Survival Of Inoculum Research Articles

Microencapsulation by spray drying of nitrogen-fixing bacteria associated with lupin nodules.

Plant growth promoting bacteria and nitrogen-fixing bacteria (NFB) used for crop inoculation have important biotechnological potential as a sustainable fertilization tool. However, the main limitation of this technology is the low inoculum survival rate under field conditions. Microencapsulation of bacterial cells in polymer matrices provides a controlled release and greater protection against environmental conditions. In this context, the aim of this study was to isolate and characterize putative NFB associated with lupin nodules and to evaluate their microencapsulation by spray drying. For this purpose, 21 putative NFB were isolated from lupin nodules and characterized (16S rRNA genes). Microencapsulation of bacterial cells by spray drying was studied using a mixture of sodium alginate:maltodextrin at different ratios (0:15, 1:14, 2:13) and concentrations (15 and 30% solids) as the wall material. The microcapsules were observed under scanning electron microscopy to verify their suitable morphology. Results showed the association between lupin nodules of diverse known NFB and nodule-forming bacteria belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes. In microencapsulation assays, the 1:14 ratio of sodium alginate:maltodextrin (15% solids) showed the highest cell survival rate (79%), with a microcapsule yield of 27% and spherical microcapsules of 5-50 µm in diameter. In conclusion, diverse putative NFB genera and nodule-forming bacteria are associated with the nodules of lupine plants grown in soils in southern Chile, and their microencapsulation by spray drying using sodium alginate:maltodextrin represents a scalable process to generate a biofertilizer as an alternative to traditional nitrogen fertilization.

Immunotherapeutic organoids

Therapeutic monoclonal antibodies have revolutionized the treatment of cancer and other diseases. However, several limitations of antibody-based treatments, such as the cost of therapy and the achievement of sustained plasma levels, should be still addressed for their widespread use as therapeutics. The use of cell and gene transfer methods offers additional benefits by producing a continuous release of the antibody with syngenic glycosylation patterns, which makes the antibody potentially less immunogenic. In vivo secretion of therapeutic antibodies by viral vector delivery or ex vivo gene modified long-lived autologous or allogeneic human mesenchymal stem cells may advantageously replace repeated injection of clinical-grade antibodies. Gene-modified autologous mesenchymal stem cells can be delivered subcutaneously embedded in a non-immunogenic synthetic extracellular matrix-based scaffold that guarantees the survival of the cell inoculum. The scaffold would keep cells at the implantation site, with the therapeutic protein acting at distance (immunotherapeutic organoid), and could be retrieved once the therapeutic effect is fulfilled. In the present review we highlight the practical importance of living cell factories for in vivo secretion of recombinant antibodies.

Climate change impacts on plant canopy architecture: implications for pest and pathogen management

Climate change influences on pests and pathogens are mainly plant-mediated. Rising carbon dioxide and temperature and altered precipitation modifies plant growth and development with concomitant changes in canopy architecture, size, density, microclimate and the quantity of susceptible tissue. The modified host physiology and canopy microclimate at elevated carbon dioxide influences production, dispersal and survival of pathogen inoculum and feeding behaviour of insect pests. Elevated temperature accelerates plant growth and developmental rates to modify canopy architecture and pest and pathogen development. Altered precipitation affects canopy architecture through either drought or flooding stress with corresponding effects on pests and pathogens. But canopy-level interactions are largely ignored in epidemiology models used to project climate change impacts. Nevertheless, models based on rules of plant morphogenesis have been used to explore pest and pathogen dynamics and their trophic interactions under elevated carbon dioxide. The prospect of modifying canopy architecture for pest and disease management has also been raised. We offer a conceptual framework incorporating canopy characteristics in the traditional disease triangle concept to advance understanding of host-pathogen-environment interactions and explore how climate change may influence these interactions. From a review of recent literature we summarize interrelationships between canopy architecture of cultivated crops, pest and pathogen biology and climate change under four areas of research: (a) relationships between canopy architecture, microclimate and host-pathogen interaction; (b) effect of climate change related variables on canopy architecture; (c) development of pests and pathogens in modified canopy under climate change; and (d) pests and pathogen management under climate change.

