Spread of Phoma exigua var. foveata to healthy potato plants

Contamination of stem-cutting bait plants exposed at various locations in and around a potato crop was greatest within the crop, less outside it and least at a site c. 20 km from commercial crops. Within the crop about half the stem infection and tuber contamination detected had occurred before haulm destruction. The incidence of contamination on tubers of bait plants placed within the crop was similar to that on tubers from the surrounding plants. Phoma exigua var. foveata was recovered from green leaves sampled at intervals between June and early September. In one year contamination by P. exigua var. foveata of tubers sampled from healthy plants surrounding a diseased plot was greatest in samples adjacent to the plot and decreased progressively away from it. This pattern was less apparent in two other years. These results are discussed in relation to the spread of airborne inoculum.     

Differentiation of Phoma foveata from P. exigua using a RAPD Generated PCR-RFLP Marker

Phoma foveata and P. exigua variety exigua both infect potatoes and are morphologically very similar. P. foveata produces a pigment which allows differentiation from P. exigua in culture. Discrimination of the two species based on the production of a secondary metabolite, which is dependent on the growth conditions, is not reliable. Therefore, there is a need to develop nucleic acid based identification markers. A 482bp random amplified polymorphic DNA (RAPD) fragment from P. foveata was isolated and sequenced. Polymerase chain reaction (PCR) primers, developed from the sequence of the RAPD product, amplified a 474bp fragment for P. foveata and P. exigua varieties exigua, diversispora, inoxydibilis and sambuci-nigrae. The similarity of the PCR fragments was demonstrated by sequence analysis and by using the restriction enzymes DdeI and DpnII. P. foveata was distinguished from the four varieties of P. exigua on the basis of the RFLP patterns of the PCR fragment. Ten isolates of P. foveata and nine of P. exigua var. exigua from different geographic locations were tested and all isolates but one showed the restriction digest pattern of the PCR fragment (PCR-RFLP) specific to each species. One isolate of P. foveata demonstrated a PCR-RFLP pattern similar to P. exigua var. exigua leading to the conclusion that the isolate had been previously misidentified as a strain of P. foveata lacking the ability to produce pigment.     

Laboratory assessments of the susceptibility of potato tubers to gangrene (Phoma exigua var. foveata)

Disease incidence was recorded in tubers of various cultivars following inoculation with macerated mycelium of Phoma exigua var. foveata and wounding in various ways. A method which produced predominantly crush wounds gave most rots and the most consistent results. The length of time between inoculation and wounding did not significantly affect the incidence of disease but disease severity did increase with damage infliction time. Inoculated, undamaged tubers produced rots but the incidence was low and discrimination between cultivars was poor when compared with damaged tubers. In some tests the resistance to gangrene could be measured by the proportion of tubers affected, in others all tubers had lesions but the extent of infection was different, The latent period of the disease differed between cultivars. Rate of lesion development was constant over a period of 12 wk within cultivars but differed between them.     

The influence of cultivar and mechanical damage on the relationship between inoculum concentration of Phoma exigua var. foveata on potato tubers and gangrene development

In an experiment to examine the influence of cultivar and tuber damage on the relationship between inoculum concentration and potato gangrene development, the extent to which cultivars differed in their response to increased inoculum concentration depended on the type of damage which the tubers received. There was considerable variation in cultivar response in terms of lesion frequency when tubers were inoculated by damaging them on a commercial grader, whereas when they were uniformly wounded or left undamaged cultivar differences were small. The relationship between the number of detectable wounds on tubers and gangrene incidence was not consistent for the three cultivars. Lesion size was shown to increase linearly with increasing concentrations of pycnidiospores when tubers of cultivars of different susceptibilities were point-inoculated.     

