Secondary leaf fall of Hevea brasiliensis: meteorological and other factors affecting infection by Colletotrichum gloeosporioides

The lower leaf surface of Hevea brasiliensis was more susceptible to infection by Colletotrichum gloeosporioides than the upper. Few lesions were produced if spore drops on susceptible leaves were allowed to dry. Lesion development after 72 h was quickest at 21 ^oC, slower at 26.5 ^oC and was stopped at 32 ^oC, probably because of bacteria in the inoculation drop. On leaflets aged 7 days from bud-burst, the effective spore dose for 50% of leaflets infected (ED₅₀) after 16 h incubation, was 260 spores and after 46 h, 120 spores/infection droplet; the minimum ED₅₀ for the upper leaf surface was about 4 spores/mm². Leaflets 15 days old, which are normally resistant, were rendered susceptible by abrading the surface with carborundum powder. Spores caught in a Hirst spore trap reached a daily maximum at 23 h, at rates of up to 440 spores/m³ air/h, but fell to low concentrations as the humidity dropped during the daytime, and also during rain. There was some correlation between disease severity and duration of 97–100% relative humidity, and moderate to severe defoliation of clone PB 86 occurred when this reached 13.5 h/day. Rainfall increases infection by prolonging the period of atmospheric saturation and leaf wetness.     

Stemphylium botryosum f. lactucae- its developmentjn vifro and its pathogenicity to lettuce leaves

Stemphylium botryosum f. lactucae, incitant of a leaf-spot disease of stored lettuce, was found to be relatively restricted in its host range. Cross-inoculations with spore suspension of this fungus failed to induce symptoms in any of the host plants tested, except carrot. Among isolates of S. botryosum from various hosts, only the isolate from carrot induced slight symptoms on lettuce. While mycelial growth of the lettuce isolate was confined to the range 13–37 ^oC spores germinated at more extreme temperatures. The optimum temperature for germination and for radial growth on PDA was found to be between 25 and 30 ^oC. Wet spores were quickly inactivated at 50 ^oC, whereas more than 40 % of dry spores withstood a 24 h exposure to that temperature. Only the outer leaves of lettuce responded readily to inoculation with a spore suspension, the required incubation period being 3 days at 25 ^oC. Symptoms developed less readily on bruised leaves. Relative humidity approaching saturation was necessary for prompt and typical infection, notably during the 24 h following inoculation. Short dry periods (60 % r.h.) interposed at a later stage, while somewhat inhibitory, did not prevent infection.     

Studies of the infection of the winged bean by Synchytrium psophocarpi in Papua New Guinea

Infection of leaves and stems of Psophocarpus tetragonolobus by Synchytrium psophocarpi only occurred following spray inoculation of motile zoospore suspensions and incubation for a minimum of 12 h in polyethylene bags or a mist chamber. The incubation period was 7 days and generation time 22 days at temperatures of 31 ^oC max, 24 ^oC min and r.h. of 90% max, 70% min. Young, 1–2 day-old leaves were most susceptible; there was no infection on 10 day-old leaves and susceptibility was not increased by the removal of leaf waxes. No infection occurred when plants were grown from seed from infected pods, seed inoculated with zoospores or sporangia and seed sown in soil containing infected leaf debris. Resting spores were not found in infected tissues stored for 12 wk or in plant debris. S. psophocarpi did not infect Arachis hypogaea, Glycine max, Phaseolus aureus, P. coccineus, P. vulgaris, Pisum sativum, Psophocarpus scandens, Vicia faba, Vigna sesquipedalis and V. unguiculata. S. minutum did not infect winged bean. Inoculation confirmed the susceptibility of the winged bean lines UPS 62, UPS 122, UPS 126 and resistance of two Thai winged bean lines 1602/1 and 1611/2.     

The effect of temperature on viability of imbibed weed seeds

Imbibed seed of 10 common arable weeds were placed in trays in initially moist soil and, after imbibing for 2h, heated in ovens/incubators set to 31^oC, 42^oC, 56^oC, 75^oC or 100^oC for 0.5, 1, 2, 4, 8 or 16 days or at 102^oC, 155^oC, 204^oC or 262^oC for 0.5, 1, 2, 5, 7.5 or 10 min. After heating, seeds were incubated for 28 days at 10/20^oC or 20/30^oC on a 12 h dark/light regime, depending on species, and germination recorded. At the lower temperatures, germination of all species was prevented by temperatures of 75^oC or higher for periods of 0.5 days or more. Germination was lower after treatment at 56^oC than at 31^oC or 42^oC for all species except Rumex obtusifolius. The maximum temperature required to prevent germination varied among species and was of greater importance than the duration of heating. Germination was variable with duration of heating. At the higher temperatures, there was very little germination of any species after heating at 204^oC for 7.5 min or 262^oC for 5 min or more. Seeds were greatly buffered from the air temperature by 3 mm of soil, throughout the shorter duration of heating. The average temperature of the soil, over the 10 min heating required to prevent over 90% germination, varied among species and ranged from 48^oC for Avena fatua to 65^oC for R. obtusifolius. This work implies that composting systems maintained at 65^oC are unlikely to provide an efficient method of weed control. Recommendations for improvement of the laboratory technique are suggested.     