Impact of anaerobic soil disinfestation combined with soil solarization on plant–parasitic nematodes and introduced inoculum of soilborne plant pathogens in raised-bed vegetable production

A two-year field study was established in August 2008 at the USDA-ARS, U.S. Horticultural Research Laboratory in Fort Pierce, FL to examine the impact of anaerobic soil disinfestation (ASD) combined with soil solarization as an alternative to methyl bromide (MeBr) fumigation for control of plant–parasitic nematodes and introduced inoculum of soilborne plant pathogens. A complete factorial experiment in a split-split plot was established to evaluate three levels of applied initial irrigation (10, 5, or 0cm), two levels of partially-composted poultry litter (CPL; amended or unamended), and two levels of molasses (amended or unamended) in combination with solarization in a raised-bed bell pepper-eggplant double crop production system. Untreated and MeBr controls were established in each block for comparison to ASD treatments. Survival of Phytophthora capsici inoculum, introduced prior to ASD treatment, was equal to that with MeBr and less than the untreated control for all solarized treatments regardless of applied soil amendments during both seasons. Survival of introduced inoculum of Fusarium oxysporum f. sp. lycopersici was least (and equivalent to the MeBr control) in treatments with applied molasses during the second season. While endemic plant-parasitic nematode populations were generally low throughout the first season, by the end of the second eggplant double crop, root-knot nematode (Meloidogyne incognita) populations in treatments not receiving molasses and/or CPL (solarization only) or not receiving irrigation at treatment averaged more than 200 nematodes per 100cm3 of soil compared to an average of 10 nematodes per 100cm3 in ASD treatments where molasses or molasses+CPL was applied and irrigated with 5 or 10cm of water. Anaerobic soil disinfestation combined with solarization may provide an alternative to chemical soil fumigation for control of soilborne plant pathogens and plant–parasitic nematodes in Florida raised-bed vegetable production systems.

The effect of winter weather conditions on the ability of pseudothecia of Leptosphaeria maculans and L. biglobosa to release ascospores

Leptosphaeria maculans and L. biglobosa are damaging pathogens of oilseed rape. The infection of plants occurs predominantly in early autumn or spring by spores produced in pseudothecia. The aim of this study was to investigate whether pseudothecia formed in the autumn are still viable in the spring and to what extend they are destroyed by winter frosts. The studies presented here demonstrated that winter frosts can render pseudothecia unable to release spores. Nevertheless, ascospores present in pseudothecia unable to discharge ascospores, were fully capable of germination, regardless of the incubation temperature. No significant differences were found between the studied Leptosphaeria species in their response to frost. A multiple regression equation has been elaborated to forecast the ability of pseudothecia to release ascospores, based on winter temperatures. Considerable correlation was found between the ascospore release in the autumn and the ability of pseudothecia to release ascospores over the winter period and the subsequent symptoms of stem canker before harvest. We have demonstrated that the potential and the survival of inoculum can have a large impact on the success of the pathogen. This may be particularly important in the light of forecasted climate change. Higher winter temperatures may increase the ability of pseudothecia to release ascospores and the discharge of ascospores of L. maculans and L. biglobosa into the air, and cause early plant infections. This in turn will increase the number of infected plants, the disease incidence at harvest, and reduce the yield of oilseed rape.

Comparison of the environmental survival characteristics of Salmonella Dublin and Salmonella Typhimurium

To examine possible correlations in bovine Salmonella isolates between environmental survival and serovar-associated epidemiological patterns, bovine field isolates of Salmonella serovars Typhimurium and Dublin (two each) were inoculated into bovine faeces slurry and tested monthly by culture for survival during a six-month period of storage at a variable ambient temperature in a disused animal transporter. Low moisture conditions, where the slurry was dried onto wooden dowels, increased detectable survival of a low-level inoculum by up to five months, compared with wet slurry. A more modest increase of survival time was seen with storage of wet slurry under refrigeration at 4°C. Under both dry and wet conditions, the concentration of culturable Salmonella Typhimurium declined at a slower rate than did that of Salmonella Dublin. Salmonella that was naturally contaminating bovine faeces from farms with Salmonella Typhimurium did not show superior survival times compared with Salmonella Typhimurium that had been artificially inoculated into samples. The differing survival characteristics of the two serovars that was observed in environmental faeces may complement their different modes of infection in cattle. Salmonella Dublin, being a bovine host-adapted strain that establishes chronic infection in some animals, may have less need to survive for a prolonged period outside of its host than does Salmonella Typhimurium.