Mass spectrometric techniques as aids in the diagnosis of gangrene in potatoes caused by Phoma exigua var. foveata

Current diagnosis of gangrene in potato tubers, caused byPhoma exigua var.foveata, is based on isolation and identification of the causal organism or on thin-layer chromatographic (TLC) detection of the anthraquinone pigment pachybasin (1-hydroxy-3-methylanthraquinone). In this paper alternative diagnostic approaches are described based on detection of specific ions by pyrolysis mass spectrometry (Py-MS), direct probe mass spectrometry (DIP-MS) and gas chromatography-mass spectrometry (GC-MS). The advantages and disadvantages of these methods are discussed in comparison with the TLC approach. GC-MS is a promising technique because of its high specificity, sensitivity and suitability for automation. Moreover, the MS approaches may lead to a reduction of total expertise time.Samenvatting De huidige diagnostiek van gangreen in aardappelknollen, veroorzaakt doorPhoma exigua var.foveata, is gebasserd op de isolatie en identifikatie van de schimmel of op de dunnelaag-chromatografische (TLC) bepaling van het pigment pachybasine (1-hydroxy-3-methylanthrachinon).In dit artikel worden nieuwe diagnostische benaderingen beschreven die gebaseerd zijn op de detectie van specifieke ionen door middel van pyrolyse-massaspectrometrie (Py-MS), direct-probe-massaspectrometrie (DIP-MS) en gaschromatografie-massaspectrometrie (GC-MS). De voor- en nadelen van deze methoden worden behandeld en vergeleken met de dunnelaag-chromatografische methode.GC-MS is een veelbelovende methode vanwege de grote specificiteit, gevoeligheid en geschiktheid voor automatisering. Op den duur kunnen de MS-methoden leiden tot een verkorting van de totale expertisetijd.     

The effect of temperature before or after inoculation with Phoma exigua var. foveata on the development of gangrene in potato tubers

Potato tubers of five cultivais were incubated at five temperatures between 2 and 20°C for 1 month and then inoculated with Phoma exigua var. foveata and stored at 5°C. Larger rots usually developed on tubers previously incubated at the higher temperatures but cultivar differences were greatest where tubers had been incubated at 2°C. In another experiment, rot size was related to the duration of incubation at 20°C before inoculation.
In a series of experiments, tubers were stored at different temperatures after inoculation; other factors (e.g. wound type or r.h.) were also investigated in some of these. Combining the results of all experiments a linear decrease of % wounds infected (logit transformation) with storage temperature was demonstrated. The size of rots was not simply related to temperature, but tended to increase over the range 2–10°C and then stabilized or declined at higher temperatures. Relative humidity treatments of 75% v. 95% did not consistently affect either the incidence of rotting or rot size. The other factors investigated did not usually interact significantly with temperature.
The relevance of the results for resistance testing is discussed and a simple equation relating disease incidence to inoculum levels, type of damage and temperature is proposed.     

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Variation in stem infections caused by aggressive and non-aggressive isolates of Leptosphaeria maculans on Brassica napus var. oleifera

Development of stem cankers in seedlings of Brassica napus var. oleifera inoculated with Leptosphaeria maculans was studied in growth cabinets. As the pathogen progressed from the petiole into the stem, hyphae invaded parenchyma cells for the first time, initiating a necrotrophic phase which resulted in the formation of a lesion. Ultimate containment of the pathogen was associated with a sequence of host reactions including lignification, cambium formation and callose deposition at the perimeter of the lesion. Two isolates of L. maculans differed in the pattern of stem infection. One was confined to the cortex and the other passed through the leaf gap into the pith. Variation in response between cultivars Rapora, Primor and Jet Neuf modified these two colonization patterns but did not eliminate the main differences. There were qualitative and quantitative differences in host cell reactions. The significance of these findings is discussed in relation to differences in resistance of the cultivars in the field.     

