = 13) were due to P. falciparum, and the rest 1.1% (n = 1) were mixed infections due to both vivax and falciparum malaria. Similarly, 24.5% (n = 392) of people were malaria infected among febrile cases visited health centres. Among them, 78.6% (n = 308) were P. vivax, 20.4% (n = 80) were P. falciparum, and the rest 1.0% (n = 4) were mixed infections due to both vivax and falciparum malaria  of people examined were Plasmodium positive confirmed by microscopy. Similarly, 3.3% (n = 24) in household and 17.2% (n = 208) in health facility-based surveys were detected false positive. No invalid test results were observed  in household and 4.3% (n = 17) in health facility-based surveys among 3.3% n = 4 in housPlasmodium infection varied among different age groups, and males were more infected than females in both survey settings. Higher proportion of the malaria infection occurred during peak malaria transmission in both survey settings. About 67% of the total positives cases were screened during October-November 2009 and about 20% during October-November 2008 in household surveys. Moreover, above half of the positives during October 2009 and one-fifths during August and November 2009 each, in the health facility-based surveys  and P. falciparum (87.5%) in household survey; and in P. vivax (92.8%) in health facility surveys. Similarly, low specificity observed in overall Plasmodium (82.7%), and P. falciparum (92.8%) in health facility surveys, and in P. vivax (87.5%) in household surveys.Table Plasmodium (64.3%) and P. falciparum (77.2%) in health facility; and overall Plasmodium (76.7%) and P. falciparum (87.5%) in household surveys. Negative predictive value of the test was good in both overall Plasmodium and P. falciparum. However, lowest NPV was found in P. vivax in both health facility (77.2%) and household (87.5%) surveys. The test and microscopy showed almost perfect agreement for household and health centres surveys  of P. falciparum with asexual parasite count of ≥ 500 p/μl were not detected by CareStart™ RDT. Moreover, the RDT failed detecting 10.3% (n = 7) of vivax malaria, five of those with ≥ 500 p/μl and the rest two with <200 p/μl in household survey. Only a single (1 of 13) falciparum case was missed by RDT in household survey (Table P. vivax (n = 15) (data not shown).In health facilities, 4.5% (n = 10) of microscopy confirmed P. falciparum and P. vivax in “active” and clinical settings of highland-fringe areas of south-central Ethiopia. A greater proportion of RDTs than slides gave positive result for Plasmodium. In the survey, the test showed low sensitivity and high specificity for P. falciparum, but vice versa for P. vivax. The test revealed lower PPV for P. falciparum and lower NPV for P. vivax. Overall, season substantially influences RDT precision with good sensitivity at health facilities compared to household survey. However, specificity was found to be low in both settings with comparable results. Interestingly, better NPV obtained unlike lowest PPV in both survey settings. RDT showed low sensitivity in children aged below 10 years. No strong evidence for contribution of low parasite density for missed Plasmodium detection by the test product.This study found varying precision of CareStart™ RDT to detect This study has got some limitations. This study used light microscopy for comparison of performance of RDT, in spite of its shortcomings related to variable techniques in specimen preparation and slide reading, and mainly the level of expertise of the examining microscopist . Thus, PP. falciparum (87.5%) and P. vivax (92.8%) using CareStart™ RDT is comparable with a study conducted in south-west  and North-West  Ethiopian highland areas [P. falciparum and P. vivax); North-East ; and South-West  Ethiopia [The present finding of low sensitivity for nd areas ,24. But Ethiopia ,23,25. TEthiopia , and in Ethiopia .The varying RDT performance in different seasons in the present household survey is generally consistent with another study in hypoendemic highland zones in Kenya and Uganda . MoreoveVarious factors such as clinical and epidemiologic characteristics of the study populations, reference standards and products of different lots influence the results of RDT-based diagnosis ,13,16. TP. vivax  and P. falciparum (survey) disagrees with other studies that consistently found multi-species RDTs to better detect P. falciparum infection than non-falciparum infection, most probably P. vivax[P. vivax may be interpreted as due to low performance of multi-species RDTs for this species. The low sensitivity obtained in this study may be explained by several factors pertinent to the manufacturing process and environmental conditions [The present finding of low sensitivity of  P. vivax,42. The nditions ,16,20. TIn addition, low-prevalence, or hypoendemic, malaria poses particular diagnostic challenges since low population prevalence reduces the PPV of tests . MoreoveP. falciparum and P. vivax for survey and health facility might be interpreted as the difference in clinical condition of patients. Study participants visiting health facilities might be clinically more specific than otherwise. Review evidences showed that higher RDT sensitivity in studies involving patients seeking relief from moderate to severe disease in clinical settings while low RDT sensitivity in studies that enrolled patients by means of “active” case finding [The varying performance of sensitivity for  finding ,41.P. vivax at both survey and health centres. A past study showed that less sensitive for non-P. falciparum than for P. falciparum[. There is rich evidence that extremely low sensitivity for both HRP-2 and p-LDH tests and batch specific problems were suspected [P. falciparum is likely due to sequestration [The RDT had low NPV for alciparum. There iuspected . This imuspected . The prestration . False pstration ,20. Crosstration .P. falciparum and P. vivax co-exist. CareStart™ RDT could be used for epidemiological studies and results interpreted cautiously by considering its seasonal variation. Future research targeted to RDT evaluation should consider the use of a more sensitive reference standard such as PCR.In conclusion, it appears plausible to put malaria RDT quality control system in place for health systems operating in highlands with low endemicity and seasonally varying transmission where The author(s) declare that they have no competing interests.AW contributed to conception and design, data acquisition, analysis and interpretation, and drafting the manuscript. WD substantially contributed to conception and design of the study and reviewing the manuscript, revisiting it critically for important intellectual content. AA substantially contributed to conception and design of the study, reviewing the manuscript, and revisiting it critically for important intellectual content. BL substantially contributed to conception and design, analysis and interpretation of data, reviewing the manuscript, revisiting it critically for important intellectual content. BL, AA, WD and AW reviewed the paper and all authors approved the final version.


