Wind Dispersal of Alternaria alternata, a Cause of Leaf Blight of Cotton

Introducing Alternaria alternata, the cause of blight disease of cotton plants, into a field of young healthy plants growing in rows cross-wind, yielded disease foci which were spread downwind up to 7 m from the infection sources. Only light disease incidence was found in the remainder of the field. When the disease was introduced into a field of mature cotton plants grown in rows cross-wind, randomly scattered disease foci occurred. In mature plantations where rows were parallel to the average wind direction, only limited size disease foci developed downwind, up to 16 m from the source. These foci did not developed further during the season. The number of air-borne spores of A. alternata was significantly increased by the presence of diseased cotton plants, being highest close to the diseased plants. The spores were transferred to a distance of at least 20 m. However, the number of air-borne spores significantly decreased 6 m from the infection source. Periodical trapping of air-borne spores of A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata in a cotton growing region for 2 years, revealed that their air dispersal is local, probably at the field level. A. alternata air-borne spores were also trapped in rather low numbers regardless of the presence of infected cotton plants. However, the number of the air-borne spores trapped was dependent mainly on the average wind direction and on the Alternaria blight epidemics occurring in the fields twice a year. It is suggested that A. alternata spores are transferred by wind for short distances but are constantly present in small numbers in the atmosphere throughout the whole year. The two peaks recorded for the number of spores present in the air above cotton crops correlate with the annual two outbreaks of Alternaria blight epidemics. In addition, both wind and plant row direction affect disease development in the fields.     

Spore Dispersal and Plant Disease Gradients; a Comparison between two Empirical Models

Power law and exponential models were fitted to 325 sets of observations which described decreases with distance in deposition of air-borne or splash-borne spores, or pollen, or in amounts of plant disease caused by fungi, bacteria or viruses. There, was generally little difference between the models in the goodness of fit to these data, although deposition gradients for spores borne in splash droplets were fitted better by exponential models and gradients for fungi with air-borne spores less than 10 μm in diameter were fitted better by power law models. The exponential model has the property that the observed variable decreases by half as the distance from the source increases by a constant increment (the half-distance); this provides a measureof the gradient that is more easy to visualize than the exponent in power law model. The half-distances of gradients for air-borne pathogens were greater than those for splash-borne or soil-borne pathogens. The exponential model is easier to incorporate into models of disease development than the power law model because the boundary condition at the source (the estimated number of spores or amount of disease at the source) is finite rather than infinite. However, both these empirical models have limitations and should not be extrapolated to distances outside the observed range.     

Seasonal variation in the spore bank of ferns in grasslands on dolomite rock

Composition and seasonal patterns of the fern spore bank were compared to the surface vegetation of grasslands on dolomite rock in Hungary. Viability and potential dormancy of spores were tested through storage experiments. Although Asplenium ruta-muraria L. was the only species found at the study sites, five others, probably originating from air-borne spores from nearby areas, emerged from the soil samples. Considerable seasonal variability was detected in the number of prothallia emerging from soil samples from different sampling dates, with a peak after spore dispersal. The increased number of emerging prothallia after 1 year of storage suggests that a part of the spores stored in the soil samples were presumably dormant. Investigations on the dormancy of fern spores might be of great interest, especially in species adapted to seasonally unfavourable habitats.     

Gone with the wind — a second blow against spontaneous generation

Christian Gottfried Ehrenberg can be considered to be the founder of the science of air-borne micro-organisms and thus aerobiology. The bicentennial of his birth (he was born on Easter Sunday, April 19th, 1795), however marks the beginning of several sciences and fields of science. He is the founder of geomicrobiology, protistology, co-founder of microbiology, scientific microscopy, neurobiology, and last not least of the theory of the Earth as a living entity, including rocks and rock deposits. The number of genera and species of bacteria, spirochaetes, fungi, diatoms, radiolaria, protozoa, and rotifera which were first described by him is in the hundreds. He is known as the discoverer and detailed describer of some of the most common bioluminescent marine organisms (Noctiluca miliaris andPeridinium), as well as the person who first deciphered the secret of the bloody hostia (Monas orBacterium prodigiosum, or more correctlySerratia marcescens), of the red snow and of the ‘meteor paper’ or ‘paper meteorites’. In this commemorative paper his work on air-borne dust and air-borne micro-organisms is reviewed.     

The fungal Flora of the air near the ground in San Antonio,Texas

Summary Daily exposure of petri dishes containing Littman's oxgall medium were used to survey the incidence of air-borne fungi in San Antonio. A total of 7026 colonies was obtained. Hormodendrum andAlternaria were found to be the most common fungi present. About 80 genera were isolated, including the yeast-like fungi.No direct correlation was found between the seasonal pattern of theHormodendrum, Alternaria or the total counts, and any single environmental condition in the area. It seems as if all factors combine including the long summer months of the area to control the number of viable spores in the air.This study was supported by U.S.P.H.S. grant 5-ROL-HE-3834-07.     