黄土高原马铃薯晚疫病发生发展与气象条件关系的研究—以甘肃定西市为例

应用1991-2005年马铃薯晚疫病发生地点、面积、发展趋势等资料,结合气象要素观测资料,采用数理统计方法,分析马铃薯晚疫病发生发展及其与气象条件的关系。结果表明:研究区域马铃薯晚疫病历年发病面积比例呈显著上升趋势,发病面积比例以每年3.5437%的速度增加。马铃薯晚疫病发病面积比例与5~10月马铃薯生长季相对湿度、降水量、气温等呈正相关,而与日照时数、平均风速呈负相关。当可控条件一定时,气象条件是马铃薯晚疫病发生发展的主导因素。本文建立了马铃薯晚疫病发病面积比例气候预测模型,为马铃薯晚疫病的积极防治及科学决策提供了依据。     

马铃薯块茎和土壤样品中粉痂病菌快速检测方法的建立

马铃薯粉痂菌(Spongospora subterranea f.sp.subterranea)是引起马铃薯粉痂病的病原.本研究根据粉痂菌内部转录间隔区和线粒体DNA的保守区域,分别设计了 2对适用于普通PCR的引物A5/A9、C3/C8和1对适用于荧光定量PCR的引物QF/QR,用于检测块茎和土壤样品中的粉痂菌.特异...     

Monitoring for resistant populations of <Emphasis Type="BoldItalic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis> and epiphytic bacteria on citrus trees treated with copper or streptomycin using a new semi-selective medium

Streptomycin has been tested as an alternative to copper bactericides, which are routinely used for the control of citrus canker (Xanthomonas citri subsp. citri, Xcc) in citrus producing areas where the disease is endemic. A major concern is that excessive use of copper as a bactericide may lead to development of copper-resistant strains of Xcc. In this study, we developed a semi-selective medium to recover copper or streptomycin-resistant strains of Xcc from citrus leaves. The newly developed semi-selective medium was used to monitor the effect of a 21-day-interval copper or streptomycin spray program on Xcc for three consecutive seasons and on citrus epiphytic bacterial populations for two seasons in a commercial grapefruit grove. Although, no copper- or streptomycin-resistant strains of Xcc were isolated after three seasons, we observed a significant increase over time in the frequency of citrus epiphytic bacteria resistant to these chemicals. Overall, the proportion of epiphytic bacteria resistant to streptomycin on treated and untreated leaves was proportionally lower than the copper-resistant bacterial population. When application of each bactericide was suspended for the season, the proportion of bactericide-resistant bacteria in the epiphytic population decreased to that of the non-treated bacterial population. Availability of an alternative bactericide, such as streptomycin, to integrate into a copper-based program would reduce the amount of each bactericide sprayed in citrus orchards and possibly lower the selection pressure for bacterial resistance to these chemicals.     

The effect of a mixture of seed-borne Microdochium nivale var. majus and Microdochium nivale var. nivale infection on Fusarium seedling blight severity and subsequent stem colonisation and growth of winter wheat in pot experiments

Greenhouse experiments were conducted in order to determine the impact of seed-borne Microdochium nivale var. nivale and var. majus inoculum, and seed treatment with a carboxin+thiram mixture, on the development of seedling blight, and on subsequent stem colonisation and growth of winter wheat (cv. Cadenza). Experiments were conducted at temperatures favourable (3°C) and unfavourable (22°C) to M. nivale. Seed-borne inoculum resulted in seedling blight symptom development when plants were grown at 3°C, but not when plants were grown at 22°C. For seedlings grown at 3°C, plants arising from heavily blighted seedlings developed more severe symptoms of stem colonisation, when compared with those arising from seedlings from carboxin+thiram treated seeds. In addition, the vigour of such plants (assessed by determining the number of tillers and ears per plant, stem length, green leaf area, dry weight and yield) was also significantly lower than for plants arising from carboxin+thiram treated seeds. Microdochium nivale var. majus and var. nivale appeared to have little effect on plant vigour from seedlings grown at 22°C. This is the first recorded incidence of seedling blight affecting subsequent plant growth. Microdochium nivale var. majus and var. nivale stem colonisation increased from growth stage (GS) 40–49 to harvest in plants raised from seedlings grown at both temperatures. Microdochium nivale var. majus and var. nivale were isolated from the second node at GS 40–49 and the third node at harvest of plants from seedlings grown at 3°C. For plants from seedlings raised at 22°C, M. nivale var. majus and var. nivale were isolated from the first node at GS 40–49 and the second node at harvest. Carboxin+thiram seed treatment decreased the extent and severity of stem colonisation on plants from seedlings grown at 22°C.     