Verbena officinalis, Thymus vulgaris and Origanum vulgare), previously found active against some phytopathogenic Fungi and Stramenopila, have been tested in vitro against five etiological agents of post-harvest fruit decay, Botrytis cinerea, Penicillium italicum, P. expansum, Phytophthora citrophthora and Rhizopus stolonifer. The tested compounds were β-fellandrene, β-pinene, camphene, carvacrol, citral, o-cymene, γ-terpinene and thymol. Citral exhibited a fungicidal action against P. citrophthora; carvacrol and thymol showed a fungistatic activity against P. citrophthora and R. stolonifer. Citral and carvacrol at 250 ppm, and thymol at 150 and 250 ppm stopped the growth of B. cinerea. Moreover, thymol showed fungistatic and fungicidal action against P. italicum. Finally, the mycelium growth of P. expansum was inhibited in the presence of 250 ppm of thymol and carvacrol. These results represent an important step toward the goal to use some essential oils or their components as natural preservatives for fruits and foodstuffs, due to their safety for consumer healthy and positive effect on shelf life extension of agricultural fresh products.Eight substances that are main components of the essential oils from three Mediterranean aromatic plants ( Among them, fungi (s.l.) constitute the more numerous group of plant pathogens and can often cause severe diseases in vegetable and fruit species [The most important plant pathogens,  species . Over se species . These p species . Sometim species . Further species . A notic species .Research on plant-derived fungicides is now being intensified, as it became evident that these substances have enormous potential to improve the future agrochemical technology. In fact, there are good reasons to suppose that secondary plant metabolism has naturally evolved to actively protect vegetable and fruit species from microbial pathogen attacks . Since ss.l.), since they are not only a fragrance and flavour source for food and beverages but are also being discovered as bioactive substances tanks [Among secondary plant metabolites, plant essential oils (PEOs) may provide potential alternatives to active substances currently used to control phytopathogenic fungi  pathogens of orange fruits, were accomplished by our research group [Verbena officinalis L., Thymus vulgaris L. and Origanum vulgare L. could be used as a possible source of ecofriendly botanical fungicides for controlling important postharvest fungal pathogens without phytotoxicity risk. Furthermore, it seemed believable that PEO volatile components could be employed to uniformly permeate storeroom air and protect, maybe better than fungicides commonly used for this goal, fruits that are in storage. Therefore, it seemed opportune to assay the in vitro fungicidal activity of the above mentioned PEO main components against five fungi (s.l.) well known as postharvest fruit decaying agents.Several investigations have been carried out abroad in this field toward antimicrobial and fungicidal PEOs properties ,7–11. Inet al.  and Zamb [et al. , more rech group . Resultsin vitro activity of the tested compounds against the five post-harvest fruit decaying agents, registered ten days after experiment beginning, is summarized in Phytophthora citrophthora did not show any mycelium growth in presence of citral and carvacrol, at concentration of 150 and 250 ppm, and in presence of thymol, at 250 ppm  in Petri dishes containing 150 ppm thymol. Its mycelium appeared white in colour and completely lacking of conidiophore and conidia, when observed at light microscope. The same fungus showed scanty conidiophores production and almost none conidium differentiation in Petri dishes containing citral, at 150 ppm concentration. The growth of Penicillium expansum , R. stolonifer (which seemed killed by 250 ppm thymol) and B. cinerea,  showed a weak colony growth four days after the respective corn meal agar (CMA) and potato dextrose agar (PDA) plugs were transferred from Petri dishes on CMA or PDA. This demonstrated that they were still alive although subjected for some days to exposure to the above PEO components.Among microorganisms which did not show any mycelium growth in presence of some PEO components, only P. citrophthora, exposed to 150 and 250 ppm of citral, and to 250 ppm of thymol, and P. italicum, in presence of thymol at 250 ppm, did not show any micelium growth after being transferred on CMA or PDA, respectively. This demonstrated that these two microorganisms have been effectively killed by the higher concentrations of tested compounds. Colony growth of the last two microorganisms from PDA plugs and CMA plugs exposed to the lower PEO component concentrations was rather slow and then did not differ from that observed in control colonies. All tested microorganisms from basic colonies grew normally in control plates containing only PDA or CMA.Conversely, s.l.) is thought to depend on specific toxicity of their single main active constituents or by their synergic effect [in vitro a fungistatic and/or, in some instances, a fungicidal action against the tested organisms and, therefore, could be employed to control food-stuff decaying agents. In fact, among the eight compounds tested, only β-pinene is reported as toxic for humans [in vivo tests. The last two compounds resulted fungicides against P. citrophthora, whereas thymol at 250 ppm killed P. italicum and markedly inhibited mycelium growth of P. expansum, the well-known and dangerous patulin producer.PEO efficacy against postharvest fruit decaying fungi  was also reported [Fusarium moniliforme J. Sheld., Rhizoctonia solani J.G. Kühn, Sclerotinia sclerotiorum (Lib.) de Bary and Phytophthora capsici Leon. was due to different concentrations of the phenolic fraction (thymol and/or carvacrol) present in the PEOs different aromatic plants. These two compounds were also reported for their in vitro antifungal activity against Ophiostoma novo-ulmi Brasier, causal agent of Dutch elm disease [The available literature reported PEOs as active drugs against phytopathogenic fungi, but very few researches demonstrated the possible fungitoxicity of their constituents. In particular, γ-terpinene, assayed  cinerea . Camphen cinerea . Carvacr cinerea . The strreported ,25. Muelreported  showed t disease .Thymus oils and the presence of phenol components, such as thymol and carvacrol. The high antifungal activity of these essential oils could be explained by the high percentage of phenol components. It seems possible that phenol components may interfere with cell wall enzymes like chitin synthase/chitinase as well as with the α- and β-glucanases of the fungus.Moreover, recently, Soković and coworkers  reportedB. cinerea [Colletotrichum strains [Agaricus bisporus (J.E. Lange) Imbach var. bisporus [Mycogone perniciosa (Magnus) Delacr., another pathogen of this mushroom, was affected by thymol [Colletotrichum acutatum J. H. Simmonds, which is considered as responsible for the major postharvest tomato tree disease in Colombia [P. expansum and B. cinerea [Carvacrol, well known for its important fungitoxic activity against  cinerea ,30 showe strains  and agaibisporus . Mycogony thymol . The samColombia . Thymol  cinerea ,35.P. italicum [Aspergillus niger Tiegh. and R. stolonifer [B. cinerea [Colletotrichum acutatum [Aspergillus flavus Link: after it penetrates cell wall, irreversibly damages plasma membrane and DNA with consequent spore loss germination. Also in A. niger, the primary citral site action seems to be cell membrane [In our assays, citral resulted as one of more active compounds. In literature, it has been reported for its good antimicrobial activity against italicum , Aspergiolonifer , B. cine cinerea ,25 and Cacutatum . Inhibitacutatum –40. Luo acutatum  suggestemembrane . Moreovemembrane .o-Cymene was reported to inhibit growth of the root pathogenic fungi Phytophthora cinnamomi Rands and Fomes annosus (Fr.) Cooke [.) Cooke .o-cymene, γ-terpinene and thymol were purchased from Sigma Aldrich, Co. . All the above compounds were kept at −20 °C.