Survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity

Aims: To determine survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity (r.h.). Methods and Results: Spinach leaves were inoculated with suspensions of E. coli O157:H7 in distilled water (DW) and 0·1% peptone water (PW) and incubated at 4, 12 and 25°C and 43, 85 and 100% r.h. The number of E. coli O157:H7 on leaves (5·6 or 1·9 log CFU per leaf) inoculated using DW as a carrier medium increased significantly at 25°C and 100% r.h. within 120 h but remained constant or decreased significantly under other test conditions. E. coli O157:H7 on leaves (5·4 log CFU per leaf) inoculated using PW as a carrier increased significantly within 72 and 24 h, respectively, at 12 or 25°C and 100% r.h.; counts using a low inoculum (2·2 log CFU per leaf) increased significantly within 24 h at 25°C. Conclusions: Escherichia coli O157:H7 can colonize on spinach leaves at 12 or 25°C in a 100% r.h. environment. Organic matter in the inoculum carrier may provide protection and nutrients which enhance survival and colonization. Significance and Impact of the Study: Colonization of E. coli O157:H7 on spinach leaves as affected by organic matter in the inoculum, temperature and r.h. was determined. These observations will be useful when developing strategies to prevent growth of E. coli O157:H7 on pre‐ and postharvest spinach.     

The effect of two biological control agents, the weevil Neochetina eichhorniae and the mirid Eccritotarsus catarinensis on water hyacinth, Eichhornia crassipes , grown in culture with water lettuce, Pistia stratiotes

We assessed the effect of two biological control agents, the mirid Eccritotarsus catarinensis (Carvalho) and the weevil Neochetina eichhorniae (Warner), singly or in combination, on the competitive ability of their host plant, water hyacinth, Eichhornia crassipes (Mart.) Solms-Laub., grown in a screen house, in competition with another aquatic plant (Pistia stratiotes L.). Water hyacinth plant growth characteristics measured included fresh weight, leaf and petiole lengths, number of inflorescences produced, and new shoots. Without herbivory, water hyacinth was 18 times more competitive than water lettuce (across all experimental combinations of initial plant densities), as estimated from fresh weights. Both insect species, singly or in combination, reduced water hyacinth plant growth characteristics. E. catarinensis alone was less damaging than the weevil and under normal conditions, i.e., floating water hyacinth, is not expected to increase control of water hyacinth beyond that of the weevil. When combined with the weevil, half the inoculum of weevils and half the inoculum of mirids produced the same growth reduction as the full inoculum of the weevil. Under conditions where the weevils are not effective because water hyacinths are seasonally rooted in mud, the mirid, which lives entirely on leaves, should become a useful additional biological control agent. Handling Editor: John Scott.     

Observations on the vapour phase activity of some foliage fungicides

By means of a Botrytis fabae/Vicia faba bio-assay technique it has been demonstrated that phenyl mercury chloride, maneb, mancozeb, dichlo-fluanid and oxythioquinox protect areas of leaf beyond the visible limits of the fungicide deposits. The evidence suggests that the extended areas of protection are due to the release of fungicidal vapours. For a given dose of mancozeb the area of protection was related to the number of conidia of B. fabae dusted on to the leaves and for a given inoculum density it extended with increasing fungicide dose applied in standard drop sizes. When the same dose of fungicide was applied in increasing volumes of water, producing widening areas of deposit, the area of protection also increased. Fungicide deposits aged on leaves for up to 4 weeks continued to release toxic vapours. Contact between the fungicides and leaves or between fungicides and spores was not necessary for the demonstration of the phenomenon since vapours diffused from deposits on glass and inhibited the germination of spores in water droplets placed at a distance from the fungicide source. For a given distance separating the fungicide and the spores inhibition increased with increasing fungicide dose. For a standard fungicide dose, inhibition decreased with increasing distances between the fungicide and the spores. The fungicidal vapours inhibited the germination of spores of test fungi other than B. fabae. The practical implications of these observations are examined in the light of evidence that vapour phase protection can occur on leaves incubated in large cabinets; on leaves pre-incubated at unsaturated humidities; and on leaves incubated in a moving stream of air.     