Inhibitory effect of isoprothiolane on metabolism of a phosphoramidate by isolates of Pyricularia oryzae cav. in relation to fungicide sensitivity

The inhibitory effect of isoprothiolane(diisopropyl 1,3-dithiolan-2-ylidenemalonate), a fungicide for rice blast control, on the metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by 20 isolates of Pyricularia oryzae was examined in relation to sensitivity of the isolates to the reference fungicide IBP(S-benzyl diisopropylphosphorothiolate). The isolates were divided into five groups based on the modes of BPA metabolism and the inhibition of BPA metabolism by isoprothiolane. Every isolate in groups I and II, which was either a field isolate or a stock culture, decomposed BPA rapidly and produced both hydroxylated and N-demethylated BPA as metabolites. BPA decomposition by these isolates was strongly inhibited by isoprothiolane, resulting in the decreased production of both metabolites in group I and of the hydroxylated metabolite in group II. These isolates were almost equally sensitive to isoprothiolane. Isolates in groups III, IV, and V were all obtained from selection of the fungus mutants found growing on media containing isoprothiolane. Isolates in group III, derived by plating large numbers of conidia, did not decompose BPA to any extent. Mutants of groups IV and V were obtained from fast-growing sectors on agar containing isoprothiolane. Both these groups decomposed BPA, but isolates belonging to group IV produced copious amount of N-demethylated BPA whereas isolates in group V did not. BPA metabolism by these in vitro mutants in groups III, IV, and V was not inhibited by isoprothiolane. Thus, the inhibitory effect of isoprothiolane on BPA metabolism was correlated with sensitivity of an isolate to isoprothiolane. The inhibitory effect of IBP on BPA metabolism was not always correlated with the sensitivity of an isolate to IBP.     

Influence of amylovoran production on virulence of Erwinia amylovora and a different amylovoran structure in E. amylovora isolates from Rubus

The amylovoran structures of five Erwinia amylovora isolates from Malaceae sp. and four isolates from Rubus sp. host plants were fully established, mainly by NMR. The structural data on one E. amylovora isolate from a Malaceae sp. host, which had been previously suggested by mass and NMR (Nimtz et al., 1996), were completed. E. amylovora strains infective on Malaceae sp. host plants had an amylovoran composed of pentasaccharide and 30–40% hexasaccharide repeating-substructures, whereas amylovoran from E. amylovora isolates from Rubus sp. host plants had only the pentasaccharide substructures. On the other hand, the exopolysaccharide (EPS) production differed in wild-type E. amylovora strains. Data on in vitro amylovoran production per cell could account for the differences in aggressiveness found in E. amylovora strains, as deduced from a pilot test with highly, moderately, and weakly aggressive strains. This correlation was confirmed with several other wild-type E. amylovora strains from different origin.     

Colonization by two isolates of Plasmodiophora brassicae with differing pathogenicity on a clubroot-resistant cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis)