β-Fellandrene, β-pinene, camphene, carvacrol, citral, Phytophthora citrophthora (R.E. & E.H. Smith) Leonian, isolate number 22 from lemon, Botryotinia fuckeliana (de Bary) Whetzel, used as its anamorph Botrytis cinerea Pers., isolate number 234 from grapevine; Penicillium italicum Wehrner, isolate number 333 from orange, Penicillium expansum Link, isolate number 335 from apple and Rhizopus stolonifer (Ehrenb.) Vuill., isolate number 238 from orange, were employed as basic 7–10-day-old colonies which were grown on PDA for P. italicum, P. expansum, B. fuckeliana, and R. stolonifer and on CMA, in the case of P. citrophthora.The species of plant pathogenic Fungi and Stramenopila and relative isolates used in this study and hereafter listed, were derived from a pure culture-maintained collection of the mycotheca of Department of Biology, Plant Protection and Agro-Forestry Biotechnology, Basilicata University , kept on potato destrose agar (PDA) or corn meal agar (CMA) at 4 °C: P. citrophthora, B. cinerea and R. stolonifer and (b) P. italicum and P. expansum was determined in two ways. (a) By putting single 3-mm-thick and 0.5-cm-diameter CMA or PDA plugs, axenically taken away from peripheral portion of basic colonies onto central part surface of 9 cm diameter Petri dishes containing PDA or CMA previously added, at 40 °C, of 0.2% Tween-20 and 50, 150 or 250 ppm of the single PEO components; (b) By dropping, under axenic conditions, 10 μL aliquots of single suspensions containing 1 × 104 conidia/mL of the single Penicillium species onto surface centre of Petri dishes containing PDA and prepared with the same percentage of Tween-20 and the single three PEO component concentration. Three replicates for each tested PEO active compound dose were provided in presence of opportune controls, i.e., PDA and CMA plugs from basic colonies of P. citrophthora, B. cinerea and R. stolonifer and of the two Penicillium species transferred or dropped, respectively, as above into Petri dishes containing either sole PDA or CMA or PDA or CMA added of 0.2% tween 20.The possible fungistatic or fungicidal activity of the single PEO components against (a) The inhibitory effects of PEO components against each tested microorganism belonging to Stramenopila or Fungi kingdoms was determined after a 3–10 days incubation period at room temperature, e.g., when control colonies margins reached plate edges. The shortest and longest colony diameters were measured for each fungal or stramenopilus culture and the average value obtained, considered as a growth index, was subjected to statistical analysis.B. cinerea and R. stolonifer and CMA plugs of P. citrophthora from Petri dishes, prepared with PEO compound concentrations which seemed to have completely inhibited growth of tested microorganism isolates, were axenically transferred into Petri dishes containing only PDA, in the case of the first two fungi or CMA for P. citrophthora. Similarly, plugs axenically excised from central portion of dishes in which growth of P. expansum and P. italicum seemed to have been stopped by some PEO component concentrations, were in the same way put on PDA. Inoculated plates were then transferred in a climatic chamber at 20 ± 1 °C, along with the opportune controls, i.e., Petri dishes containing PDA or CMA inoculated with agar plugs taken from basic colonies of the above phytopathogenic microorganisms. All plates were daily observed for 30 days to ascertain eventual mycelium growth.PDA plugs of p < 0.01.Data were statistically processed and subjected to analysis of variance (ANOVA). Means significantly different were separated by the least significant difference (LDS) with in vivo studies to verify the possible phytotoxic effects of the above PEO active compounds and to set up an application method able to maximize their effect against target post-harvest rot causal agents [The achieved results, along with fact that essential oil poisoning is documented for a few of these volatile compounds often inappropriately used , constitl agents . On the


Pseudomonas aeruginosa is an important opportunistic pathogen in recreational waters and the primary cause of hot tub folliculitis and otitis externa. The aim of this surveillance study was to determine the background prevalence and antimicrobial resistance profile of P. aeruginosa in swimming pools and hot tubs. A convenience sample of 108 samples was obtained from three hot tubs and eight indoor swimming pools. Water and swab samples were processed using membrane filtration, followed by confirmation with polymerase chain reaction. Twenty-three samples (21%) were positive for P. aeruginosa, and 23 isolates underwent susceptibility testing using the microdilution method. Resistance was noted to several antibiotic agents, including amikacin (intermediate), aztreonam, ceftriaxone, gentamicin, imipenem, meropenem (intermediate), ticarcillin/clavulanic acid, tobramycin (intermediate), and trimethoprim/sulfamethoxazole. The results of this surveillance study indicate that 96% of P. aeruginosa isolates tested from swimming pools and hot tubs were multidrug resistant. These results may have important implications for cystic fibrosis patients and other immune-suppressed individuals, for whom infection with multidrug-resistant P. aeruginosa would have greater impact. Our results underlie the importance of rigorous facility maintenance, and provide prevalence data on the occurrence of antimicrobial resistant strains of this important recreational water-associated and nosocomial pathogen. P. aerup system –4. Additoduction ,4. P. ae regimen . The oveesistant . This refections .P. aeruginosa and is a meaningful exposure pathway for bacterial transmission, where wet skin and occlusion provide optimal conditions for P. aeruginosa to thrive [P. aeruginosa has been implicated in numerous nosocomial and community outbreaks, with therapy tanks and whirlpools frequently acting as the environmental reservoir [P. aeruginosa contamination; in nosocomial and household settings, contaminated sinks and shower heads have been a common reservoir for P. aeruginosa, where the inaccessible armature is nearly impossible to adequately decontaminate [P. aeruginosa proliferation, which routinely grows in water 4–42 °C [Indoor recreational water is an important reservoir for o thrive . P. aerueservoir –10. Comptaminate ,11. Swim 4–42 °C ,12,13. F 4–42 °C ,15.P. aeruginosa contamination, the background prevalence of P. aeruginosa in indoor recreational water has not been well evaluated during non-outbreak periods, where adequate chlorine levels are expected to prevent contamination [P. aeruginosa in indoor swimming pools and hot tubs during non-outbreak periods may have important infection control and facility maintenance implications. Our first study goal was to examine the prevalence of P. aeruginosa contamination in both of these indoor recreational waters, through the collection of water and swab samples.While conditions are ideal for mination ,16. DeteP. aeruginosa isolates from swimming pools and hot tubs. The presence of multidrug-resistant P. aeruginosa in a recreational environment may be important for immune-suppressed or other at-risk individuals, for whom treatment difficulties have greater implications [P. aeruginosa often result in increased cost of treatment, lengthy stay, and overall morbidity and mortality [P. aeruginosa to proliferate rapidly when disinfectant levels fall below recommended levels, monitoring the prevalence of resistant strains may be important for the prevention of future outbreaks.The second goal of the study was to evaluate the level of antimicrobial resistance among the ications . Indeed,ortality ,17,18. I2.2.1.A convenience sample of 50 water and 58 swab samples were obtained from eight indoor swimming pools and three hot tubs from March, 2009 through April, 2010 from throughout the central Ohio area in the United States. All swimming pools were public Class A or B competition or large recreation pools . One hot tub was a small residential model and two were public, large capacity systems. All public hot tubs and pools were operated by either an educational institution or municipality. Water samples were collected in sterile, polypropylene tubes, at approximately 0.30–0.50 meters below the water surface to best simulate an exposure zone; this depth is similar to a sampling regime recommended for beach water . SterileWater samples were analyzed for free chlorine, total chlorine, and turbidity. Free and total chlorine levels were measured immediately according to DPD Method 10069 and 10070 using a Hach 2800 