Extended type 1 chain glycosphingolipids: dimeric Lea (III4V4Fuc2Lc6) as human tumor-associated antigen

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Lea, with the structure as follows. [formula: see text] Antigens containing this structure and various analogous structures (including enzymatically synthesized Lea/Lex hybrid antigen) were tested with ST-421. While the mAb was equally reactive with dimeric Lea and Lea/Lex, only the former was chemically detectable as the slow-migrating glycolipid from the tumor extract. ST-421 showed less reactivity with simple Lea (III4FucLc4) or extended Lea (V4FucLc6, and/or IV3Gal beta 1----3[Fuc alpha 1----4]GlcNAcnLc4), and was not reactive with Lex/Lex (dimeric Lex). It was concluded, therefore, that the major tumor-associated slow-migrating glycolipid reacting with ST-421 has the dimeric Lea structure shown above. Since extension of lacto-series structure has been shown to be limited to type 2 chain in normal cells and tissues, extended elongation of type 1 chain as shown in this structure represents a novel tumor-associated epitope.     

Pollen and Spore Incidence and Phenology in the Stockholm Area during 1972

Records of air-borne pollen and spores were made during a six month period in the Stockholm area in 1972. This paper presents phenological data and volumetric measurements obtained by means of a Burkard trap. Attention was paid to both arboreal pollen and non-arboreal pollen and spores, identified to family, genus or species level. Spores of certain common Pteridophytes were also included in the investigation, but not Bryophytes or fungal spores.     

Control of Chenopodium album by soil application of Ascochyta caulina under greenhouse conditions

Effects of soil application of Ascochyta caulina spores on seedlings of Chenopodium album and five cultivated plant species were investigated under greenhouse conditions as a part of a study on biological control of C. album. Application of A. caulina spores to soils resulted in disease development on C. album and to a lesser degree on Spinacia oleracea seedlings, but not on Beta vulgaris subspecies vulgaris, Zea mays, Triticum aestivum and Pisum sativum seedlings. Affected C. album seedlings had an abnormal olive-green colour or necrotic spots on cotyledons and hypocotyls, and were stunted or died. Affected S. oleracea seedlings were pale in colour or had necrotic spots on the cotyledons, but did not die. Time courses of disease incidence and of mortality of C. album could be described by a monomolecular model. Effects of spore density, sowing depth, soil water content, soil type and time of sowing on disease development were examined. Disease incidence and mortality were influenced by spore density, soil water content and soil type, but not by sowing depth. Spores in a moist soil maintained infectivity at least 2 wk. Spore densities of 10⁹ to 10¹⁰ spores m^-2 were required for 50% mortality of emerged C. album plants. Aspects of the development of A. caulina into a soil-applied mycoherbicide are discussed.     

Fine structure of the knobby spore type of Streptomyces torulosus

The type strain of Streptomyces torulosus Lyons and Pridham (1971) was studied by scanning- and transmission electron microscope. Spore chains were formed in spirals by aerial mycelium. The spores were connected by nozzles in which small channels could be observed. The knobby ornamentations of the spores arised on a thin fibrous sheath, enveloping the spore chains. These irregular blunt projections, called knobs, had varying diameters of 100 to 250 nm. The base of the knob, consisting of globose to flattened electron dense material, was sitting directly on the sheath. It was covered by several small vesicles of the same material. Each hollow vesicle beared a thin bowlshaped shell of electron transparent material. In general, the cupular bowls and their supporting vesicles became easily depressed on their base, but not detached from the surface of the spores. This type of knobby spore ornamentation was suggested to be designated as a verrucate spore type.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