A commercial clubroot-resistant F₁ cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis), Kukai 70, is resistant to an isolate of populations of Plasmodiophora brassicae from Hagi (HG) city and is susceptible to another from Yamaguchi (YMG) city. The degree and frequency of primary and secondary cycle colonization by the isolates in the root hairs and root tissues of cv. Kukai 70 were compared. Seedlings of cv. Kukai 70 were grown in soils amended with inoculum of either HG or YMG and harvested 10 days after inoculation to observe the primary cycle (number of root-hair infections) and 20, 30, and 40 days after inoculation to observe of the secondary cycle (frequency of infected cells and degree of plasmodial development based on the number of nuclei in infected cells). Although more root hairs were infected in HG than in YMG, fewer cells in root tissues including the cortex and medullary rays were infected in HG than in YMG. In addition, YMG developed plasmodia with many nuclei and formed resting spores, whereas plasmodia remained immature with a small number of nuclei in HG and did not form resting spores even by 40 days after inoculation. These results suggest that suppression of plasmodial development during secondary colonization is associated with resistance mechanisms to HG in cv. Kukai 70. Starch did not accumulate (i.e., development of amyloplasts) in HG-infected cells. This may be involved in the suppression of secondary colonization of P. brassicae in the cultivar.     

Relationships between chemical structure and phloem mobility in Ricinus communis var. Gibsonii with reference to a series of ω-(1-naphthoxy)alkanoic acids

The phloem translocation of an homologous series of ω-(1-[1-⁴C]naphthoxy)alkanoic acids relative to that of applied [6,6′-³H] sucrose was measured in Ricinus communis var. Gibsonii. Relative phloem mobility decreased with increase in octan-l-ol/water partition coefficients. However, non-ionisable compounds with partition coefficients similar to the readily translocated acids were not phloem mobile. These results are explained in terms of an ion-trap mechanism subject to limitations on the partition coefficients of the acids and possibly their anions.     

Biological control of Rhizoctonia solani on potatoes by antagonists. 5. The effectiveness of three isolates of verticillium biguttatum as inoculum for seed tubers and of a soil treatment with a low dosage of pencycuron

In field experiments in 1983, inoculation of seed tubers with three isolates ofVerticillium biguttatum, separately or mixed, was successful in reducing the amount of sclerotia ofRhizoctonia solani formed on new tubers. In holocene marine soils the sclerotium index (s.i.) was on average 50% of that of the harvest from untreated tubers.In two slightly acid pleistocene sands an additional soil treatment with pencycuron at 20% of the recommended dosage improved the results of seed tuber inoculation in one soil (further reduction of the s.i. from 60 to 32%), but not in the other (reduction to 55%). The three isolates ofV. biguttatum used in these experiments did not differ in their efficacy to suppressR. solani.Lowering the number of spores in the inoculum fluid to 20% of the initial density (6×10⁵ per ml instead of 3×10⁶ did not influence the results.Samenvatting Het beënten van voorgekiemde pootaardappelen met drie isolaten vanVerticillium biguttatum, elk apart of als mengsel, bleek in veldproeven in 1983 gunstige resultaten te geven: de hoeveelheid lakschurft gevormd op nieuwe knollen was duidelijk minder dan in de onbehandelde controle. In zavelgronden werd de sclerotiumindex (maat voor de hoeveelheid lakschurft) gemiddeld teruggebracht tot de helft van de hoeveelheid die voorkwam op de oogst uit onbehandelde, niet ontsmette, poters.In twee licht zure zandgronden werd een aanvullende grondbehandeling gegeven (volvelds) met pencycuron in een concentratie die 20% was van de aanbevolen dosering. In één grond werden de resultaten van de enting hierdoor verbeterd en trad een verdere reductie van de sclerotiumindex op van 60% (met alleenV. biguttatum) tot 30%. In de andere grond bleef de reductie beperkt tot het niveau van 55%. De dosering was hier waarschijnlijk te laag.Het effect van enting metV. biguttatum op het verlagen van de hoeveelheid sclerotiën op de jonge knollen bleek minstens even goed als een chemische ontsmetting van het pootgoed en in de meeste gevallen zelfs beter.De drie isolaten vanV. biguttatum die in deze proeven gebruikt werden waren statistisch niet verschillend in hun vermogenR. solani terug te dringen. Evenmin was een verlaging van de sporendichtheid in de entvloeistof tot 20% (6×10⁵ sporen ml^–1) van de oorspronkelijk gebruikte dichtheid van invloed op de resultaten.