spectrophotometer . The method reporting range is 0.1–10.0 mg/L . Turbidity readings were measured using a Hach 2100P portable turbidimeter .2.2.P. aeruginosa samples, and confirmed using polymerase chain reaction . Negative and positive controls (ATCC 27853) were utilized for all reactions. All end-point PCR was conducted using a MultiGene Thermal Cycler , under the following conditions: 95 ºC for 1 min; 40 cycles of denaturation at 95 ºC for 15 s, annealing at 58 ºC for 20 s; final extension at 68 ºC for 40 s; with primer pa722F (5′-GGCGTGGGTGTGGAAGTC-3′) and pa899R (5′-TGGTGGCGATCTTGAAC TTCTT-3′). For the real-time PCR, we used ABI Pseudomonas detection kit  with a 48-well StepOne™ Real Time System  following the manufacturer’s instructions.Water samples were concentrated by membrane filtration using a sterile 0.45-μm-pore-size, 47-mm-diameter cellulose membrane filter  . Swab sa2.3.P. aeruginosa-positive sample) were chosen for analysis and processed according to the manufacturer’s recommended protocol and the Clinical and Laboratory Standards Institute methods [P. aeruginosa-positive samples; isolates from three samples failed to be subcultured sufficiently for microdilution analysis.The minimum inhibitory concentration (MIC) to 22 antimicrobial agents was determined using a TREK Sensititre Sensitouch microdilution system . Twenty-three randomly selected isolates ; all samples from the residential hot tub were positive. Sixty-seven percent (2/3) of hot tubs and 63% (5/8) of swimming pools were positive at least once for P. aeruginosa. P. aeruginosa-positive samples were much more likely to come from swabs than from water samples. The positive swab locations were diverse, including side-wall tiles, gutter drains, jets, and strainer baskets which may indicate that these are biofilm-rich locations. A total of 108 water  and swab samples  were collected from eight swimming pools and three hot tubs. Twenty-three samples (21%) were positive for P. aeruginosa-positive and negative samples with respect to water temperature and free chlorine. Turbidity levels were very similar between the groups. Water quality data were not available for the residential hot tub (which comprised 63% of the P. aeruginosa positive hot tub samples).Water analysis indicated low turbidity overall (0.1–0.6 NTUs), and a wide range of free (FC) and total chlorine (TC) levels . Swimming pool temperature ranged from 25.9 °C to 32.4 °C, and hot tub temperature 38.7 °C to 39.3 °C. Differences were noted between etc.; See Pseudomonas aeruginosa infections. Resistance is defined as an MIC result above the susceptibility upper bound, and intermediate resistance is defined as an MIC result above the susceptibility lower bound, but below the susceptibility upper bound.All 23 isolates analyzed were resistant to at least one antimicrobial, and several were multidrug-resistant. Of the clinically relevant antibiotics included in the screening panel, there was resistance to amikacin , aztreonam (22%), ceftriaxone , gentamicin (9%), imipenem (26%), meropenem , ticarcillin/clavulanic acid (4%), tobramycin , and trimethoprim/sulfamethoxazole . P. aeruginosa contamination is common in swimming pools and hot tubs, even where chlorine concentrations are well above recommended levels. Recommendations for adequate chlorine levels are 2 to 4 mg/L for swimming pools, and 3 to 5 mg/L for hot tubs, measured as free chlorine [P. aeruginosa may return with a level of 104–106 cells/mL within 24 to 48 hours when adequate chlorine levels are not maintained [P. aeruginosa was routinely isolated, the regularly high chlorine levels found here likely reduced prevalence. This confirms the importance of not only adequate disinfectant levels, but also constant monitoring and adjustment to ensure chlorine levels never drop below recommended levels. It should be noted that the facilities in this study conducted hourly monitoring of pool and hot tub chlorine concentrations, except the residence, which performed weekly monitoring. The less frequent monitoring of the residential hot tub may have resulted in greater halogen fluctuations, and thus higher prevalence of P. aeruginosa.The findings of this study indicate that chlorine . Accordiintained . In the versus water samples, which are more likely to represent planktonic bacteria), and the presence of P. aeruginosa, was noted. LeChevallier et al. (1988) have described several mechanisms leading to disinfectant resistance, of which attachment to surfaces was most important [P. aeruginosa in pools/hot tubs with higher chlorine levels. There was also a relationship between higher water temperature and the presence of P. aeruginosa. This finding is consistent with the thermo-tolerant nature of P. aeruginosa.Consistent with previous research, a relationship between high chlorine levels, biofilm growth ; this may act to concentrate microbial load present in the water [There are several possible reasons why ormation . In addihe water . FinallyP. aeruginosa has considerable levels of antibiotic resistance. Isolates demonstrated resistance to a wide range of clinically relevant antimicrobial agents, including antipseudomonal agents aztreonam (22%), gentamicin (9%), and imipenem (26%); and intermediate resistance to amikacin (9%), meropenem (4%), and tobramycin (9%). Resistance was also noted to alternate agents ceftriaxone , ticarcillin/clavulanic acid (4%), and trimethoprim/sulfamethoxazole . Although P. aeruginosa is a model of antimicrobial resistance (due to a number of intrinsic factors), past research has found that the degree of resistance to antipseudomonal agents varies considerably. The current results are similar to past studies, which have found analogous prevalence levels of resistant P. aeruginosa in both the hospital and community [The results of this study also indicated that environmental eropenem %, and toommunity .P. aeruginosa is intrinsically resistant, it is possible that the frequency with which we isolated resistant P. aeruginosa is representative of natural background levels. Alternately, in some cases resistant P. aeruginosa strains may ultimately be traced to a human source, either direct shedding from colonized humans or indirect transmission through the contamination in upstream aquatic reservoir. P. aeruginosa is not a predominant component of normal human flora  [P. aeruginosa strains which infect patients and those in the immediate patient environment. Rogues et al. [P. aeruginosa by patients was “both the source and the consequence of (tap) water colonization”. That is, a colonized or infected patient may lead to in-room and neighboring area contamination; and contaminated water, fomites, and aerosols may infect susceptible individuals [The origins of multidrug-resistant strains in the swimming pool and hot tub environment are unknown, but may have developed through several mechanisms. Since  matter) . However matter) . Also, i matter) ,26. In as et al.  pointed-ividuals . A similIn some cases, there may be selective pressure for resistance by the presence of low-level antibiotic concentrations in either sweat secretions, which may enter recreational water through bather use, or from supply water. Prior research has demonstrated the presence of a range of antimicrobial agents in arm and axilla sweat secretions after oral or intravenous dosing . While iP. aeruginosa has both intrinsic resistance and a dynamic ability to develop resistance during the course of infection, a high frequency of resistance is now expected in hospitals. However, in the non-clinical environment, the absence of selective pressure may reduce antimicrobial resistance levels. The presence of resistance to front-line antipseudomonal drugs may have important clinical and prevention implications. Given how common swimming pool and hot tub use is, immune-suppressed or other high-risk individuals may wish to exercise caution before use, especially hot tub use. Exposure to resistant P. aeruginosa results in even greater risk for individuals with cystic fibrosis.While the levels of resistance among study isolates are similar to those previously reported, it is important to draw the distinction between a nosocomial setting and the non-clinical, non-outbreak, recreational setting of the current study. In clinical scenarios, there is constant selective pressure