Behaviour and survival of Phytophthora cambivora inoculum in soil‐like substrate under different water regimes

SummaryPhytophthora cambivora is a soil‐borne pathogen responsible for root and collar rot of woody species including Castanea spp., on which it causes Ink disease. P. cambivora does not produce chlamydospores, and the prevalence of a single mating type in nature limits the production of oogonia and oospores. Thus, survival of P. cambivora, in the absence of suitable hosts, relies mainly on sporangia, zoospores and mycelium. However, inoculum survival in soils or substrates represents a major factor in disease epidemics and is probably dependant on environmental factors. The aim of this article was to study the behaviour of P. cambivora in a peat‐based substrate (PbS) in the absence of a host, under controlled conditions, and by varying the substrate water regimes. Total inoculum in the substrate was quantified by qPCR, using customized rDNA primer sets, whilst zoospores were quantified after elution, and their vitality was determined by a specific staining procedure. Inoculum infectivity was assessed using chestnut root baits. Results indicated that during the initial 48 h after infestation (matric potential, pF 1.2; 70% water content), the inoculum level increased producing microsporangia and mycelium before decreasing, regardless of the water regime applied. The number of free zoospores increased over a period of (maximum) 7 days after substrate inoculation. Vitality of zoospores declined to 45% at day 18, with no statistical differences amongst water regime treatments. Both rDNA and zoospore number increased in flooded samples at day 23, illustrating the vitality of the inoculum. Inoculum was able to infect chestnut root baits up to 45 days at pF 1.2 and 3.5, before and after flooding, but at pF 6.3, only in the absence of flooding. Although based on experiments carried out in microcosms, these results highlight an unexpected resistance of P. cambivora zoospores to environmental constraints. Furthermore, the infectivity ability of the inoculum to survive in PbS over time even in the absence of the host adds relevance to the risk of movement of soil and substrates associated with plant trading worldwide.

Conditions Affecting Shelf-Life of Inoculated Legume Seed

Microbial inoculants are becoming more available as sustainable alternatives to fertilizers and other agrichemicals in broad-acre cropping. However, with the exception of legume inoculants little is understood about effective delivery and survival of the inoculum. Legume inoculants are applied to both seed and soil but seed inoculation is the most economical technique. Large quantities of pasture seed in Australia are inoculated by commercial seed coating companies, but the long-term survival of seed-applied inoculum is variable and monitoring of viability requires specialist microbiology skills and facilities. The aim of our research was to define optimum storage conditions for survival of rhizobia on legume seed and evaluate water activity as a means of monitoring shelf-life. The relationship between survival and water activity varied according to seed species, inoculum preparation, coating ingredients, initial water activity and time suggesting that storage conditions would need to be defined for each different combination. Although drying seeds after coating significantly reduced viable numbers of rhizobia, survival of rhizobia on dried commercially coated lucerne seed after 11 weeks was less variable than seeds that had not been dried. The highest numbers were maintained when seeds remained dry with water activities of between 0.47 and 0.38. The quality of inoculated seed could be improved by reducing the death rate of inoculum during preparation and providing optimum storage conditions for long-term survival.

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

This study estimated the incidence of non-O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non-O157 VTEC in clay and sandy loam soils. Twenty farms were tested over a 12-month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR-positive samples were plated onto Chromocult tryptone bile X-glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non-O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non-O157 serotypes had highest D-values in the sandy loam soil with D-values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60-48·25 days. This study shows that non-O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. This is the first such study of non-O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non-O157 VTEC should be seen as an emerging risk to be controlled within the food chain.