Environmental factors influencing the infection of wheat by Puccinia graminis

Germination rate and total germination of Puccinia graminis uredospores were directly related to pustule age and duration between spore collections. Partial drying of the spores enhanced germination rate; keeping them for 18 h at 100% r.h. reduced both rate and total germination. Spores germinated in polystyrene dishes between 4 and 29 °C and optimally between 15 and 23 °C Light (3 cal/cm²/h) had little effect on germination on moist surfaces but inhibited germination on the leaf. In Hybrid 229/8 wheat this effect was more pronounced than in var. Little Club. The number of primary infections increased linearly with duration of surface wetness with a narrow temperature optimum at 23.5 °C. Two phases of infection could be distinguished: germination (requiring darkness and capable of taking place over a wide temperature range) and penetration (requiring light and slightly higher temperature than for germination). Stomatal closure caused by subjecting the plants to water stress led to proporational reductions in infection. The results are discussed in relation to dew formation.     

Epidemiology of Cladosporium allii and Cladosporium allii-cepae, leaf blotch pathogens of leek and onion.

Conidia of Cladosporium allii and C. allii-cepae germinated over the temperature range 2–30°C on agar with optimal responses at 15–20°C (C. allii) and 20°C (C. allii-cepae). Conidia of both fungi germinated in water and at c. 100% relative humidity (r.h.) but not at lower humidities on leaf and glass slide surfaces. Germination was more rapid when spores were applied dry to agar or leaves than when applied in water or nutrient solution. More lesions developed when conidia of C. allii-cepae were deposited dry on onion leaf discs or leaf surfaces than when they were applied suspended in water. Conidia of both fungi required 18–20 h at c. 100% r.h. to germinate and infect when applied dry to leaves. Damaging the leaves or the addition of nutrients to the leaf surface increased the incidence of infection by C. allii-cepae compared to controls. Inoculated onion bait plants placed out-of-doors developed infection after at least 17 h at c. 100% r.h. or with leaf wetness. Similar conditions were necessary for infection of bait plants exposed in onion and leek crops infected by C. allii-cepae and C. allii respectively. Disease development and spread of infection occurred at different rates over the same period in two different cultivars of leeks, with spore concentrations increasing in proportion to disease. Spore numbers in the air fell considerably when infected leeks were ploughed under.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Nutritional requirements of keratinophilic fungi and dermatophytes for conidial germination

Germination of spores of Chrysosporium crassitunicatum, Nannizzia fulva (+), Nannizzia fulva (–) and Trichophyton equinum was studied in the presence of various carbon and nitrogen sources. Effect of different temperatures on spore germination was also determined. Maximum spore germination within 24 hours was recorded when glucose was used as a carbon source for all the test fungi. Except sodium nitrate all the inorganic nitrogen sources enhanced the spore germination at 0.05% concentration. Most of the organic nitrogen sources used were found to be stimulatory for the spore germination of test fungi. Optimum temperature i.e. 28 °C supported maximum spore germination of all the test fungi within 24 hours. C. Crassitunicatum, N. fulva(+), N. fulva(–) could germinate upto 35 °C but beyond that no spore germination was noticed in these fungi. T. equinum could germinate at a higher temperature of 40 °C but the percentage of germination was very low.     

Effect of temperature, light and host on prepenetration development of Puccinia graminis avenae and Puccinia coronata avenae

The influence of temperature and light on prepenetration development of single and mixed isolates of Puccinia graminis avenae and Puccinia coronata avenae was studied on 0–2% water agar and on leaves of three oat cultivars and on three non-cultivated species of Avena. Germination of uredospores of P. graminis avenae and P. coronata avenae occurred best at 10–30^oC and at 20^oC respectively. The optimum temperature for germ-tube growth and for appressorial formation was 20^oC for both rusts. An inverse relationship was observed between light intensity and prepenetration development with maximal germination of uredospores, germ-tube growth and appressorial formation occurring in darkness. Under optimum conditions maximum percentage germination and appressorium formation of both rusts was attained within 4 and 12 h after inoculation respectively. The proportion of germinated uredospores of crown rust which gave rise to appressoria was about twice that observed for stem rust. No significant differences were observed in prepenetration development between the single and mixed race inocula of the two rusts. Although germination of uredospores was significantly greater on water agar than on oat leaves, there were no significant differences in prepenetration development of the rusts on the various oat cultivars and species examined. Consequently, the data failed to indicate the presence of resistance mechanisms operating during the prepenetration phase of the infection process on the cultivars and species examined.     