to enhance the proliferation of multidrug-resistant strains. Given that P. aeruginosa and the degree of antimicrobial resistance. The residential hot tub owner did not experience previous skin diseases related to the hot tub and the hot tub did not undergo thorough maintenance and flush-fill cycles as did the public hot tubs, which were cleaned daily, drained completely once per week, and have fresh water introduced continually. Further study is required with expanded sample size to determine if the frequency of multidrug-resistant strains remains steady over time. Additional studies should focus primarily on hot tubs, where 53% of samples were positive. Also, isolates from three P. aeruginosa-positive samples were unable to be revived for antimicrobial resistance analysis.Study limitations include a relatively small sample size and the convenience design. Although only one residential hot tub was accessible for sampling in this study, there was a desire to broadly evaluate whether differences existed in the prevalence of 4.Pseudomonas aeruginosa can be regularly isolated from both swimming pools and hot tubs, with substantial prevalence. In addition, isolated P. aeruginosa is resistant to a wide variety of antimicrobial agents, including front-line antipseudomonal drugs. These results are despite the high disinfectant levels noted in water samples. We believe that these findings underlie the critical importance of regular, thorough, aggressive maintenance to ensure a clean, healthy swimming pool and hot tub environment. The results confirm that high chlorine levels are not alone sufficient to prevent P. aeruginosa growth, and that notable levels of antimicrobial resistant P. aeruginosa are endemic in both hot tubs and swimming pools.The results of this study indicate that


Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions.The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS) putative auxin carriers localize to the endoplasmic reticulum (ER) and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and, subsequently, affect nuclear auxin signaling. Here, we investigate sequence diversification of the PILS family in  Plant development is particularly flexible due to its postembryonic growth behavior, allowing individual adjustment of the body plan according to the environment  auxin transport also determines cellular auxin levels  or PhytozomeSequences were downloaded from PLAZAin silico analyses of PILSes are found at: http://www.arabidopsis.org , http://bioinformatics.psb.ugent.be/plaza/ , www.genevestigator.com (expression), http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi (expression), http://www.enzim.hu/hmmtop/index.php (prediction of protein topology), http://biophysics.biol.uoa.gr/TMRPres2D , http://weblogo.berkeley.edu/logo.cgi (sequence logo), http://www.cbs.dtu.dk/services/NetPhos/ (prediction of phosphorylation sites), http://www.cbs.dtu.dk/services/NetPhosK (prediction of phosphorylation sites), http://www.ebi.ac.uk/Tools/msa/clustalw2/ , http://blast.ncbi.nlm.nih.gov/ (sequence information), http://www.phytozome.net/ (sequence information), http://www.r-project.org/ .The online available servers used to perform A. thaliana PILS genes by comparing 200 surrounding translated genes for each PILS. The comparison was performed for pairs of PILS genes by using blastp program  for analysis of PILS amino acid sequences or on the Whelan and Goldman model with discrete Gamma distribution  for analysis of PIN-PILS dataset. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. The PILS analysis involved 42 amino acid sequences and 322 positions in the final dataset. The PIN-PILS analysis involved 67 amino acid sequences and 354 positions. Evolutionary analyses were conducted in MEGA5 , Zea (10), Medicago (13), or Populus (18), have 6 to 18 PILS genes , Zea 0, MedicaPhyscomitrella, Selaginella, Picea, Brachypodium, Oryza, Medicago, Arabidopsis, and Populus sequences .The available green algae genomes from the lineage e Figure . We coulPhyscomitrella, Selaginella, Brachypodium, or Oryza  localize to the ER and regulate the subcellular compartmentalization of auxin and auxin metabolism . Only very weak or no collinearity was detectable between PILS5 and PILS7  PILS1/PILS3/PILS4, (ii) PILS5/PILS7, and (iii) PILS2/PIL6.Next we addressed the sequence diversifications among the in silico PILS gene organization and expression. A. thaliana PILS gene transcripts organization is pretty well-conserved regarding the number and size of the exons , 3 (125 nucleotides), and 4 (122 nucleotides) is largely kept in AtPILS genes and encode for a highly conserved region of the predicted transmembrane helices 2–4 . Also a C-terminal transmembrane domain seems to be encoded by the last exon (125 nucleotides) in almost all AtPILS genes  and 7–12 exons , medium (PILS6) or high  expression levels (see text footnote 5). PILS2-to-AtPILS6 are expressed in seedlings, leaves, and flowers . Based on these evidences it seems that PILS genes show specific and partially overlapping expression patterns in all plant tissues.PILS gene activity. PILS3 and PILS5 appear to bear two and four alternative transcripts, respectivelyPILS3 and PILS5 function. However, the importance of PILS transcript splicing remains to be demonstrated.Alternative splicing might furthermore contribute to the regulatory complexity and diversity for The pronounced differences in the expression levels and tissue distributions might indicate that PILS-mediated regulation of plant growth and development may be largely determined by gene regulation.PILS genes will give rise to tissue specific distribution of distinct PILS proteins. Next we analyzed predicted PILS protein organization and searched for domains to speculate on PILS function. PILS proteins range in size from 390  to 472  amino acids. However, the predicted protein topology is highly similar for all PILS proteins. PILS proteins are presumably characterized by two hydrophobic transmembrane regions found at N- and C-termini , kinase-specific phosphorylation sites and isoform variations could be predicted for PILS proteins by available online servers such as NetPhos ]. The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 75 nucleotide sequences. All positions with less than 0% site coverage were eliminated. That is, fewer than 100% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 1113 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., Click here for additional data file.Supplementary Figure S2Alignment of PILS amino acid sequences. The multiple amino acid alignment of PILSes was generated by using Muscle in MEGA5 software (Tamura et al., Click here for additional data file.Supplementary Figure S3Alignment of PILS and PIN amino acid sequences. The multiple alignment was generated by using Muscle in MEGA5 software (Tamura et al., Click here for additional data file.Supplementary Figure S4Arabidopsis thaliana PILS amino acid sequencesAlignment of . A multiple sequence alignment generated by ClustalW server (Larkin et al., Click here for additional data file.Supplementary Table S1Sequence information.Click here for additional data file.