Comparison of conventional and miniaturized biochemical techniques for identification of animal streptococcal isolates

Human clinical streptococcal isolates can be identified rapidly by means of commercially available miniaturized biochemical systems, in contrast to animal and environmental isolates which may require extensive characterization using conventional methods. Streptococcal isolates (n=548) from fresh animal feces of cattle, swine, and broiler chickens were tested by means of conventional biochemical and physiological techniques, and also with a miniaturized technique in which conventional formulations were dispensed in 0.1 ml volume into microtiter plates. Agreement of the positive feature frequencies of the two methods were compared. Results from the tolerance tests in the two methods were generally in good agreement. However, the miniaturized method tended to give false negative results in some carbohydrate fermentation tests. Agreement between the 2 methods ranged from 100% for bile esculin tests to 71% for raffinose fermentation. Cluster analysis of the conventional method data indicated that there were 11 biochemically related groups of isolates, 2 of which were identified asStreptococcus faecalis, andS. morbillorum. Half of the isolates biochemically resembledS. faecium. Errors of miniaturized tests occurred mainly in certain tests and in certain biochemically related clusters of isolates. The data indicate that further investigation of experimental conditions such as medium formulation and inoculum size could lead to a successful miniaturized technique for testing animal streptococcal isolates.     

Detection of Latent Infection of Wheat Leaves Caused by Blumeria graminis f.sp. tritici Using Nested PCR

Wheat powdery mildew caused by Blumeria graminis f.sp. tritici (Bgt) is an important and destructive disease worldwide. Detection of latent infection of wheat seedlings is critical to estimate initial inoculum potential of epidemics in the fields. To improve the conventional method, a nested PCR approach had been established in this study to detect latent infections of wheat leaves caused by Bgt. The DNA primer sets including external and internal primer pairs for the nested PCR were designed followed by testing their specificities to Bgt by using Bgt and other fungal species of wheat. Sensitivity test demonstrated that the nested PCR could detect as low as 0.1 fg template DNA and about 10,000 times more sensitive than the standard PCR. Results of artificial inoculation experiments showed that the nested PCR assay can detect a low level of latent infection of wheat seedlings 2 days earlier than did standard PCR. The incidences of latent infection of wheat seedlings determined by the nested PCR linearly correlated with those by the conventional incubation method (r² = 0.66, P = 0.0023). The incidences of latent infection detected with nested PCR were higher than that with the conventional method. This study provides an accurate method to efficiently estimate the initial inoculum potential of wheat powdery mildew epidemics in the fields.     

Dispersal of the conidia of Colletotrichum gloeosporioides by rain and the development of anthracnose on onion

The conidia of Colletotrichum gloeosporioides were found to be dispersed during rainfall by wash-off and splash mechanisms. The initiation and development of onion anthracnose was found to depend on the frequency of rainfall and the movement of conidial inoculum during rainfall. Experiments conducted under controlled conditions in the laboratory employing splash and wash-off assemblies showed that impacting incident water drops (splash) and flowing water (wash-off) liberated the conidia from the anthracnose lesions of the onion leaf/peduncle. Peak liberation of conidia occurred with 3 to 5 water drops and most of the conidia were removed from the source within 90 seconds. A possibility of the dispersal of conidia of C. gloeosporioides from soil to lower leaf by splash mechanisms and then from the leaves to the neck of the onion bulb and to the bulb by wash-off mechanisms is indicated.     

Performance and survival of different races of Puccinia graminis f.sp. tritici on detached leaves of wheat

The detached leaf culture of Puccinia graminis f.sp. tritici was tested with eight races and two biotypes of the pathogen. The inherent characteristics of the races were similar to those in intact plants. Continuous maintenance of races in pure culture on detached leaves showed the usefulness of the technique for multiplication of inoculum as well as for maintenance of races in other basic studies.     

Effects of Long‐Term Storage Methods on the Infectivity of Urediospores of Phakopsora pachyrhizi

A method for long‐term storage of spores of Phakopsora pachyrhizi was optimized. Three methods with different procedures for spore harvest and four different reactivation methods (varying in hydration or using heat shock) were analysed for the suitability for long‐term storage at ?80°C. All conservation methods as well as all reactivation methods lead to the infection of soybean leaves after 1 year of storage. Regarding efficiency and labour input, the most recommended method is to tap off spores from infected and sporulating leaves with subsequent dehydration before storage at ?80°C. Because hydration or heat shock steps did not provide any advantages, spores can be suspended in Tween water directly after storage and used as inoculum.     