Forest Type Influences Transmission of Phytophthora ramorum in California Oak Woodlands

The transmission ecology of Phytophthora ramorum from bay laurel (Umbellularia californica) leaves was compared between mixed-evergreen and redwood forest types throughout winter and summer disease cycles in central, coastal California. In a preliminary multisite study, we found that abscission rates of infected leaves were higher at mixed-evergreen sites. In addition, final infection counts were slightly higher at mixed-evergreen sites or not significantly different than at redwood sites, in part due to competition from other foliar pathogens at redwood sites. In a subsequent, detailed study of paired sites where P. ramorum was the main foliar pathogen, summer survival of P. ramorum in bay laurel leaves was lower in mixed-evergreen forest due to lower recovery from infected attached leaves and higher abscission rates of infected leaves. Onset of inoculum production and new infections of bay laurel leaves occurred later in mixed-evergreen forest. Mean inoculum levels in rainwater and final infection counts on leaves were higher in redwood forest. Based on these two studies, lower summer survival of reservoir inoculum in bay laurel leaves in mixed-evergreen forest may result in delayed onset of both inoculum production and new infections, leading to slower disease progress in the early rainy season compared with redwood forest. Although final infection counts also will depend on other foliar pathogens and disease history, in sites where P. ramorum is the main foliar pathogen, these transmission patterns suggest higher rates of disease spread in redwood forests during rainy seasons of short or average length.

Gaseous emissions from soil biodisinfestation by animal manure on a greenhouse pepper crop

Soil solarisation together with the application of animal manure has been described as an alternative process for control of Phytophthora capsici root rot in pepper crops. A mixture of fresh sheep manure and dry chicken litter (SCM) and a semi-composted mixture of horse manure and chicken litter (HCM) were applied at 5.1 kg m −2 (dry weight) under plastic sheets to reduce Phytophthora inoculum survival rate and disease incidence. Non-solarised (C) and solarised (S) soils were used as control treatments. Mean NH 3 concentration increased in SCM during biodisinfestation process (14.8 mg NH 3 m −3) compared with HCM (9.1 mg NH 3 m −3), accounted for the higher organic N content and potential N mineralisation. The higher NH 3 concentration in SCM could have contributed to reduce the inoculum survival rate (30.6% and 75.0% in SCM and HCM plots, respectively). Inoculum survival rate was not reduced in S (94.4%) as temperature was below 33 °C throughout the experimental period. After biodisinfestation treatment, N 2O and CO 2 emissions tended to be higher in SCM, despite high spatial variability. Cumulative N 2O emissions were 1.31 and 0.42 g N 2O-N m −2 in SCM and HCM after 43 days. The larger N application and organic N mineralisation rate on fresh manure amended soils might have contributed to higher N 2O emissions during and after soil biodisinfestation by denitrification and nitrification, respectively. Cumulative CO 2 emission averaged 211.0 and 159.9 g CO 2-C m −2 in SCM and HCM, respectively. The soluble organic C, more abundant in fresh manure, might have favoured soil respiration in SCM. Disease incidence decreased in SCM and HCM plots (disease incidence, 2%–8%) in relation to solarised soils (42%) after 4 months. Microbial suppressiveness might have contributed to minimise Phytophthora disease incidence in SCM and HCM plots. Pepper fruit yield increased with manure amendment in SCM and HCM, which averaged 4.6 and 4.3 kg m −2, respectively. Further research will be necessary to guarantee an effective Phytophthora biodisinfestation by fitting manure N and organic matter applications, improving crop yield and reducing greenhouse gas pollution.

Survival, distribution and genetic variability of inoculum of the strawberry red core pathogen, Phytophthora fragariae var. fragariae, in soil

An experimental field infested with Phytophthora fragariae var. fragariae (Pff) and used for strawberry red core fungicide and cultivar resistance trials until 1981 was surveyed for the presence of inoculum of the pathogen 11 and 12 years later. Alpine strawberries, highly susceptible to all races of Pff, were grown from true seed and planted as a bait crop on a 0·5 m‐spaced grid. Rapid and widespread red core infection was observed, which provided good evidence that oospores had survived in soil for this extended period. Site elevation and the distribution of red core infected plants showed a strong correlation, with a higher frequency of infected and dead plants in the lowest areas of the field. The race designation of 18 recovered isolates were determined and AFLP fingerprint patterns of some of these and their single‐spore derivatives were analysed. The isolates differed little in race type, and the majority were genetically identical at 433 AFLP loci. Races used to inoculate the site in the 1970s were recovered. The fingerprints of the single variant isolate matched that of an isolation made by Hickman in the 1950s, originally used to inoculate the site. Clearly Pff is a very stable and long‐lived pathogen able to retain its genetic integrity and lie dormant in soil for many years, ensuring its survival between epidemiologically favourable conditions which occur erratically.

