Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman

Seventy three isolates of Pythium aphanidermatum obtained from cucumber from four different regions of Oman and 16 isolates of muskmelon from the Batinah region in Oman were characterized for aggressiveness, sensitivity to metalaxyl and genetic diversity using AFLP fingerprinting. Twenty isolates of P. aphanidermatum from diverse hosts from different countries were also included in the study. Most isolates from Oman were found to be aggressive on cucumber seedlings and all were highly sensitive to metalaxyl (EC₅₀ < 0·80 µg mL^?1). Isolates from cucumber and muskmelon were as aggressive as each other on both hosts (P > 0·05), which implies a lack of host specialization in P. aphanidermatum on these two hosts in Oman. AFLP analysis of all isolates using four primer–pair combinations resolved 152 bands, of which 61 (~40%) were polymorphic. Isolates of P. aphanidermatum from Oman and other countries exhibited high genetic similarity (mean = 94·1%) and produced 59 different AFLP profiles. Analysis of molecular variance indicated that most AFLP variation among populations of P. aphanidermatum in Oman was associated with geographical regions (F_ST = 0·118; P < 0·0001), not hosts (F_ST = –0·004; P = 0·4323). These data were supported by the high rate of recovery (24%) of identical phenotypes between cucumber and muskmelon fields in the same region as compared to the low recovery (10%) across regions in Oman, which suggests more frequent movement of Pythium inoculum among muskmelon and cucumber fields in the same region compared to movement across geographically separated regions. However, recovering clones among regions and different countries may imply circulation of Pythium inoculum via common sources in Oman and also intercontinental spread of isolates.     

Effect of salinity on pythium damping-off of cucumber and on the tolerance of Pythium aphanidermatum

A study was conducted to investigate the effect of salinity on pythium damping-off and tolerance to salinity among Pythium aphanidermatum isolates from different geographical origins, genetic backgrounds and saline/non-saline soils. Increasing irrigation-water salinity from 0·01 to 5 dS m⁻¹ significantly increased mortality in cucumber seedlings inoculated with P. aphanidermatum and reduced dry weight of non-inoculated seedlings. In vitro tests in culture media amended with different concentrations of NaCl showed that growth of P. aphanidermatum, P. spinosum and P. splendens isolates was stimulated or unaffected at salinity levels stressful for cucumber (electrical conductivity = 5 dS m⁻¹). Significant differences were found in tolerance to salinity among 47 P. aphanidermatum isolates obtained from different greenhouses. The concentration of NaCl required to reduce growth of P. aphanidermatum isolates by 50% varied from 23 to 62 dS m⁻¹, with an average of 46 dS m⁻¹, however, oospore production was more sensitive to salinity and no oospores were produced above 20 dS m⁻¹. Analysis of tolerance of P. aphanidermatum isolates to salinity showed no relationship between tolerance to salinity and geographical origins or genetic data. Isolates of P. aphanidermatum from greenhouses with no salinity problems were as tolerant to salinity as isolates obtained from salinity-affected greenhouses, suggesting lack of evidence for ecological adaptation within P. aphanidermatum in Oman. Increased mortality in cucumber seedlings at higher salinity levels may imply a synergistic interaction between salinity stress and salinity-tolerant Pythium species on cucumber seedlings, resulting in greater seedling losses.     

Evaluation of a method for quantification of Pythium aphanidermatum in cucumber roots at different temperatures and inoculum densities

Journal of Plant Diseases and Protection - A method for the quantification of Pythium aphanidermatum density in cucumber roots based on an indirect enzyme-linked immunosorbent assay (Elisa) was...     

Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum

Root and crown rot of cucumber caused by Pythium aphanidermatum can be suppressed by various rhizobacteria or PGPR (plant growth-promoting rhizobacteria). When cucumber roots were treated with Pseudomonas corrugata 13 or Pseudomonas aureofaciens 63–28, phenylalanine ammonia-lyase (PAL) activity was stimulated in root tissues in 2 days and this activated accumulation lasted for 16 days after bacterization. Peroxidase (PO) and polyphenol oxidase (PPO) activities were increased in roots 2–5 days after bacterization with P. corrugata strain 13. After bacterized cucumber roots were challenged with P. aphanidermatum, the enzyme activities of PAL, PO and PPO increased as the disease developed on the roots. These accumulations peaked 4–6 days after pathogen inoculation. A split root system demonstrated that the three enzymes could be systemically induced by the Pseudomonas strains 63–28 and 13, as well as P. aphanidermatum. Furthermore, isoperoxidase native PAGE (polyacrylamide gel electrophoresis) analysis indicated that the peroxidase isomer forms in cucumber roots induced by rhizobacteria were different from that in roots infected with P. aphanidermatum. These results suggest that the plant defense enzymes could be stimulated in cucumber roots which have been colonized by non-pathogenic rhizobacteria or in a compatible interaction between cucumber and P. aphanidermatum. The mechanisms of PO activation by the rhizobacteria may be different from those of pathogen infection.     

Mycoparasite-like behaviour of the plant pathogen Pythium aphanidermatum in vitro

Pythium aphanidermatum antagonized several other fungi after hyphal contact on films of water agar, recorded by video microscopy. It sometimes coiled round, penetrated and caused cytoplasmic coagulation of hyphae of the mycoparasites P. oligandrum, P. acanthophoron, P. mycoparasiticum and P. periplocum , but was not affected by them. It also coiled round, penetrated and coagulated P. ultimum, P. vexans, P. catenulatum, P. dissotocum and (infrequently) P. graminicola. It caused cytoplasmic coagulation of Fusarium oxysporum and Trichoderma aureoviride after contact, but without coiling. Six isolates of P. aphanidermatum behaved similarly, with a mode of action like that of the mycoparasite P. oligandrum but less rapid and consistent than this species. The results are discussed in relation to possible ecological roles of P. aphanidermatum.     

海南发生木瓜茎腐病

木瓜 (ＣａｒｉｃａｐａｐａｙａＬ .)是重要的热带、亚热带水果 ,从播种到采收只需 9个月 ,可周年结实 ,味美且富含ＶＡ 和ＶＣ,深受生产者和消费者的亲睐。 2 0 0 2年 6月 ,在海南省乐东一木瓜生产基地发现一种木瓜新病害 ,发病率高达 5 0 % ,严重影响木瓜产量。经初步鉴定认为是一种由瓜果腐霉引起的木瓜茎腐病。1　症状木瓜幼苗期和成株期均可受害。发病初期 ,在茎基部近地面处产生水渍状斑 ,并流出白色胶状物 ,后组织崩解、缢缩折倒 ,整株萎蔫。湿度大时 ,病部产生白色棉絮状菌丝体 ,即为病菌菌丝、孢囊梗和孢子囊。2　病原菌菌丝发达 …     

Thick root of cucumber: other susceptible plants and the effect of pH

Thick root is a relatively new disorder of cucumber grown in artificial substrates. Plants of cucumber, tomato, sweet pepper, lupin, anthurium, Cucurbita ficifolia , C. maxima and two lines from crosses between C. maxima and C. moschata were grown in thick root disease (TRD)-infested nutrient solutions containing the TRD agent. Plants from each species or line, except anthurium, developed TRD symptoms. Growth of diseased plants, except those of the line C. maxima  × C. moschata RS841, was significantly reduced compared with the nondiseased controls. Two weeks after infestation, by adding TRD-affected nutrient solution to fresh nutrient solution, the presence of the infective TRD agent was shown in each of the nutrient solutions in which the plants had been grown, including anthurium. No infective TRD agent could be shown in nutrient solution that had not been in contact with roots of living plants. The minimum pH for thick root formation was shown to be between 5·0 and 5·5. Nutrient solutions with pH values ranging from 4·0 to 6·5 and infested with the TRD agent lost their infectivity within 8 days. The infective period decreased with decreasing pH (pH 4·0–6·5) and a pH effect on the infectivity of a nutrient solution was shown within 1 h of infestation. The data show that the risk of TRD can be reduced by decreasing the pH of the nutrient solution.     