Survival of inoculum of the leaf blight fungus Phytophthora colocasiae infecting taro, Colocasia esculenta in the Solomon Islands

Phytophthora colocasiae was successfully isolated by baiting with detergent-treated taro leaf discs 8 cm diameter placed on water slurries of soil, on suspensions of macerated infected leaf lesions or on the washings from petioles of harvested plants. Taro root tips, detached or left on corms, were not susceptible to zoospores of P. colocasiae nor were detached root tips of Lupinus angustifolius. Cubes of taro corm used as baits, and agar selective for Phycomycetes which was inoculated directly with soil, both became too heavily overrun by Phythium splendens to allow detection of P. colocasiae. Investigations indicated that inoculum on lesions of detached leaves and in soil remains viable for only a few days. Petiole bases which comprise the bulk of the ‘tops’ used for vegetative propagation, lost detectable natural inoculum rapidly (2 days) if stored dry, but less rapidly (14 days) if planted immediately in the field. Artifically augmenting surface inoculum with naturally produced sporangia considerably extended the periods of detectability, probably by increasing the chances that a few propagules would survive, especially during dry storage. Incubation of inoculated tops in high humidity led to active infection and sporulation on petioles, especially on cut ends, a situation that might be paralleled under suitable moisture conditions in the field. Of several aroid species tested by artificial inoculation only Alocasia macrorrhiza was susceptible. Natural infection of this plant has not been seen, making it an unlikely alternate host of P. colocasiae under field conditions. Thus perennation between taro crops is effected by shortlived surface propagules and possibly also by mycelium within lesions on petioles. Reduction of the former and prevention of the latter might be achieved by dry storage of tops for 2 to 3 wk.     

Fungal species diversity in juvenile and adult leaves of Eucalyptus globulus from plantations affected by Mycosphaerella leaf disease

In recent years, Mycosphaerella leaf disease (MLD) has become very common in Eucalyptus globulus plantations in Galicia, northwest Spain. The aetiology of MLD is complex and is associated with several species of Mycosphaerella and Teratosphaeria. A survey of the fungal mycobiota associated with juvenile and adult leaves and with leaf litter of the same trees in MLD‐affected plantations was made. The goal was to identify pathogens and endophytes, to determine whether the mycobiota of each leaf type differed and whether leaf litter might be a reservoir of MLD inoculum. Fungi belonging to 113 different species were isolated from the leaves of juvenile and adult trees sampled at 10 locations; 81 species occurred in juvenile and 65 in adult leaves. The average number of species obtained from juvenile leaves was significantly greater (P > 0.01) compared to adult leaves. This difference suggested that juvenile leaves are not only more susceptible to a group of pathogens, but to a wide range of fungi. Therefore, a general resistance mechanism might be lacking or be less effective in juvenile than in adult leaves. Several pathogenic species were identified in both leaf types. Leaf litter and living leaf mycobiotas were very different. However, some of the species they shared were MLD pathogens, suggesting that leaf litter could contribute to the inoculum of MLD.     

Seasonal changes in primary and secondary inoculum during epidemics of leaf blotch (Rhynchosporium secalis) on winter barley

Seasonal changes in numbers of conidia of Rhynchosporium secalis on debris from previous barley crops infected with leaf blotch (primary inoculum) were monitored in 1985–86 and 1986–87. In 1986–87, changes in numbers of conidia on leaves of plants in the new winter barley crop (secondary inoculum) were also recorded. The greatest increases in production of primary inoculum were in early spring after rain, when temperatures were increasing after periods of sub-zero temperatures when there was little conidial production. Subsequently, more conidia were recovered from this debris after cycles of drying and rewetting than when it remained wet. After January 1987, amounts of secondary inoculum produced on the crop were much greater than amounts of primary inoculum on debris. Most spores were produced on the basal leaves and more spores were present on the September-sown than on the November-sown crop. Thus, while primary inoculum was a source of disease when plants were emerging, secondary inoculum on basal leaves was the main source of disease at stem extension, especially on early-sown crops.     

Effect of leaf age and inoculum concentration on the symptoms produced by Gymnosporangiumjuniperi-virginianae on apple

Gymnosporangium juniperi-virginianae, the incitant of cedar apple rust, induced the greatest number of lesions (flecks, pycnial lesions, aecial lesions) on 6- and 8-day-old leaves of single-shoot potted apple plants inoculated and incubated under controlled conditions. The largest pycnial lesions developed on 2- and 4-day-old leaves and the largest aecial lesions on 6-day-old leaves. The number of lesions was positively correlated with the concentration of basidiospores in the inoculum, but lesion size was constant at different inoculum levels. Elevated inoculum concentrations failed to induce pycnia on cv. McIntosh which normally bears only flecks; very low concentrations induced the development of pycnia but not aecia on cv. Rome Beauty, which normally bears aecia. An ‘infection rating’, log₁₀ (10 nd²), where n is the highest number of pycnial lesions per leaf and d is the largest mean diameter of pycnial lesions on any leaf, calculated for four cultivars in greenhouse tests showed a positive correlation with a ‘susceptibility rating’ developed from observations of natural infection of these cultivars in the field.     