Efficient perception of attacking pathogens is essential for plants. Plant defense is evoked by molecules termed elicitors. Endogenous elicitors or damage-associated molecular patterns (DAMPs) originate from plant materials upon injury or pathogen activity. While there are comparably well-characterized examples for DAMPs, often oligogalacturonides (OGAs), generated by the activity of fungal pathogens, endogenous elicitors evoked by bacterial pathogens have been rarely described. In particular, the signal perception and transduction processes involved in DAMP generation are poorly characterized.Xanthomonas campestris pv. campestris deficient in exbD2, which encodes a component of its unusual elaborate TonB system, had impaired pectate lyase activity and caused no visible symptoms for defense on the non-host plant pepper (Capsicum annuum). A co-incubation of X. campestris pv. campestris with isolated cell wall material from C. annuum led to the release of compounds which induced an oxidative burst in cell suspension cultures of the non-host plant. Lipopolysaccharides and proteins were ruled out as elicitors by polymyxin B and heat treatment, respectively. After hydrolysis with trifluoroacetic acid and subsequent HPAE chromatography, the elicitor preparation contained galacturonic acid, the monosaccharide constituent of pectate. OGAs were isolated from this crude elicitor preparation by HPAEC and tested for their biological activity. While small OGAs were unable to induce an oxidative burst, the elicitor activity in cell suspension cultures of the non-host plants tobacco and pepper increased with the degree of polymerization (DP). Maximal elicitor activity was observed for DPs exceeding 8. In contrast to the X. campestris pv. campestris wild type B100, the exbD2 mutant was unable to generate elicitor activity from plant cell wall material or from pectin.A mutant strain of the phytopathogenic bacterium C. annuum and X. campestris pv. campestris. The bacterial TonB-system is essential for the substrate-induced generation of extracellular pectate lyase activity. This is the first demonstration that a TonB-system is involved in bacterial trans-envelope signaling in the context of a pathogenic interaction with a plant.To our knowledge, this is the second report on a DAMP generated by bacterial features. The generation of the OGA elicitor is embedded in a complex exchange of signals within the framework of the plant-microbe interaction of  Arabidopsis thaliana[oxidative burst, and a programmed cell death [Erwinia[Xanthomonas[Pseudomonas[Pseudomonas syringae[Plant cells are permanently monitoring their immediate environment to identify attacking pathogens and subsequently initiate defense. Highly conserved molecular structures termed microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) are obvious targets for this recognition process. The term PAMP-triggered immunity (PTI) is increasingly used for this innate immunity . Recogni thaliana. In olde thaliana. Plant dll death . Example [Erwinia,6, Xanthnthomonas,8, or Pseudomonas, syringo syringae or lipop syringae.Erwinia carotovora has been reported to induce the generation of a DAMP [A. thaliana wall-associated kinase 1 (WAK1) was identified as a candidate for a PRR that specifically recognizes OGAs. While the receptor-like kinase WAK2 was shown to be involved in pectin-dependent signaling [In addition to monitoring for pathogen-derived MAMPs, plants recognize endogenous molecules that are released upon injury or infection as alarm signals. Such molecules are termed damage-associated molecular patterns (DAMPs) . Often Df a DAMP , which af a DAMP . Upon thf a DAMP , the A. ignaling , a recenignaling , therebyXanthomonas species are members of the γ subdivision of the Gram-negative Proteobacteria, which have adopted a plant-associated and usually plant pathogenic lifestyle [Xanthomonas campestris pv. campestris is a pathogen of Brassicacea including A. thaliana. Upon infection of cabbage plants it causes the black rot disease. In non-host plants like pepper (Capsicum annuum) and tobacco (Nicotiana tabacum), however, it induces an HR. For X. campestris pv. campestris, LPSs [X. campestris pv. campestris seems to be a very complex situation, where multiple elicitors are active in parallel [X. campestris pv. campestris in host plants and to the induction of a resistance response in non-host plants. Protein secretion systems, in particular the type III secretion system, have an important role in the pathogenic interactions with plants [X. campestris[Xylella fastidiosa, where a polygalacturonase has been characterized that is similar to the pglA2 gene product of X. campestris pv. campestris B100 [X. campestris[X. campestris two-component systems signal-transduction systems are characterized experimentally [ifestyle ,25. Xantis, LPSs -29, as wis, LPSs , fragmenparallel ,31. The h plants -35. Furth plants ,36. Theyh plants -40, or ph plants . Pectateampestris-47. Moreris B100 . Rapid pampestris-52. Concampestris and a diampestris, respectmentally -58.X. campestris pv. campestris tonB gene cluster showed some very interesting characteristics. TonB systems of Gram-negative bacteria are multi-component transport systems that perform the specific active uptake of various compounds across the outer membrane [X. campestris pv. campestris, an unusual high number of diverse TonB-dependent receptors has been identified in a profound analysis [X. campestris pv. campestris [N-acetylglucosamine, but their specific role remained unclear [X. campestris pv. campestris genes tonB, exbB, and exbD1, which code for the TonB system core components, are essential for iron uptake [Brassica oleracea, to induce an HR in the interaction with the non-host plant C. annuum, and they are involved in the infection of X. campestris pv. campestris by the lytic bacteriophage ΦL7 [X. campestris pv. campestris there is a similar second exbD gene, termed exbD2, which is located in the same gene cluster in tandem directly downstream of exbD1[exbD genes located in tandem has only been described for the fish-pathogenic Flavobacterium psychrophilum, where again the exbD2 gene, which was also not required for iron uptake, was involved in pathogenicity [X. campestris pv. campestris exbD2 gene is not well understood in detail, there are hints that the gene product is involved in the export of X. campestris pv. campestris exoenzymes.In previous analyses, the membrane . These smembrane . TonB ismembrane . For X. analysis . Functiompestris . A gene mpestris . TonB-de unclear . The conn uptake . They arhage ΦL7 . Differi of exbD1. This ge of exbD1. A similgenicity . AlthougexbD2 gene in more detail. In the course of the analyses, we discovered that exbD2 is involved in the induction of bacterial pectate lyase activity, which then releases OGAs from plant-derived pectate that are subsequently recognized as a DAMP by the plant.In this study, we have analyzed the tonB gene cluster of X. campestris pv. campestris is unusual, and the role of the second exbD gene located in this cluster is still puzzling. Differing from the genes tonB, exbB, and exbD1, exbD2 is not required for iron uptake [C. annuum[The structure of the n uptake , but it C. annuum. Hence, X. campestris pv. campestris. This led already to the discovery of an unexpected wealth of TonB-dependent receptors [tonB, two copies of exbB and four copies of exbD were identified within the genome. Downstream of the previously characterized tonB-exbB-exbD1-exbD2 genes, which are located close to the chromosomal origin of replication, a third exbD gene was identified  cassettes in tonB1, exbB1, exbD1, and exbD2[tonB1, exbB1, exbD1, and exbD2, while there were substantial halos caused by pectate degradation around colonies of the wild-type and a positive control , B100-7.03 (exbB1), B100-9.01 (exbD1), B100-11.03 (exbD2), and the wild-type were grown in liquid medium under inducing conditions. The pectate lyase activity was determined in a photometric assay [X. campestris pv. campestris mutations analyzed, it seemed likely that the mutations in the genes tonB1, exbB1, exbD1, and exbD2 affected the induction of pectate lyase genes.These first results were checked in a more elaborate approach. The strains B100-5.05 . This plasmid, which could not replicate in X. campestris pv. campestris, was integrated into the chromosomes of the X. campestris pv. campestris wild-type strain B100 and of the exbD2 mutant, which was not affected in iron uptake [X. campestris pv. campestris strains carrying the constitutively expressed pglI gene, the wild-type as well as the exbD2 mutant, were then infiltrated into C. annuum leafs. Here, the complemented exbD2 mutant induced an HR with symptoms similar to the wild-type, although with a delay of one day , centrifuged again and dialyzed with a molecular-weight cut-off of 1000 Da. These samples were tested for elicitor activity in tobacco suspension cell cultures by measuring H2O2 generation, the so-called oxidative burst. While the supernatant of incubated cell walls induced no activity, the cell walls co-incubated with X. campestris pv. campestris gave rise to an oxidative burst that indicated the presence of a soluble elicitor  are MAMPs that induce pronounced elicitor activity [oxidative burst caused by the X. campestris pv. campestris supernatant. To purify the supernatants from LPS, polymyxin B agarose was employed, which binds LPS with high affinity. By this method, essentially all elicitor activity could be removed