Indirect effect of 6-azauracil on Pythium debaryanum in cucumber

Protection of cucumber seedlings against damping off, caused byPythium debaryanum, was obtained by soaking the seeds in a solution of 1 ppm 6-azauracil (AzU), but not by treatment with a 10 or 100 ppm AzU solution. After the 1 ppm AzU treatment of the seeds, the rhizosphere microflora of the developing seedlings was changed; an increase of the bacterial population and a decrease of the fungal population was observed. SinceP. debaryanum is rather insensitive to AzU in vitro, it is suggested that the control of damping off is obtained in an indirect way, possibly via the microflora of the roots.Samenvatting Bescherming van komkommerzaailingen tegen aantasting doorPythium debaryanum werd verkregen na weken der zaden in een oplossing van 1 ppm 6-azauracil (AzU); 10 en 100 ppm AzU waren niet werkzaam. Na behandeling der zaden met 1 ppm AzU traden veranderingen in de rhizosfeermicroflora der kiemplanten op; de bacteriepopulatie nam toe en de schimmelpopulatie nam af. AangezienP. debaryanum in vitro vrijwel ongevoelig is voor AzU, wordt verondersteld dat de bescherming tegen aantasting door deze schimmel op indirecte wijze verkregen wordt, mogelijk via de microflora van de wortels.     

黄瓜嫁接苗与接种哈茨木霉菌黄瓜直根苗对根腐病防效及根系生理响应

为探明黄瓜嫁接栽培与木霉菌使用提高黄瓜幼苗抗根腐病的能力及生理调节的差异,利用哈茨木霉菌DQ002和根腐病病原菌孢子悬浮液对黄瓜直根苗根部接种与根腐病病原菌孢子悬浮液对黄瓜嫁接苗进行根部接种处理,测定黄瓜直根苗和黄瓜嫁接苗病害发生率及根系生理变化。结果表明：哈茨木霉菌DQ002通过激发黄瓜直根苗根系中POD、PPO、SOD活性而抑制了H₂O₂积累和O^-₂的产生速率,并促进了黄瓜直根苗根系PAL、几丁质酶、β-1,3-葡聚糖酶活性以及类黄酮含量升高,提高了抗病性;接种根腐病菌,黄瓜嫁接(T2)提高了根系中POD活性、培养前期PPO活性及培养后期SOD的活性,降低了H₂O₂的积累和O^-₂产生速率,促进了PAL、几丁质酶及培养后期的β-1,3-葡聚糖酶活性,但H₂O₂的积累和O^-₂产生速率显著高于哈茨木霉菌DQ002处理的黄瓜直根苗,而PAL、几丁质酶、β-1,3-葡聚糖酶活性以及类黄酮含量明显低于木霉菌处理的直根苗;T3(先接种病原菌后接种木霉菌)和T4(先接种木霉菌后接种病原菌)处理的黄瓜直根苗根腐病的发病率分别为22.39%和17.87%,病情指数分别为23.03%和14.33%,明显低于CK1(清水处理直根苗)和T1（单独病原菌处理直根苗）处理的发病率(35.82%和57.39%)和病情指数(37.10%和46.97%),以及黄瓜嫁接苗(T2)的发病率(42.90%)和病情指数(40.47%),但哈茨木霉菌使用时间不同则效果有异。黄瓜嫁接和哈茨木霉菌DQ002处理直根苗能提高黄瓜苗对根腐病的抗性,但是二者对黄瓜苗根系生理的调节作用存在差异,可能是导致根腐病发生存在差异的生理原因之一。     

Characterization of the Microbial Community Involved in the Suppression of Pythium aphanidermatum in Cucumber Grown on Rockwool