Germination of concentrated suspensions of spores from Aspergillus niger

Summary Germination of condiospores fromA.niger in very concentrated suspension was required to inoculate solid fermentation media, but a germination self-inhibitor was detected in spores. It was found that the inhibitory effect depended on the composition of the medium and was reduced when glucose was used as a carbon source. The effect of the self-inhibitor was eliminated by washing the spores and using glucose and a protein nitrogen source in the germination medium. By this method it was possible to increase about 100 times (10⁶ to 10⁸) spore concentration, keeping more than 90% germination.     

Hirsutella thompsonii, a fungal pathogen of mites. I. Biology of the fungus in vitro

Hirsutella thompsonii, a moniliaceous fungus pathogenic to mites, grew and sporulated on sterilised wheat bran. The effects of environmental factors were studied on the fungus grown on potato-dextrose-agar (PDA). The fungus was mesothermophilic. Growth, sporulation and conidial germination were best at 25^o-30 ^oC. Conidia kept at 37 ^oC for 5 days on PDA died, but those held at 5 ^oC germinated upon a subsequent removal to 25 ^oC. Almost all conidial germ tubes survived an 8 h exposure to 3–5% r.h. and to 60% r.h., but subsequently the former grew poorly at 100% r.h. H. thompsonii sporulated equally well in continuous darkness or light, and produced typical chlorinous to light olive-green mycelium and conidia under all conditions. A 2 h exposure of naked mycelium and conidia (which have melanised walls) to u.v. irradiation failed to kill the fungus.     

Some factors affecting germination of Peronospora farinosa conidia in water

The germination of conidia of Peronospora farinosa f. sp. betae, collected from sugar beet and suspended in deionized water, was inhibited by dilution with 10% solutions of glycerol, glucose or sucrose and with sap from sugar-beet leaves. Germination was stimulated by diluting with deionized water but not with tap water or biological saline. Substances that diffused from excised buds of sugar-beet plants into deionized water also stimulated germination of conidia but diffusates from leaves did not. This may partly explain why buds are more susceptible to downy mildew than leaves in sugar beet. Germination of conidia was apparently stimulated more by diffusates from buds of seedlings than by those from buds of older plants; this may help to explain why sugar-beet seedlings are more susceptible to downy mildew than older plants. Diffusates from plants of four sugar-beet stocks, that differed from each other in susceptibility to downy mildew, had very similar effects on germination of P. farinosa conidia. Stimulation of spore germination on the surfaces of buds and leaves did not seem, therefore, to be an important factor in determining resistance or susceptibility to downy mildew in these stocks.     

Release of elicitors from rice blast spores under the action of reactive oxygen species

Effects of reactive oxygen species (ROS) on the release of putative elicitors from spores of rice blast causal fungus Magnaporthe grisea (Hebert) Barr were studied. While studying the influence of exogenous ROS, the spores were germinated for 5 h in the presence of 50 μM H₂O₂ and then treated with catalase to decompose hydrogen peroxide. The spore germination fluid was then boiled to inactivate catalase. When the resulting diffusate was applied onto rice (Oryza sativa L.) leaves, it caused necroses and stimulated superoxide (O₂⁻) production. Both effects were observed with the resistant rice cultivar but not with the cultivar susceptible to the fungal strain. The susceptible cultivar did not acquire resistance to challenge with fungal spores, which were applied one day after the treatment. The fractionation of the spore diffusate showed that both low- and high-molecular compounds (mol wt < 3 kD and >3 kD, respectively) should be present in combination to induce O₂⁻ production by leaves. The diffusates from spores germinated in water also caused necroses and stimulated O₂⁻ generation, though to a weaker extent than diffusates from spores germinated in H₂O₂. The effect of diffusates from spores germinated in water was abolished by catalase or superoxide dismutase added initially to the spore suspension. The results suggest that germinating spores of M. grisea are able to release elicitors and this ability depends on ROS formation by spores. Presumably, the yield of elicitors is increased additionally if fungus M. grisea is stressed or subjected to exogenous ROS. The described phenomena may be involved in incompatibility mechanisms.     