from the X. campestris pv. campestris supernatant . This was in good accordance with an elicitor composed of carbohydrates like oligosaccharides or polysaccharides. To further characterize the elicitor, the monosaccharide composition of the supernatant was determined by total hydrolysis with trifluoroacetic acid. The resulting monosaccharide sugars were identified by HPAEC  with subsequent centrifugation should remove or inactivate proteins from the supernatant. Considering these preliminary facts and that X. campestris pv. campestris contained more galacturonate than the controls (data not shown). As Xanthomonas has extracellular pectate lyases, it seemed reasonable that the elicitor-active compound could be a pectate fragment from the plant cell wall and hence a DAMP, as it was reported for E. carotovora[Another major component of the plant cell wall is galacturonate, which is the building block of pectate and which in combination with rhamnose. To monitor also this compound, compositional analyses of the charged sugars were carried out using HPAE chromatography. These experiments gave evidence that the co-incubation of plant cell wall material and arotovora. The eliX. campestris pv. campestris was analyzed by MALDI-TOF MS  was measured. The cultures showed a specific oxidative burst reaction  have a completely different function as they act as suppressors of the plant pathogen defense and thereby promote the growth of pathogenic fungi . In A. treceptor ,23, onlyarotovora.avr gene products interactions with host plants [hrp gene products [Xanthomonas. Obviously, all these MAMPs and DAMPs are part of the very complex and specific damage- and microbe-associated molecular signal, where individual elicitors contribute in a complex manner [Plants need to permanently monitor whether there are indications for pathogen attack, a task that is not trivial as it requires to efficiently filter pathogen-related signals from others, like those generated by commensal or symbiotic microorganism. For each plant it is of fundamental importance to decide correctly whether to initiate defense or not, as defense includes expensive measures like sacrificing plant tissue by intentional cell death at the assumed infection site, while mistakenly omitted defense can be lethal . Analyzit plants , lipopolt plants ,27, murot plants , hrp genproducts , secreteproducts  and the x manner  to obtaiA. thaliana OGA receptor WAK1 was recently identified [E. coli[exbD2 gene product of X. campestris pv. campestris B100 seems involved in trans-envelope signaling via the TonB system, while the exbD1 gene is also required to import substances like ferric iron [exbD2 might also be involved in uptake of cell wall degradation products, and as exbD1 might be involved in further so far unidentified signaling processes. Currently there is no evidence that the products of both genes are involved in both functions, transportation and signaling. But likewise, so far there is no reason to assume strict task sharing, where the exbD1 gene product is exclusively required for transport, while ExbD2 is specialized on signaling.While the entified ,23, it ientified ,84 besidentified , which c [E. coli. The exbric iron . HoweverX. campestris pv. campestris B100 includes six genes of enzymes that cleave the glycosidic bonds between adjacent glucuronic acid residues  is supposed to be involved in signaling exclusively by contributing to energizing the outer membrane TonB-dependent transducer via TonB. The specific involvement of ExbD2 in signaling may indicate a more direct role of this ExbD isoform in signal transduction.Against the emerging background of TonB-related signal transduction  it is noX. campestris pv. campestris was grown at 30°C on solid TY medium  plants derived from seedlings were grown in the greenhouse at 21°C with 12/12 day/night hours. Cell wall material was isolated from 6 weeks old plants.X. campestris pv. campestris cultures were grown for 24 h in M9 medium supplemented with pectate and FeSO4. The resulting values were calibrated to the activity of glucose-6-phosphate dehydrogenase. For the tests on agar plates [X. campestris pv. campestris strains were cultivated for 2 days on M9 medium supplemented with pectate and FeSO4 as described elsewhere [Extracellular pectate lyase activity was monitored by an agar plate test and quantified in a photometric assay . For ther plates , X. camplsewhere .Xanthomonas genomes that were available from public databases [http://www.cazy.org/) has been considered [Genome data were analyzed and visualized by means of the GenDB annotation system . The EDGatabases ,46,96-99nsidered .BamHI fragment of X. campestris pv. campestris 8004 carrying the pglI gene in cosmid pIJ3051 [BamHI-ClaI sub-fragment with the pglI gene was then transferred to the cloning vectors pBCKS+ and pBCSK+, resulting in the plasmids pHGW261 and pHGW262, respectively. In pHGW262, pglI was constitutively expressed in E. coli from the lac promoter of the pBCSK+ multiple cloning site. To express pglI also in X. campestris pv. campestris, pHGW267 was constructed by cloning the 3.8 kb BamHI-ClaI sub-fragment with the X. campestris pv. campestris 8004 pglI gene into the multiple cloning site of pUC6S (Apr) [out promoter of the aacC1 gene from pMS246 [BamHI fragment into the BamHI site upstream of pglI.All cloning was performed applying standard methods  and as d pIJ3051  was inse6S (Apr) , where im pMS246 , which wC. annuum were employed to obtain cell wall material. Leafs (30 g) were homogenized in 150 ml sodium acetate  for 3 min and filtered with a fluted filter. After the filtration, the cell wall material was washed with 1 l sodium acetate (4°C), 1 l ethanol (4°C) and with 1 l acetone (−20°C). The washed material was then air dried at room temperature and stored under inert atmosphere at -20°C.Leafs of X. campestris pv. campestris over-night liquid culture was centrifuged. After removal of the supernatant, the sediment was re-suspended in 5 ml phosphate buffer . 100 mg of the isolated cell wall material was then added to this solution and incubated over night at 28°C. The sample was then centrifuged and the pellet discarded. After heating , centrifugation  and dialysis (molecular weight cut off 1000), the sample was freeze-dried. Resuspended lyophilized material was then used for further experiments.5 ml X. campestris pv. campestris lipopolysaccharides (LPSs) were removed from the elicitor preparation using a batch technique by adding an excess amount of polymyxin B agarose [ agarose  as descr agarose . Upon adThe analyses of oligosaccharides was performed by HPAEC using a DIONEX GP-40 gradient pump; a Merck-Hitachi D-2000 Chromato Integrator; a DIONEX pulsed amperometric detector and a DIONEX UV detector.Monosaccharide composition of isolated oligosaccharides was analyzed upon acid hydrolysis in trifluoroacetic acid . Neutral sugars were separated and identified using an isocratic elution  with amperometric detection on a CarboPac® PA-100 column. For charged sugars a linear sodium acetate gradient ranging from 0.02 M to 0.5 M under alkaline conditions (0.1 M NaOH) with a flow rate of 1 ml/min was used .Pectate fragments were separated using a sodium acetate gradient . For the identification of pectate fragments a pectate standard was generated by digestion of pectin  by pectate lyase (Sigma P-7052). The isolation of pectate fragments was carried out under the conditions described above, but a semi-preparative column  was used.