Impact of Cropping Sequences and Alternative Hosts on Take-all Management of Winter Wheat in Arkansas

Cultural practices are the principle means for managing take-all of wheat caused by Gaeumannomyces graminis var. tritici. This research identified cropping sequences that can be used to manage take-all in Arkansas. For dryland fields where the opportunity to grow rotational crops is limited, summer fallow was the best option for managing take-all. For irrigated fields, rotation out of wheat for at least one year reduced incidence and severity of take-all, and rice was the most effective rotational crop. Summer fallow or a rice crop was more detrimental to survival of take-all inoculum compared to corn or soybean. Reductions in inoculum were associated with elevated soil temperatures during the summer in fallow fields and with soil anoxia in flooded rice fields. Managing grassy weeds is important during rotations out of wheat. Rescuegrass was the most susceptible grassy weed to G. graminis var. tritici in this study. Although Italian ryegrass was only moderately susceptible, it likely plays a major role in maintaining inoculum because of its wide distribution and large population size.Accepted for publication 11 April 2009. Published 12 May 2009.

Effects of crop rotation, residue retention and sowing time on the incidence and survival of ascochyta blight and its effect on grain yield of field peas ( Pisum sativum L.)

Ascochyta blight is endemic in many regions where field pea ( Pisum sativum) is grown, causing significant losses in grain yield. Crop management is the main method used to reduce the severity of disease and minimise yield losses, but with varying degrees of success. This is likely to be due to variation in the production and survival of inoculum. A 6-year experiment was conducted in which disease pressure was manipulated by crop rotation, residue retention, time of sowing and fungicide treatment. The experiment measured the amount and persistence of inoculum on crop residues and in soil as well as its dispersal in the air during the growing season, and assessed disease development and yield. Aerial inoculum from infected pea residues was the main source of infection, followed by soil-borne inoculum. Soil-borne inoculum declined slowly. The average rate of decline in soil inoculum was 15% per year, suggesting that a break of 6 years is required to reduce soil inoculum by 90%. This slow decline meant that increasing the disease break from 2 to 4 years did not consistently reduce the severity of disease or improve grain yield. By comparison, inoculum on pea crop residues declined rapidly after one season. Aerial inoculum was released from residues in two main periods: in autumn, before the pea crop was sown, and also in spring. Very little aerial inoculum was measured in winter. Rainfall in summer and autumn influenced inoculum release prior to sowing and reduced the disease pressure during establishment of the crop. Aerial inoculum was exhausted once 193 mm of rainfall had been received after 1 December. Delayed sowing was the best strategy for reducing the level of disease by avoiding the autumn peak in aerial inoculum. Disease severity was more than halved and grain yield increased by 6% by delaying sowing by 3–4 weeks. However, in the absence of disease, grain yield of early-sown crops was 20% greater than that of late-sown crops. A strategy of early sowing and the strategic use of fungicide may enable the dual goals of reduced disease and improved yields to be achieved. Early sowing without the disease penalty may be possible in seasons that have high summer and autumn rainfall, which results in the release of ascospores prior to sowing.

Enhancing biohydrogen production from chemical wastewater treatment in anaerobic sequencing batch biofilm reactor (AnSBBR) by bioaugmenting with selectively enriched kanamycin resistant anaerobic mixed consortia