ABSTRACT The root pathogen Pythium aphanidermatum induced lower levels of disease in cucumber (Cucumis sativus) plants on unsterilized, re-used rockwool slabs than on heat-sterilized, re-used rockwool. Several recolonization treatments of the sterilized rockwool enhanced the suppressiveness of the rockwool. Microbial community structures in the different rockwool treatments were investigated by plate counts on selective media. Disease suppressiveness in the different rockwool treatments showed the highest correlation with the culturable number of filamentous actinomycetes in both experiments (r = 0.79 and 0.94), whereas the numbers of Trichoderma spp. correlated with suppression only in the first experiment (0.86). The numbers of total culturable bacteria, fluorescent pseudomonads, Bacillus spores, and fungi all showed lower correlations with disease suppressiveness. The filamentous actinomycetes enumerated with the plate counts were mainly Streptomyces spp., of which 10% were antagonistic toward P. aphanidermatum in dual culture. The composition of the bacterial and actinomycete populations was studied with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Multivariate analyses of these patterns with canonical correspondence analysis showed significant correlations between the microbial composition and the disease suppressiveness. However, none of the bands in PCR-DGGE patterns occurred exclusively in the treatments that had enhanced disease suppressiveness. Bands extracted from the actinomycete-specific DGGE gels showed closest similarity with members of several actinomycete genera, i.e., Streptomyces, Mycobacterium, Microbacterium, Rhodococcus, Curtobacterium, and Tsukamurella. The possible mechanism of disease suppressiveness in used rockwool slabs, based on the results obtained with culture-dependent and culture-independent detection methods, is discussed.     

Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers

A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum.     

Protection of cucumber against Pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and antibiosis

Four Pseudomonas strains were evaluated for their intrinsic properties conferring their ability to protect long English cucumber against Pythium aphanidermatum in hydroponic culture. Two of the strains, BTP1 and its siderophore-negative mutant M3, increased plant yield as compared with the non-inoculated control plants. Strain BTP7 was intermediate in its biocontrol activity while strain ATCC 17400 failed to reduce disease development. The role of pyoverdines could not be confirmed since treatment with either BTP1 or its siderophore-negative mutant M3 provided similar suppression of Pythium disease. In addition, no siderophores were detected in the nutrient solution. BTP1 did not inhibit pathogen growth in vitro on several media, suggesting that antibiosis was not a mechanism of suppression. Quantification of root bacterial populations did not indicate differences among the strains. On the other hand, roots treated with either BTP1 or its sid⁻ mutant M3 contained more antifungal phenolics than roots from any other treatments including controls. These results suggest that antifungal compounds induced by inoculation of cucumber roots with the fluorescent Pseudomonas strains BTP1 and M3 participate actively in the protection of cucumber plants against P. aphanidermatum     

Comparison of an in vitro and a damping-off assay to test soils for suppressiveness to Pythium aphanidermatum

Testing of soil samples in greenhouse assays for suppressiveness to soilborne plant pathogens requires a considerable investment in time and effort as well as large numbers of soil samples. To make it possible to process large numbers of samples efficiently, we compared an in vitro growth assay with a damping-off assay using Pythium aphanidermatum as the test organism on tomato seedlings. The in vitro test compares the radial growth or relative growth of the fungus in soil to that in autoclaved soil and reflects suppressiveness of soils to the pathogen. We used soils from a field experiment that had been farmed either organically or conventionally and into which a cover crop (oats and vetch in mixture) had been incorporated 0, 10, 21, and 35 days previously. We obtained a significant, positive correlation between damping-off severities of tomato seedlings in damping-off assays and both relative and radial growth in vitro. In addition, radial and relative growth of P. aphanidermatum in the in vitro assay were positively correlated with several carbon and nitrogen variables measured for soil and incorporated debris. We did not find differences between the two farming systems for either growth measures of P. aphanidermatum or disease severities on tomato at different stages of cover crop decomposition. The in vitro assay shows potential for use with any fungus that exhibits rapid saprophytic growth, and is most suitable for routine application in suppressiveness testing.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

不同土壤处理剂对设施蔬菜黄瓜根结线虫防治技术研究

为了筛选蔬菜根结线虫的防治技术,在赤峰市进行了7种不同土壤处理药剂对黄瓜根结线虫的防治效果田间试验,结果表明,应用7种不同土壤处理药剂进行土壤处理,揭膜放气5d后定植黄瓜,对黄瓜生长无不良影响,而且对根结线虫均有较好的抑制作用,其中98.1％1,3-二氯丙烯-氯化苦复配胶囊16 kg/667 m2、95％1,3-二氯丙烯液剂65 kg/667m2及10％液体石灰氮65 kg/667m2对黄瓜根结线虫的防治效果达90％左右,优于目前生产中常用药剂42％威百亩水剂6 kg/667m2和10％噻唑磷颗粒剂2 kg/667m2的防治效果,并且可以显著地提高黄瓜产量,适于在设施蔬菜生产上推广使用.     