–1 crude extract lyophilisate dissolved in demineralized water. One microliter of this mixture was dropped onto the MALDI target.Crude extracts were analyzed on a Bruker ultraflex I MALDI-TOF mass spectrometer  in the negative–ion mode. Samples were analyzed in the linear and in the reflector TOF. Gentisic acid was used as matrix. For sample preparation, 1 μl saturated gentisic acid solution was mixed with 1 μl of 50 mg mloxidative burst was performed using the H2O2-dependent chemiluminescence reaction described by Warm [oxidative burst 200 μl aliquots of these suspensions were mixed with phosphate buffer  and 1.2 mM luminol in the same phosphate buffer. The reaction was started by the addition of 100 μl of 14 mM potassium hexacyanate. The luminescence was measured with a Luminometer 1250 . The intensity of luminescence was calibrated for hydrogen peroxide concentrations of 0.01 mM, to 0.05 mM.The detection of the  by Warm . Three t by Warm ) and incPolygalacturonic acid , pectin and polymyxin B agarose was from Sigma-Aldrich, Taufkirchen, Germany. Unless otherwise specified, other chemicals were obtained from Merck, Darmstadt, Germany.Ap: Ampicillin; bp: Base pair; CBM: Carbohydrate-binding Module; CDS: Protein-coding Sequence; Cm: Chloramphenicol; DAMP: Damage-associated Molecular Pattern; DF: Diffusible Factor; DP: Degree of Polymerization; DSF: Diffusible Signal Factor; Gal: Galactose; Glc: Glucose; Gm: Gentamicin; HPAE: High-performance Anion Exchange; HPAEC: High-performance Anion Exchange Chromatography; HPLC: High-performance Liquid Chromatography; HR: Hypersensitive Response; Kb: Kilobase; Km: Kanamycin; LPS: Lipopolysaccharide; MALDI-TOF: Matrix-assisted Laser Desorption/ionization time-of-flight; MAMP: Microbe-associated Molecular pattern; min: Minute(s); MS: Mass Spectroscopy; OGA: Oligogalacturonide; PAMP: Pathogen-associated Molecular Pattern; PGA: Polygalacturonide; PRR: Pattern Recognition Receptor; PTI: PAMP-triggered Immunity; r: Resistant; Rha: Rhamnose; ROS: Reactive Oxygen Species; Sm: Streptomycin; UV: Ultraviolet.The authors declare that they have no competing interests.X. campestris pv. campestris strains on C. annuum. HS carried out in large part the OGA-related analyses and composed an early draft of the manuscript. VKS characterized the isolated pectate fragments by HPAE chromatography. KM carried out oxidative burst measurements with suspension cell cultures of the non-host plant N. tabacum. HK supervised experiments carried out by HS. AP provided infrastructure and advice, in particular related to the genes of the TonB system. KN supervised the whole project, and provided part of the manuscript’s discussion section. All authors read and approved the final manuscript.FJV has performed genomic analyses, compiled the experimental results, and wrote the main part of the manuscript. HGW initially suggested the study, provided genetic constructs, and analyzed the pectate lyase activity and its effect on the HR of Multiple alignment of Xanthomonas exbD2 gene products.Click here for fileFigure displaying the recovery of extracellular pectate lyase activities in complemented X. campestris pv. campestris strains originally deficient in genes of the TonB system.Click here for fileTable S1 with pectate lyase activity in X. campestris pv. campestris and E. coli strains.Click here for fileFigure displaying oxidative burst reactions in heterologous N. tabacum cell suspension cultures upon elicitation with supernatants of X. campestris pv. campestris cultures deficient in genes of the TonB system.Click here for fileTable S2 with genes of pectin-degrading enzymes in X. campestris pv. campestris B100.Click here for file


AbstractDayus Mahmood are described from China: D. bifurcatussp. n., D. trifurcatussp. n. and D. serratussp. n. A key to distinguish all Chinese species of the genus is provided.Three new species of the Oriental empoascine leafhopper genus  Dayus was established by Mahmood in Dayus elongatus Mahmood (Singapore) as its type species. Subsequently, Dayus takagii Dworakowska (Japan) and transferred Dayus upoluanus  (Western Samoa) and Dayus euryphaessus  (Fiji) to the geuns, Dayus formosus from China (Taiwan) and Dayus lii Qin & Zhang, Dayus membranaceus Qin & Zhang and Dayus lamellatus Qin & Zhang.The Oriental typhlocybine leafhopper genus Dayus from China and provide a key for the separation of all known Chinese species. The specimens examined are deposited in the Institute of Entomology, Guizhou University, Guiyang, Guizhou, China (GUGC) and The Natural History Museum, London (BMNH).Here we describe three new species of The methods and terminology follow GenusMahmoodhttp://species-id.net/wiki/DayusDayus Mahmood, 1967: 39.Dayus elongatus Mahmood, 1967 by original designation.rd apical cell petiolate, cua cell broad distally; veins RP, MP’ and MP’’+CuA’ arise from m cell. Hindwing .The new speciesnamealludes to the trifurcate processes of the aedeagus.Thenew species can be distinguished mainly by the strongly sinuate posterior margin of the pygofer and shape of the aedeagal shaft and configuration of its process as noted in the description.Yu & Yangsp. n.http://zoobank.org/A163BC19-73A4-459A-BA07-125F00EF9D8Ahttp://species-id.net/wiki/Dayus_serratusLength, male 3.9mm.General color yellowish.Male ventral abdominal apodemes reaching segment 3 . Male pyHolotype male. China: Hainan Province, Wuzhi mountain, 13 April 2009, coll. Zaihua Yang.The new species name is derived from the serrationsat midlength of the aedeagal shaft and base of the aedeagal processes.Thenew species can be distinguished mainly by the relatively rather uniform width of the subgenital plate and shape of the aedeagal shaft and configuration of its process as noted in the description.


AbstractMurdannia saddlepeakensis (Commelinaceae), a new species from the Andaman and Nicobar Islands, India, is described and illustrated. The new species is remarkable for its narrowly linear leaves, two fertile stamens, single seeded locule and scorbiculate seeds. Murdannia is represented by 54 species . forests , 13°09'NPageBreak, Ophiorrhiza mungosL.(Rubiaceae) and Gomphostemma javanicum(Blume)Benth. (Lamiaceae). It was observed flowering and fruiting from October to February.It grows in an open scrub forest in rocky situations at an elevation of 508 m. The common associates are Etymology.Murdannia saddlepeakensis is named after the type locality Saddle Peak National Park. It is the highest peak of the entire archipelago, reaching an altitude of 732 m.Murdannia saddlepeakensis was collected only once from the Saddle Peak National Park (North Andaman Islands). At this site c. 25 individuals in an open scrub forest in rocky situations were observed and hence it is assumed to be rare. However, larger part of the National Park is unexplored due to human inaccessibility. Therefore, the species can be accessed as “Data Deficient” (DD), using the criteria of Murdannia saddlepeakensis belongs to the group Terminatae G. Brückn. (173: 1830) . In India, series Terminatae is represented by eight species viz. Murdannia dimorpha G. Brückn., Murdannia divergens (C.B. Clarke) G. Brückn., Murdannia hookeri (C.B. Clarke) G. Brückn., Murdannia japonica (Thunb.) Faden, Murdannia gigantea (Vahl) G. Brückn., Murdannia loriformis (Hassk.) R.S. Rao & Kammathy, Murdannia nudiflora (L.) Brenan and Murdannia simplex (Vahl) Brenan (modified after Brückn. 1930). The first four species have more than two seeds per locule and rest four have two seeds per locule while Murdannia saddlepeakensis has single seed per locule.Murdannia saddlepeakensis closely resembles Murdannia simplex but can be easily distinguished by its narrow linear leaves, terminal flowering shoot in the basal rosette, glabrous leaf sheaths, single seeded locule, elliptic seed with scorbiculate surface. In addition, Murdannia saddlepeakensis is restricted to northern Andaman Island whereas Murdannia simplex is much more wide spread in Tropical Africa and Asia. Murdannia gigantea with a terminal flowering shoot also is similar to Murdannia saddlepeakensis but differs in having thick, fibrous roots, broad leaves, three stamens and seeds with dorsal embryotega. Murdannia saddlepeakensis can also be mistaken for the widespread Murdannia loriformis in general. However, the presence of erect, terminal flowering shoot and scorbiculate, single seeded locule along with anthesis by noon supports its distinctness.Murdannia from Andaman & Nicobar Islands namely Murdannia crocea (Griff.) Faden subsp. crocea, M. gigantea (Vahl) G. Brückn., M. nudiflora (L.) Brenan, M. spirata (L.) G.Brückn. and Murdannia vaginata (L.) G.Brückn.The occurrence of Murdannia crocea (Griff.) Faden subsp. crocea from Andaman seems to be erroneous as the screening of herbarium specimens at Kew PageBreakhave revealed that Murdannia crocea subsp. crocea is known only from Myanmar, Tenasserim [Herb. Helfer 5497 (K!)]. No one has reported this species till from India after Helfer’s collection. A key for the species of the genus Murdannia in Andaman and Nicobar PageBreakIslands and closely related taxa of the new species is given below to facilitate identification(modified after