Biocontrol of Ailanthus altissima: inoculation protocol and risk assessment for Verticillium nonalfalfae (Plectosphaerellaceae: Phyllachorales)

Verticillium nonalfalfae has been proposed as a biocontrol for invasive Ailanthus altissima (tree-of-heaven) in Ohio, Pennsylvania and Virginia. However, previous studies evaluating this potential biocontrol utilised a conidial suspension with a short shelf life as inoculum. Anticipating future expanded use of V. nonalfalfae, we evaluated other inoculum formulations, inoculation protocols and sensitivity of non-target (non-Ailanthus) plant species within Pennsylvania. The most effective inoculum formulation, with an extended shelf life, was prepared by mixing water with stored, refrigerated soil containing V. nonalfalfae. Less successful, but positive infections were obtained by simply using infected Ailanthus wood and leaves as inoculum. Monthly inoculation of Ailanthus trees demonstrated that the optimal time for successful inoculations was April to May, but limited infections were achieved during all months, including the winter. The health of Ailanthus and non-target species was evaluated within a decade-old natural Verticillium wilt epicentre, where all mature Ailanthus trees had been killed by V. nonalfalfae. Verticillium wilt was observed on a few small Ailanthus trees, likely newly established seedlings, whereas non-target species were asymptomatic. Our findings reveal that soil formulated and natural inocula are effective biocontrols against Ailanthus, and V. nonalfalfae appears to pose little threat to non-target plants.     

Patchy distributions of Spirogyra arcta do not affect growth of the submerged macrophyte Ceratophyllum demersum

Spatial heterogeneity is common in aquatic conditions, but few studies have examined the effects of heterogeneous distributions of biological factors on aquatic plants. Spirogyra (Spirogyra arcta) coexists with many submerged macrophytes, such as Ceratophyllum demersum, but no study has examined the effects of heterogeneous distributions of spirogyra on the growth of submerged plants. We grew the submerged plant C. demersum in three homogeneous, aquatic conditions (0, 50 and 100% of the water surface in the container was evenly covered by spirogyra, referred to as ‘control’, ‘50%’ and ‘100%’, respectively) and two patchy conditions (the left half of the water surface in the container was not covered by spirogyra and 50% and 100% of the water surface in the right half of the container was evenly covered by spirogyra, referred to as ‘50%‐patchy’ and ‘100%‐patchy’, respectively). Compared with the control, the 100% treatment greatly decreased the biomass and number of ramets of C. demersum, but the 50% treatment did not. Growth of C. demersum in the left half of the container did not differ significantly between the control and the two heterogeneous treatments (50%‐patchy and 100%‐patchy). In addition, growth of C. demersum in the right half of the container did not differ between the 100% and the 100%‐patchy treatment or between the 50% and the 50%‐patchy treatment. Our results suggest that C. demersum can tolerate shading by spirogyra to a certain extent and that heterogeneous distributions of spirogyra do not affect its growth.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT. II. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN MILK

SUMMARY: The sporicidal efficiency of an ultra-high-temperature (UHT) milk processing plant has been tested using spores of a strain of Bacillus subtilis in milk. With the inoculum and volume of milk adjusted to obtain a countable number of survivors by a conventional dilution counting method, a temperature of 130·5° with the minimum time setting of the plant was found necessary to give a destruction of 99·99999%. This temperature was lower than that found previously (135°) for spores suspended in water and evidence is produced to support the suggestion that UHT milk may be inhibitory to the germination and/or subsequent growth of heated spores.     

Dynamics of infection by Leptosphaeria maculans on canola (Brassica napus) as influenced by crop rotation and tillage

Abstract

This study, intending to understand the effects of crop rotation and tillage on blackleg disease, was conducted in a field at Carman, Manitoba, Canada, from 1999 – 2002. Canola, wheat and flax were among the rotated crops. Rotations were performed under conventional or zero-till conditions. The number of infected plants, infected leaves per plant, lesions per plant, and percentage of leaf coverage with lesions decreased when canola was rotated with wheat and flax under zero till. The number of lesions per plant and percentage of leaf coverage with lesions were strongly correlated with stem disease severity, and the number of infected plants with stem disease incidence. Ascospores and pycnidiospores of Leptosphaeria maculans were reduced by crop rotation and tillage. This study suggests that the appropriate combination of rotation and tillage may lower airborne inoculum and reduce infection of canola plants by L. maculans.     