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum

BACKGROUND: Tolerance to the oomycete‐specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root‐rot and damping‐off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. RESULTS: Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. CONCLUSION: It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target‐site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket. Copyright © 2012 Society of Chemical Industry     

黄芪根腐病病株和健株根围微生物菌群变化分析

通过分析黄芪根腐病病株和健株根围微生物菌群的变化,探究根腐病的发病机理,寻找预警病害发生的生物指示因子,为土壤微生态的生物调控提供依据。试验通过菌落计数、PCR-DGGE和16S rRNA V3区基因测序的方法,分析不同年限下黄芪根围病/健土中微生物区系、多样性及群落结构的变化。结果表明:细菌作为黄芪根围土中的优势种群,是影响病土中微生物数量升高的关键因子,其多样性降低是影响病害加重的主要原因之一。假单胞菌属Pseudomonas和无色杆菌属Achromobacter为土壤中的优势菌群,健土中的1号和4号特异条带分别为未培养的假单胞菌Uncultured Pseudomonas sp.和荧光假单胞菌P.fluorescens;6号未培养假单胞菌Uncultured Pseudomonas sp.的丰度与根腐病发病率负相关,16号无色杆菌Achromobacter sp.的丰度则在3年生土壤中显著升高,随后急剧下降。上述4个菌可作为潜在的土壤健康或发病指示因子进一步研究。     

Detection of <Emphasis Type=Italic>Pythium aphanidermatum </Emphasis>in Sludge from Drinking-water Plant

Pythium aphanidermatum causing damping off of cucumber seedlings was detected in water clarifier sludge from a drinking-water plant in the Kanto district. During December 1996 to November 1997, the pathogen was consistently isolated from the sludge at <1.9 CFU ml^-1 of the sludge. Received 25 February 2000/ Accepted in revised form 28 April 2000     

Resistance assessment and biochemical responses of sugar beet lines against <Emphasis Type="Italic"> Pythium aphanidermatum,</Emphasis> causing root rot

Wood-staining fungi, moulds and decay fungi colonize freshly cut wood. Of these, only ophiostomatoid fungi are considered major agents of sapstain on logs and freshly sawn lumber because they can cause permanent staining and discoloration. Sapstain in pine pallets significantly reduces their market value and use in the food industry. The aim of this study was to identify the sapstaining fungi that colonize Scots pine wood used in pallet production. In addition, we evaluated the growth and stain intensity of six isolates of ophiostomatoid fungi on freshly cut Scots pine billets. Fungi were isolated from samples of Scots pine and identified based on morphology and DNA sequence comparisons for three gene regions (ITS, β-tubulin, TEF-1α). A total of 1259 isolates representing 31 fungal species were obtained from the pine samples in Poland during July and September. The isolates represented different ecological and taxonomical groups and belonged to the categories of staining fungi, decay fungi and surface moulds. The most frequently isolated fungi were ophiostomatoid fungi (14 species including an unknown Leptographium sp. and Ophiostoma sp.) and moulds (mainly Trichoderma and Mucor spp.). Of the ophiostomatoid species, Ophiostoma floccosum, O. piceae, O. piliferum and Endoconidiophora pinicola were the predominant species. The results of pine billet inoculation showed that among the six fungal species tested, E. pinicola exhibited the fastest growth in all three directions on the billets. Ophiostoma minus and O. piliferum displayed moderate growth rates in pine billets, while O. floccosum, O. piceae, and Leptographium lundbergii grew very slowly, especially in the tangential and radial directions. The information provided in this paper will help develop more effective control strategies for sapstain prevention in Scots pine.