Dark lines formed between colonies of isolates of phoma exigua var. foveata on a semi-selective medium

Samenvatting In het Instituut voor Plantenziektenkundig Onderzoek (IPO) te Wageningen wordt gangreen bij aardappel, veroorzaakt door de schimmelPhoma exigua var.foveata, onderscheiden van ander droogrot door aangetast weefsel uit te leggen op een semiselectief medium. Op dit medium wordt de vorming van kristallen van voor de schimmel kenmerkende anthrachinonen bevorderd. De gemakkelijk waar te nemen geelgroene kristallen in het medium wijzen op de aanwezigheid van de betrokken schimmel. Bij een van deze toetsen vormden zich tussen de zich ontwikkelende kolonies van de schimmel donkere lijnen. Deze lijnen verschilden van reeds eerder beschreven violette lijnen, die zich vormen tussen kolonies van de twee variëteitenexigua enfoveata vanP. exigua. Onderzoek aan 27 isolaten vanP. exigua var.foveata toonde aan dat er met betrekking tot de vorming van donkere lijnen 11 verschillende typenP. exigua var.foveata onderscheiden konden worden. Isolaten van deze typen vormden bij gepaarde groei op het selectieve medium donkere lijnen met andere typen, maar niet met isolaten van het zelfde type. Er bleek geen verband met het groeitype van de cultures noch met de vorming van het antibioticum E te bestaan.     

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).     

Infection process of Rhizoctonia solani on Solanum tuberosum and effects of granular nematicides

The infection process ofRhizoctonia solani AG-3 was studied on potato sprouts, cv. Bintje, in growth chamber trials at 15 °C. Initially hyphae ofR. solani grew predominantly in the longitudinal direction of the sprouts (runner hyphae). They tended to follow the junctions between epidermis cells as was observed by SEM. The hyphae formed side-branches mainly half-way of the subterranean parts of the sprouts. They branched several times with short swollen cells to form infection cushions. Lesions developed only underneath the infection cushions and were first observed five days after inoculation. The necrotic area was proportional to the area covered with infection cushions on the sprouts. Depth of the lesions could extend up to the vascular bundle. Sprouts were colonized only in healthy tissue in the epidermal layer underneath the infection cushion and in necrotic tissue. A few days after appearance of the lesions,R. solani formed brown, uninfective mycelium on and in the circumference of these lesions.Aldicarb did not influence any part of the infection process. Ethoprophos delayed the emergence of sprouts, but increased the number of sprouts per tuber. As soon as sprouts had emerged, growth was considerably promoted by ethoprophos. Ethoprophos delayed the appearance of lesions and reduced their size. Oxamyl showed the same effects to a smaller extent.As the size of lesions appears to be proportional to the size of the infection cushions, any agents that change the size of the infection cushions, such as pesticides or antagonists, may alter the severity of the disease.Samenvatting Het infectieproces vanRhizoctonia solani AG-3 werd bestudeerd op aardappelspruiten, cv. Bintje, in een klimaatcel bij 15C. Aanvankelijk groeide de schimmel met runnerhyfen voornamelijk in de lengterichting van de spruit. Via SEM kon waargenomen worden, dat de hyfen hierbij vooral over de begrenzingen van de epidermiscellen groeiden. Het mycelium vormde veel zijvertakkingen, bestaande uit iets gezwollen korte cellen, welke voornamelijk halverwege op het ondergrondse deel van de spruit gevormd werden. Een dichte massa van deze cellen vormde een infectiekussentje. Lesies, welke vanaf vijf dagen na inoculatie werden waargenomen, bevonden zich slechts onder spruitoppervlak bezet met infectiekussentjes. De lesiegrootte was recht evenredig met het spruitoppervlak dat bezet was met infectiekussentjes. De diepte van de lesies reikte tot aan de vaatbundels. De spruit werd alleen door de schimmel gekoloniseerd in gezond epidermisweefsel onder het infectiekussentje en in necrotisch weefsel. Enkele dagen na verschijning van lesies vormde R.solani bruin, niet infectieus, mycelium op en rondom de lesies.Aldicarb had geen effect op het infectieproces. Ethoprophos vertraagde de opkomst en verhoogde het aantal tot ontwikkeling gekomen spruiten per knol in gestoomd zand. Direct na opkomst had ethoprophos echter een sterk groeistimulerend effect. Ethoprophos vertraagde de lesievorming en reduceerde de lesiegrootte, vergeleken met onbehandelde planten. Oxamyl vertoonde deze effecten in geringere mate.Daar de lesiegrootte direct gecorreleerd blijkt met de grootte van het infectiekussentje, mag verwacht worden dat elke beïnvloeding van de ontwikkeling van het mycelium van R.solani, bijvoorbeeld door pesticiden of antagonisten, een verandering van de lesiegrootte ten gevolge heeft.     