VvPIP2;4N aquaporin involvement in controlling leaf hydraulic capacitance and resistance in grapevine

Hydraulic capacitance (C) in a plant tissue buffers the xylem tension, storing and releasing water and has been highlighted in recent years as an important factor that affects water relations such as drought tolerance and embolism formation. Aquaporins (AQPs) are well known to control leaf hydraulic resistance (Rh) but their role in the control of C is unknown. Here, we assess Rh and C on detached grapevines wild‐type (WT) (cv. Brachetto) leaves and over‐expressing the aquaporin gene VvPIP2;4N (OE). For this purpose, we developed a new method inspired from the pressure‐volume curve technique and the rehydration‐kinetic‐method, which allowed us to monitor the dynamics of dehydration and rehydration in the same leaf. The recovery after dehydration was measured in dark, light non‐transpirative conditions, light‐transpirative conditions and light‐transpirative condition adding abscisic acid. Pressurizing to dehydrate leaves in the OE line, the recorded Rh and C were respectively lower and higher than those in the WT. The same results were obtained in the dark recovery by rehydration treatment. In the presence of light, either when leaves transpired or not (by depressing vapor pressure deficit), the described effects disappeared. The change in Rh and C did not affect the kinetics of desiccation of detached leaves in dark in air, in OE plants compared to WT ones. Our study highlighted that both Rh and C were influenced by the constitutive over‐expression of VvPIP2;4N. The effect of AQPs on C is reported here for the first time and may involve a modulation of cell reflection coefficient.     

Studies on the Pathogenic Potential and Life Cycle of Reniform Nematode on Broccoli

The significant reduction in the plant growth was observed at an initial inoculum level of 2 immature females of Rotylenchulus reniformis/g soil and hence this inoculum level was considered as the minimum damaging threshold level for broccoli. The population of reniform nematode increased with the increasing levels of inoculum. Though, the maximum nematode population was recorded at the highest level of inoculum, the rate of reproduction of nematode was found to be highest at the lowest level of initial inoculum. Symptoms like chlorosis, stunted growth, shedding of leaves, early sprouting of inflorescence and sparsely developed roots were found during the experimental studies. Rotylenchulus reniformis required 30 days to complete the life cycle on broccoli.     

Photothermal beam deflection: a new method for in vivo measurements of thermal energy dissipation and photochemical energy conversion in intact leaves

A novel photosynthetic technique, photothermal deflection spectroscopy, is presented which is based on the mirage effect and allows the rapid measurement of thermal deactivation of excited pigments in leaf samples placed in an open cell. Modulated heat emission from leaves illuminated with intensity-modulated light was measured via the detection of the periodic deflection of a laser beam parallel to the sample surface. Photothermal deflection signals can be monitored in vivo in leaves placed in various, liquid or gaseous, environments with a satisfactory signal-to-noise ratio close to 60–80 (in distilled water) at low modulation frequencies (below 50 Hz). Using this new and simple photothermal method, it was possible to easily obtain useful information on the leaf photochemical activity and its light-saturation characteristics under normal or stress conditions, suggesting that in vivo deflection signals could be used for assaying the photosynthetic state of health of crop plants. The beam deflection method presented in this paper appears to be a potentially useful photosynthetic tool complementary to the related photoacoustic technique.Abbreviations DCMU dichlorophenyldimethylurea - PD photothermal deflection - PL photochemical energy storage - S/N ratio signal-to-noise ratio     

Preservation of coagulation efficiency ofMoringa oleifera, a natural coagulant

In recent years, there has been an interest to useMoringa oleifera as the natural coagulant due to cost, associated health and environmental concerns of synthetic organic polymers and inorganic chemicals. However, it is known thatM. oleifera as the natural coagulant is highly biodegradable and has a very short shelf life. This research was carried out to investigate the effects of storage temperature, packaging methods, and freeze-drying on the preservation ofM. oleifera seeds powders. Non freeze-driedM. oleifera was prepared into different packaging namely open container, closed container and vacuum packing, whilst, freeze-driedM. oleifera was stored in closed container and vacuum packing. Each of the packaging was stored at room temperature (30 to 32°C) and refrigerator (4°C). The turbidity removal efficiencies of storedM. oleifera were examined using jar test at monthly interval for 12 months. The results indicated that non freeze-driedM. oleifera kept in the refrigerator (4°C) would preserve its coagulation efficiency. In addition, closed container and vacuum packing were found to be more appropriate for the preservation of non freeze-driedM. oleifera, compared to open container. Freeze-driedM. oleifera retained its high coagulation efficiency regardless the storage temperature and packaging method for up to 11 months. Besides, higher increment in zeta potential values for water coagulated with freeze-driedM. oleifera indicated the higher frequency of charge neutralization and better coagulation efficiency of freeze-driedM. oleifera, compared to non freeze-dried seeds. As a coagulant,M. oleifera did not affect the pH of the water after treatment.