Spore germination in Dictyostelium discoideum

Mutant spores of Dictyostelium discoideum, strain SG-10, differ from wild type spores in their ability to spontaneously germinate, to be activated with 5% dimethyl Sulfoxide (DMSO), and to be deactivated with 0.2 M sucrose. Both heat-activated wild type and mutant spores began to swell after a lag of 60–75 min at ambient temperature. Suspension of heat activated spores in 5% DMSO resulted in blockage of spore swelling and a concomitant severe inhibition of respiration; removal of 5% DMSO allowed resumption of respiration and the spores began to swell after a lag of only 15 min. It was concluded that 5% DMSO allowed the early reactions (M) to proceed but blocked the later reactions (R) of post-activation lag.Treatment of one day old spores with 20% DMSO solution for 30–120 min quantitatively activated the population. The post-activation lag time was directly dependent on the time of 20% DMSO treatment. Spores activated with 20% DMSO treatment could be deactivated by incubation at 0°C; the spores most quickly deactivated at 0°C were those within 10 min of swelling. Mitochondrial transport inhibitors such as azide and cyanide caused deactivation in an analogous manner. It is hypothesized that spores proceed to the second portion of the lag phase called (R) before the environment determines if dormancy is reimposed or if germination will proceed. The sensitive strain (SG-10) showed a greater degree of damage than the wild type after supraoptimal treatment with 40% DMSO. The spores became more resistant with age to the damaging action of 40% DMSO. All the observed effects of DMSO treatment were compatible with our multistate model of activation which suggests that the early portion of the lag phase (M) may involve a relative uncoupling of oxidative phosphorylation while the later portion (R) may require tight coupling.     

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non-toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.     

Viability and physiological state transitions of <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> sporangiospores in tempe starter culture

The viability and various physiological characteristics of individual sporangiospores of Rhizopus oligosporus in tempe starter cultures that had been stored for 8, 10, 16 and 30 months were examined by flow cytometry in combination with fluorescent dyes. Besides live, dead, and dormant spores we distinguished a category of sublethally damaged spores. Results indicated that the shelf-life of tempe starters was not limited by the death of spores, but by sublethal damage to spores as well as by dormancy which can be overcome by resuscitation, respiratory activation. During storage, the number of dormant and sublethally damaged spores increased: the longer the starter cultures were stored, the less dormant spores could still be activated. In contrast, the transition from sublethally damaged (spores that are not able to transform cFDA and emit green fluorescence except by activation treatment) to activated spores did not decrease with longer storage. However, after very long (30 months) storage, sublethally damaged spores could still be activated but could not germinate anymore. The shelf-life of spores in tempe starter is related to the physiological state of spores being sublethally damaged; a mechanism of physiological state transitions of R. oligosporus sporangiospores is proposed.     

Sclerotinia sclerotiorum: Epidemiological Factors Affecting Infection of Greenhouse Aubergine Crops

A series of greenhouse cultivations of Aubergine plants were studied for a three-year period which included the 1981—1982, 1984—1985 and 1985—1986 growing seasons to determine the number of infections produced by S. sclerotiorum and the plant organs infected. A time-phase difference was detected between the discharge of air-borne spores and the appearance of infections. The appearance of flowers, the plant organ most susceptible to infection, appeared to mark the end of the latent period and the beginning of infections. The distribution of the infections within the greenhouse was random for two of the three seasons studied and fitted a Poisson distribution (p < 0.01). Analysis of the tendencies of the number of infections and numbers of infected plants revealed a sigmoidal curve that was a function of the accumulated hours in which relative humidity was 80 % or more.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

Factors affecting growth from heat-treated spores of non-proteolytic Clostridium botulinum

Heat treatment of spores of non-proteolytic Clostridium botulinum at 85°C for 120 min followed by enumeration of survivors on a medium containing lysozyme resulted in a 4.1 and 4.8 decimal reduction in numbers of spores of strains 17B (type B) and Beluga (type E), respectively. Only a small proportion of heated spores formed colonies on medium containing lysozyme; this proportion could be increased by treatments designed to increase the permeability of heated spores. The results indicate that the germination system in spores of non-proteolytic Cl. botulinum was destroyed by heating, that lysozyme could replace this germination system, and that treatments that increased the permeability of the spore coat could increase the proportion of heated spores that germinated on medium containing lysozyme. These results are important in relation to the assessment of heat-treatments required to reduce the risk of survival and growth of non-proteolytic Clostridium botulinum in processed (pasteurized) refrigerated foods for extended storage.     

Fruit ripening-related expression of a gene encoding group 5 late embryogenesis abundant protein inCitrus

A cDNA and genomic clone (CuLEA5) encoding a group 5 late embryogenesis abundant protein (Lea5) was isolated from citrus fruit cDNA and genomic libraries. Sequence analysis indicated that the clone contains an open reading frame of 97 amino acids, and that the genomic structure is composed of two exons and one intron. A comparison of its amino sequence with other plant proteins showed that Lea5 proteins can be classified into two types - gymnosperm and angiosperm — based on a P-segment sequence designated by this study. Examination of its expression patterns indicated thatCuLEA5 has important roles during the development or ripening of seedless fruits and leaves inCitrus. The 5′-flanking region of the genomic DNA contains a number of putative hormonal- and stress-responsive elements. This is the first report that describes the expression ofLea5 during fruit ripening, as well as the sequence characteristics of its promoter region.