The basic aim of this study was to investigate the feasibility of bioaugmentation strategy in the process of enhancing biohydrogen (H 2) production from chemical wastewater treatment (organic loading rate (OLR)—6.3 kg COD/m 3-day) in anaerobic sequencing batch biofilm reactor (AnSBBR) operated at room temperature ( 28 ± 2 °C) under acidophilic microenvironment (pH 6) with a total cycle period of 24 h. Parent augmented inoculum (kanamycin resistant) was acquired from an operating upflow anaerobic sludge blanket (UASB) reactor treating chemical wastewater and subjected to selective enrichment by applying repetitive/cyclic pre-treatment methods [altering between heat-shock treatment (100 °C; 2 h) and acid treatment (pH 3; 24 h)] to eliminate non-spore forming bacteria and to inhibit the growth of methanogenic bacteria (MB). Experimental data revealed the positive influence of bioaugmentation strategy on the overall H 2 production. Specific H 2 production almost doubled after augmentation from 0.297 to 0.483 mol H 2/kg COD R-day. Chemical wastewater acted as primary carbon source in the metabolic reactions involving molecular H 2 generation leading to substrate degradation. The augmented culture persisted in the system till the termination of the experiments. The survival and retention of the augmented inoculum and its positive effect on process enhancement may be attributed to the adopted reactor configuration and operating conditions. Scanning electron microscope (SEM) images documented the selective enrichment of morphologically similar group of bacteria capable of producing H 2 under acidophilic conditions in anaerobic microenvironment. This depicted work corroborated successful application of bioaugmentation strategy to improve H 2 production rate from anaerobic chemical wastewater treatment.

Survival of Inoculum of Phytophthora capsici in Soil Through Time Under Different Soil Treatments.

From September 2001 until August 2002 and from September 2002 until August 2003, inoculum of Phytophthora capsici consisting of mycelium and oospores was buried in soil under three different soil treatments: soil solarization, fumigation with methyl bromide and chloropicrin, and white-on-black plastic mulch without fumigation or solarization. The effect of these soil treatments on the population and survival of P. capsici was evaluated through time after 28, 63, 119, 175, 245, and 343 days. Three techniques were used for detection of the localized inoculum in soil: soil dilution plating (SDP), a modified soil dilution plating technique with an overlay assay to allow for extra incubation (mSDPO), and lemon leaf baiting of soil (LLB). No viable inoculum was detected from any soil samples from the fumigated plots regardless of the soil detection technique used. By the last sampling date, viable oospore inoculum was still detected in both soil solarization and nontreated soils, but only using mSDPO and LLB. Overall, the mSDPO assay was the most sensitive assay, followed by LLB. Using mSDPO, populations in the last sampling date were 32.9 CFU/g soil for the untreated plots and 14.7 CFU/g soil for the solarized plots. Survival of P. capsici for a year would indicate that oospores have the potential to survive from year to year, and possibly much longer, in Florida and other locations.

Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae, is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30‐cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2‐m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR‐specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.

Eradication of Plasmodiophora brassicae during composting of wastes

Survival of infectious inoculum of the clubroot pathogen Plasmodiophora brassicae was assessed following bench‐scale flask composting experiments and large‐scale composting procedures. Clubroot‐affected material was provided by artificial inoculation of Chinese cabbage or naturally infected Brussels sprout and cabbage roots. Both sets of diseased material were used in flask experiments, and the latter in large‐scale windrow and aerated tunnel experiments. Municipal green wastes, onion waste and spent mushroom compost were evaluated in flask experiments with varying temperature, aeration and moisture conditions. Green wastes were used in larger‐scale composts. Within the limits of a Chinese cabbage seedling bioassay, both temperature and moisture content were critical for eradication of P. brassicae spores extracted from composted clubroot‐affected residues. Incubation in compost at 50°C for 7 days or 1 day at 60°C with high moisture levels (= −5 kPa matric potential or 60% w/w moisture content) eradicated inoculum from artificially inoculated Chinese cabbage roots. In large‐scale windrows and aerated tunnels, the pathogen was eradicated from naturally infected brassica wastes after 6–7 days at 54–73°C.