An investigation of uncertainty in field habitat mapping and the implications for detecting land cover change

Land cover data for landscape ecological studies are frequently obtained by field survey. In the United Kingdom, temporally separated field surveys have been used to identify the locations and magnitudes of recent changes in land cover. However, such map data contain errors which may seriously hinder the identification of land cover change and the extent and locations of rare landscape features. This paper investigates the extent of the differences between two sets of maps derived from field surveys within the Northumberland National Park in 1991 and 1992. The method used in each survey was the Phase 1 approach of the Nature Conservancy Council of Great Britain. Differences between maps were greatest for the land cover types with the smallest areas. Overall spatial correspondence between maps was found to be only 44.4%. A maximum of 14.4% of the total area surveyed was found to have undergone genuine land cover change. The remaining discrepancies, equivalent to 41.2% of the total survey area, were attributed primarily to differences of land cover interpretation between surveyors (classification error). Differences in boundary locations (positional error) were also noted, but were found to be a relatively minor source of error. The implications for the detection of land cover change and habitat mapping are discussed.     

Molecular aspects of the potato — Phytophthora infestans interaction

The fungal pathogenPhytophthora infestans is the causal organism of late blight, one of the most devastating diseases of potato. In the past, various aspects of the potato-P. infestans interaction have been studied extensively. In this paper we briefly review the current knowledge of the molecular events associated with the interaction and in addition we discuss a new approach for analyzing the molecular basis of pathogenicity ofP. infestans.     

Effect of live weight and dietary supplement of raw potato starch on the levels of skatole, androstenone, testosterone and oestrone sulphate in entire male pigs

This study evaluated the effect of supplement of raw potato starch (RPS) on the levels of skatole, androstenone, testosterone and oestrone sulphate in plasma from entire male pigs. The study also evaluated relationships between plasma levels of skatole and testicular steroids at three different live weights (LW) of approximately 90, 100 and 115 kg. A total of 111 entire male pigs of a crossbred (Yorkshire dams×Swedish Landrace sires) were used. Animals were raised either in mixed pens, with females and males, or single-sex pens. Each pen contained seven or nine pigs. The most fast-growing three pigs from the pens with nine pigs were slaughtered when they reached 90 kg LW, and the remaining pigs were slaughtered at 115 kg LW. All pigs were fed the same commercial diet until the average pen weight reached 100 kg. Then, 33 out of 80 remaining pigs received RPS, 0.6 kg per pig and day, for 2 weeks prior to slaughter. Blood samples were taken from the pigs at three occasions: first, the day prior to first slaughter occasion, low-weight group; second, the day prior to change in diet, middle-weight group; and third, the day prior to second slaughter occasion, high-weight group. Plasma was analysed for the levels of skatole, androstenone, testosterone and oestrone sulphate. Fat samples were taken at slaughter and analysed for the levels of skatole and androstenone. The levels of skatole and testicular steroids in plasma were significantly higher in entire male pigs from the high-weight group fed no RPS compared to those from low- and middle-weight groups. The levels of the investigated compounds did not differ between low- and middle-weight groups (P>0.1). The diet with RPS induced a decline in skatole levels in plasma and fat (P<0.001), but not plasma levels of testicular steroids and fat levels of androstenone (P>0.05). Skatole levels were positively correlated to testosterone and oestrone sulphate levels in the middle- and high-weight pigs fed no RPS as well as to testosterone in the low-weight group. In the high-weight group fed RPS, skatole levels were not correlated to any of the analysed compounds. Approximately 26% of the entire male pigs (11 out of 43) from the high-weight group fed no RPS produced skatole levels in fat above 0.20 μg/g, whereas the pigs from the low- and high-weight group fed RPS did not produce skatole levels above 0.20 μg/g in fat. Androstenone levels in fat were high in all groups. In total 47% (52 out of 111) pigs expressed androstenone levels above the rejection levels in fat of 1.0 μg/g and 88% (98 out of 111) had androstenone levels above 0.5 μg/g. It was concluded that a lower slaughter weight and the supplement of raw potato starch to the diet could be used to reduce skatole levels in entire male pigs. Androstenone levels in fat, however, could not be reduced by either a lower weight at slaughter or dietary manipulation.     