Activate to Eradicate: Inhibition of Clostridium difficile Spore Outgrowth by the Synergistic Effects of Osmotic Activation and Nisin

Background

Germination is the irreversible loss of spore-specific properties prior to outgrowth. Because germinating spores become more susceptible to killing by stressors, induction of germination has been proposed as a spore control strategy. However, this strategy is limited by superdormant spores that remain unaffected by germinants. Harsh chemicals and heat activation are effective for stimulating germination of superdormant spores but are impractical for use in a hospital setting, where Clostridium difficile spores present a challenge. Here, we tested whether osmotic activation solutes will provide a mild alternative for stimulation of superdormant C. difficile spores in the presence of germinants as previously demonstrated in several species of Bacillus. In addition, we tested the hypothesis that the limitations of superdormancy can be circumvented with a combined approach using nisin, a FDA-approved safe bacteriocin, to inhibit outgrowth of germinated spores and osmotic activation solutes to enhance outgrowth inhibition by stimulating superdormant spores.

Principal Findings

Exposure to germination solution triggered ∼1 log₁₀ colony forming units (CFU) of spores to germinate, and heat activation increased the spores that germinated to >2.5 log₁₀CFU. Germinating spores, in contrast to dormant spores, became susceptible to inhibition by nisin. The presence of osmotic activation solutes did not stimulate germination of superdormant C. difficile spores exposed to germination solution. But, in the absence of germination solution, osmotic activation solutes enhanced nisin inhibition of superdormant spores to >3.5 log₁₀CFU. The synergistic effects of osmotic activation solutes and nisin were associated with loss of membrane integrity.

Conclusions

These findings suggest that the synergistic effects of osmotic activation and nisin bypass the limitations of germination as a spore control strategy, and might be a novel method to safely and effectively reduce the burden of C.difficile spores on skin and environmental surfaces.     

Overwintering of Sphaerotheca humuli, the cause of hop powdery mildew

The ability of Sphaerotheca humuli to overwinter as cleistocarps in infected hop cones and leaves and in aerial buds on rootstocks was examined during the winters of 1970-1, 1971-2 and 1972-3. Periodical examination of cleistocarps, collected in October and overwintered in Terylene bags on the soil of a hop garden, consistently revealed two periods of maturation ending in November and in March, when over 50% contained eight, well-defined ascospores. In laboratory tests cleistocarps, kept either in the hop garden or dry at 4, 8 or 18^oC during the winter, could not be encouraged to dehisce earlier than April when naturally dehisced cleistocarps were first detected in the field. More ascospores were discharged from cleistocarps, and germination of ascospores in laboratory tests was greater, at 18 than at 4, 8 or 24^oC. Colonies of S. humuli arose on leaves of potted plants exposed to overwintered cleistocarps in the hop garden and were observed microscopically to originate from ascospores. However, a Burkard spore trap, operated amidst the cleistocarps in this garden in 1972 and 1973, failed to detect ascospores. Ascospores, discharged onto susceptible leaves in the laboratory, germinated but failed to produce colonies. It was demonstrated that S. humuli can perennate in aerial, dormant buds on hop rootstocks. Examination of buds in autumn revealed mycelium external to and between the bud scales. At budburst the mycelium was still present internally. Cleistocarps were occasionally associated with hibernating mycelium. Primarily infected shoots arose from plants bearing infected buds in conditions which precluded chance infection. Some evidence was obtained that conditions during the winter determine the success of survival in buds. The fungus appeared to be incapable of infecting a selection of weeds common to hop gardens and their vicinity.     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.     

Fungitoxicity of acetaldehyde vapour to some major post-harvest pathogens of citrus and subtropical fruits

In laboratory trials, fumigation with acetaldehyde vapour at 0–75 to 20 % concentration (v/v) for 0–5 to 120 min at 21 ^oC killed six species of post-harvest citrus pathogens grown on agar media. The fungicidal effect of acetaldehyde vapour at this temperature was a function of concentration and exposure. Penicillium digitatum and P. italicum were not as susceptible to inhibition of spore germination as Alternaria citri, A. tenuis, Colletotrichum gloeosporioides and Glomerella cingulata. A concentration of 10 % acetaldehyde vapour at 21 ^oC for 10 min was fungitoxic to all the pathogens tested.     

The spore‐associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild‐type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID₅₀) of spores was significantly higher (2‐logs) in the bclA1^? mutant compared to the isogenic wild‐type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild‐type spores. bclA1^? spores germinated faster than wild‐type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre‐spore germination) stages of infection. We also show that the ID₅₀ was higher in mice infected with R20291, a ‘hypervirulent’ 027 strain, that carries a truncated BclA1 protein.