Incidence and Severity of Bean Rust (Uromyces appendiculatus) in Alleys between Leucaena Hedgerows in Kenya

The effects of inter-row spacing of Leucaena leucocephala (Lam.) de Wit in an alley cropping system on the incidence and severity of rust (Uromyces appendiculatus (Pers.) Unger) on intercropped beans (Phaseolus vulgaris L.) and their yield were examined over 2 years (1993 and 1994) at Chepkoilel Campus in Kenya. Each experiment consisted of three randomized blocks with treatments of three alley widths (2 m, 4 m and 8 m) and a treeless control with two intra-row spacings of Leucaena (0.5 m and 1.0 m). Hedgerows were coppiced at 1.0 m height and pruned subsequently at 2–3 months intervals. No fertilizer was applied but Leucaena loppings were incorporated as green leaf manure. Rust on beans was assessed at three growth stages in each season, using the Centro International de Agricultura Tropical (CIAT) scales. Microclimate was monitored in treatments, in 1994. Bean growth and yield were also measured. Rust increased with increasing alley width and was less severe in bean rows adjacent to hedgerows. Bean yield was highest in the treeless control plots and declined with decreasing alley width. Beans in 2 m alleys had significantly lower (p < 0.05) yields than 8 m alleys and treeless control plots. Bean growth was greatly modified in 2 m alleys and close to hedgerows. Light availability and diurnal temperature increased with alley width but relative humidity and leaf wetness duration decreased. Proximity to hedgerows also had marked effect on microclimate. The changes in yield, rust incidence and severity were examined in relation to microclimate, inoculum survival and dispersal.

First Report of Downy Mildew of Opium Poppy Caused by Peronospora arborescens in Spain.

Opium poppy (Papaver somniferum) is an economically important pharmaceutical crop in Spain with approximately 7,400 ha cultivated annually. In the spring of 2004, severe attacks by a new foliar disease were observed approximately 500 km apart in commercial opium poppy fields in the Castilla-La Mancha and Andalusia regions of central and southern Spain, respectively. The incidence of affected fields ranged from 40 to 50%, and incidence of diseased plants ranged from 20 to 30%. Initial disease symptoms included irregularly shaped, chlorotic-to-light yellow leaf lesions (ranging in size from 0.5 to 4 cm). Affected tissues curled, thickened, and became deformed and necrotic as disease developed. Lesions expanded in size and often coalesced, eventually giving rise to large necrotic areas in leaves or death of entire leaves. In wet weather or conditions of high relative humidity, a dense felt of sporangiophores with sporangia was produced on the abaxial leaf surface and occasionally on the adaxial surface. Microscopic observations revealed sporangiophores branching dichotomically at least four to six times, ending with sterigmata bearing single sporangia. Sporangia were hyaline, elliptical to spherical in shape, and measured 18 to 24 × 14 to 18 μm (average 19 ± 1.2 × 15 ± 1.6 μm). Occasionally, oospores formed in necrotic leaf tissues. Oospores were dark brown (the surface was irregularly ridged) and measured 36 to 46 μm in diameter (average 39 ± 4.4 μm). The oospore wall was 3 to 11 μm thick. On the basis of the observed morphological features of six symptomatic plant samples from fields at Castilla-La Mancha and Andalusia regions, we identified the pathogen as Peronospora arborescens (1). Pathogenicity was confirmed by inoculating 4- to 6-week-old opium poppy plants (cv. nigrum) with an isolate collected from a field in Ecija, Andalusia. Seed of test plants was surface disinfested and germinated under sterile conditions. Plants were sprayed with a suspension of 1 to 5 × 105 sporangia per ml in sterile distilled water. Plants sprayed with sterile water served as controls. There were five replicate plants per treatment. Plants were enclosed in sealed plastic bags and kept in the dark for 24 h. This was followed by incubation in a growth chamber at 21°C, 60 to 90% relative humidity, and a 12-h photoperiod (fluorescent light: 360 μE·m-2·s-1). After 5 to 7 days, typical downy mildew symptoms developed in inoculated plants. All control plants remained symptomless. Sporulation by the pathogen on symptomatic leaves occurred when affected plants were sprayed with water, enclosed in sealed plastic bags, and incubated at 21°C in the dark for 24 h. To our knowledge, this is the first report of P. arborescens infecting opium poppy in Spain. Infestations of poppy weeds (Papaver rhoeas) and wild Papaver somniferum were also observed in affected opium poppy fields, which may bear importance in the epidemiology of the disease as alternative hosts for inoculum increase and survival of P. arborescens under field conditions.