野生朱红硫磺菌驯化栽培研究

营养试验、菌丝体培养和出菇温度试验、培养基酸碱度试验、出菇条件试验结果表明：朱红硫磺菌菌丝体适宜生长的温度范围为15-32℃，最适温度范围为22～28℃；子实体发生的最适温度为16～25℃，气温低于16℃，子实体色泽为淡橙色，16℃以上，子实体呈鲜橙红色；培养料适宜营养C／N为20-26／1；朱红硫磺菌菌丝体适宜酸碱度范围为pH3．0～8．0，最适酸碱度为pH4．0—6．5；朱红硫磺菌的产量与光照条件无关，但光照强度影响子实体的发生时间和色泽；朱红硫磺菌栽培原料广泛，凡栽培木腐菌的培养料均可用于栽培朱红硫磺菌。     

马铃薯渣酶法水解液制备单细胞蛋白饲料的研究

本文研究了酶法水解马铃薯渣制备膳食纤维后的滤液制备单细胞蛋白的可行性。试验结果表明,单细胞蛋白边糖化边发酵的摇瓶培养的较优工艺条件为:底物浓度为8%(添加8%的麸皮水)、初始pH值为4.5、接种量为15%、葡萄糖淀粉酶加入量为100U/g(原料中淀粉)、青霉素加入量为80U/g(原料)、培养温度为28℃、培养时间为6h、转速为250r/min。在此条件下,干酵母产量最高为19.920g/L,单细胞蛋白中的蛋白质含量达12.27%。     

Biological and Physical Constraints on Maize Production in the Humid Forest and Western Highlands of Cameroon

The aim was to identify biological and physical factors responsible for reducing maize yield in Cameroon. Two surveys were conducted in 137 fields in two agroecological zones in 1995–1997. In the Humid Forest (HF), Bipolaris maydis, Stenocarpella macrospora, Puccinia polysora, Rhizoctonia solani and soil fertility were factors that reduced maize production in 1995 and 1996. In the Western Highlands (WHL), Cercospora zeae-maydis, and the interaction between soil fertility and maize variety were the most important constraints to maize production in 1996. In 1997, C. zeae-maydis, S. macrospora, physiological spot and stem borer damage (Busseola fusca) were negatively related to ear weight. The combination of these biological factors (diseases and insects), and the physical parameter of soil fertility were responsible for reducing maize yield in these selected benchmarks of Cameroon. Maximum potential yield reductions were estimated at 68% due to B. maydis and 46% due to S. macrospora, respectively, in the HF in 1995. In 1996, maximum potential yield reductions in the HF were estimated at 34%, 41% and 30% due to S. macrospora, P. polysora and R. solani, respectively. In the WHL, C. zeae-maydis had the potential to cause a yield reduction of 79% in 1996. In the WHL in 1997, the interaction between C. zeae-maydis and B. fusca, stem diseases and the physiological spot caused potential reductions of 52%, 34% and 39%, respectively.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .