Infection of potato stems by the gangrene pathogen, Phoma exigua var. foveata

Limited transport of pycnidiospores of Phoma exigua var. foveata within transpiring stems was demonstrated by isolating from cut mature stems placed for 24 h in a spore suspension. Pieces of shoot dipped in spore suspension and subsequently rooted contained the pathogen which was redistributed as the plants grew but without causing symptoms until after desiccation. The fungus was only rarely detected within stems growing from rotted seed tubers suggesting that spore transport in the vascular system was relatively unimportant in these experiments. Stems were readily infected from spore suspensions applied to unwounded epidermis especially when the inoculum was held in cotton wool and secured with wax film. The progressive colonisation of growing stems was demonstrated by isolations made at different positions on the stem and at different times after inoculation. In field experiments in two years stems were inoculated with four spore concentrations on four dates from late June to late August. After desiccation the extent of pycnidial lesions was greatest from the earliest inoculation date and the largest spore concentration and these differences were reflected subsequently in the incidence of gangrene on damaged progeny tubers. Younger stems may be more susceptible and infection in early summer probably results in extensive growth within the stem before desiccation and thus in massive pycnidial production.     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.     

A rapid method to estimate the resistance of cold stored carrot roots to Botrytis cinerea

Inoculating whole carrot roots at 4°C with mycelial/agar discs of the grey mould fungus Botrytis cinerea gives a measure of their resistance and hence storage potential to this pathogen, but results are not obtained for at least 33 days. In the present investigation a more rapid method was used which involved inoculating root slices with spore suspensions containing 5 × 10^3–5 × 10⁶ spores/cm³ at 24°C. Resistance was assessed visually and by a chitin estimation 48 h after inoculation. Both methods were used to measure the resistance of cold stored carrot roots during two storage seasons, 1976/77 and 1977/78. In each season there was a particular inoculum level which most clearly recorded the increasing susceptibility of roots with time in store. In 1976/77 this was 1 × 10⁵ spores/cm³ whereas in 1977/78 it was the lower inoculum concentration of 5 × 10⁴ spores/cm³, suggesting the roots were generally more susceptible in 1977/78 than the previous season. This was in accord with the results from the whole root method of assessment. A chitin estimation proved to be the more sensitive method of assessment for inoculum potential experiments.     

Infection of Potato Tubers with the Common Scab Pathogen Streptomyces scabiei in a Soil‐less System

A novel soil‐less method was developed to define susceptibility of developing potato tubers accurately to infection with Streptomyces scabiei the causal agent of common scab disease. Hydroponic production enabled precise identification of individual tuber development. Direct inoculation of tubers with a spore suspension of S. scabiei resulted in disease development, demonstrating that infection could be initiated in a soil‐less media. Tubers were most susceptible to infection between 3 and 20 days after tuber initiation, confirming that this early period of tuber formation is critical to disease development.     

种间竞争梯度对白车轴草与其病原菌白车轴草单孢莠菌相互作用的影响

白车轴草（Trifolium repens)在与其病原菌白车轴草单孢锈菌（Uromyces trifolii-repentis)的长期相互作用中分化形成了抗病型（Resistance)无性系和易感型（Susceptibility)无性系。该研究工作旨在了解：1）在种间竞争不断增强的环境梯度中,抗型无性系和易感型无性系的生长表现有何区别？2）在同样的实验条件下,分别对抗病型无性系和易感型无性系进行接种感染后,两者的生长表现又有何区别？在一严重感病的白车轴草自然种群中,分别标定了17个抗病型无性系和14个易感型无性系,从各元性系的匍匐茎上分别剪取长5cm,具两个叶两个腋芽的小段作实验材料。接种孢子采自同一植物种群植株的病叶。实验在一个处在次生演替阶段的草本植物群落上进行,在该群落的种间竞争梯度上选定3个分别具有弱、中等和强种间竞争的生境（Habitat)建立实验站,每站设置等量的实验和对照组。结果显示：1）随着环境中种间竞争强度的增加,无论接种与否,抗病型和易感型白车轴草的叶生长量均凝减;2）在弱或强种间竞争环境条件下,无论接种与否,易感型无性系的生长均好于抗抗病型无性系;但在中等间竞争强度的环境条件下,对照实验中易感型无性系的生长显著好于抗病型无性系,而接种实验中的结果相反。这一研究结果表明,种间竞争强度可能是影响给定生境中寄主植物抗病型和易感型遗传型比率的重要因子之一,这一影响还将随着病原菌存在而否而发生改变。     

A comparison of inoculation methods for determining potato cultivar reaction to black leg

Two methods of inoculating tubers, one by dipping them in an aqueous suspension of Erwinia carotovora subsp. atroseptica, the other by inserting the end of a wooden toothpick charged with undiluted bacteria into the rose-end near a sprout, were compared over two years for their ability to produce black leg and for their effect on plant growth and yield on a number of cultivars chosen because of expected differences in susceptibility. Although both methods produced a similar rank order for cultivar reaction, the more consistent results were obtained using the toothpick method, clear differences in black leg incidence and yield being evident between the susceptible cultivars Ulster Sceptre and Maris Bard and the more resistant Pentland cultivars. As some cultivars appear more tolerant to black leg than others, yield may be as important a criterion as disease incidence when assessing overall cultivar performance. The inoculation of the base and middle of stems and the infiltration of the bacterium into tubers were also investigated as alternative methods for assessing cultivar susceptibility.     

Fate and dissemination of Bacillus subtilis spores in a murine model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

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.     

Control of Fusarium solani rot of potato tubers with fungicides

Tecnazene (up to 33 ppm) and dichloran (up to 500 ppm) had little effect on germination of spores or growth of Fusarium solani isolated from and causing a rot of potato tubers; they also did not decrease rotting when applied to wounds later inoculated with the pathogen. Benomyl and thiabendazole (up to 500 ppm) also had little effect on spore germination but did greatly decrease growth at 5 ppm. A pronounced pink coloration developed in cultures growing slowly in the presence of benomyl; a similar though less striking effect appeared in agar cultures containing thiabendazole. Benomyl suspended in water or diluted with Fuller's earth gave good control of rotting when applied to wounds inoculated later with F. solani. Still better control was obtained with thiabendazole; dusts containing 1% a.i. substantially decreased rots and those containing 10 % a.i. gave almost complete control when applied to wounds shortly before inoculation. Thiabendazole was also very effective when used 24 h after inoculation and a fair measure of control was obtained when it was applied 24 h later. Benomyl and thiabendazole placed on apparently intact surfaces of tubers caused tissue 5 mm deep to become toxic to F. solani 10 days later, and, unexpectedly, this tissue prevented spore germination. Fuller's earth alone substantially decreased rotting. The results obtained suggest that dusts containing thiabendazole have some promise for the control of Fusarium rots of potato tubers, especially of early crops.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

FIELD EXPERIMENTS ON POTATO SKIN-SPOT DISEASE CAUSED BY OOSPORA PUSTULANS OWEN & WAKEF

In experiments on skin-spot disease of potatoes, caused by Oospora pustulans Owen & Wakef., Kerr's Pink proved the most susceptible of twenty-four commercial varieties; Arran Banner, Majestic and King Edward were highly susceptible; Home Guard and Golden Wonder were highly resistant.
Thymol and tetrachlornitrobenzene, applied as dusts to pits of potatoes at lifting, did not give satisfactory control of the disease. The efficacy of an organo-mercury dip treatment at lifting time was confirmed.
The incidence of the disease was considerably decreased by storing tubers in boxes instead of in pits, by digging the crop about a month before normal harvest, or by cutting the haulms at this time and digging at the normal time.
Ware tubers showed a significantly higher infection than seed tubers of the same crop, indicating that susceptibility of tubers increases towards maturity.
The difference in eye infection between varieties may not be related to that of general superficial infection. It is suggested that, in future, the assessment of varietal susceptibility should be based upon the degree of eye infection, which is in practice the measure of the economic importance of the disease. Tubers having pustules at or near all the eyes gave markedly reduced sprout emergence and plant stand.     

THE DETERMINATION OF THE REACTION OF BARLEY VARIETIES TO LOOSE SMUT

The inoculation of barley with loose smut ( Ustilago nuda ) using various techniques has given results which do not reflect the known behaviour of varieties under natural conditions. No consistent results have been obtained, but all the varieties thus inoculated could become infected. No variety therefore possessed true physiological resistance and in the field some must escape infection. The inoculation techniques obviously overcame this escape.
A method has been devised which, with the varieties used, has reflected their behaviour in the field. Drops of coloured water or spore suspension are allowed to fall from a height on to ears at anthesis. Grains on the ears of varieties which do not become infected in the field do not become coloured or infected (depending on the drops used), while grains of varieties susceptible in the field do become coloured or infected. This is due to the glumes of the former group of varieties remaining closed, and so preventing access of smut spores under natural conditions.     

Recovery of bacillus spore contaminants from rough surfaces: a challenge to space mission cleanliness control

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load ("bioburden") of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.     

Adhesion of bacillus spores in relation to hydrophobicity

R önner , U., H usmark , U. & H enriksson , A. 1990. Adhesion of bacillus spores in relation to hydrophobicity. Journal of Applied Bacteriology 69 , 550–556.
The adhesion of spores of five different Bacillus species to solid surfaces of different hydrophobicity was evaluated. The spore surface hydrophobicity was measured using hydrophobic interaction chromatography (HIC). A large variation in hydrophobicity was found among the spores of the different species tested. The degree of adhesion of spores to the solid surfaces was consistent with the results obtained using the HIC method. The most hydrophobic spores, according to the HIC method, adhered in a much larger extent to the hydrophobic surfaces. Furthermore, spores generally adhered to a greater extent to hydrophobic and hydrophilic surfaces than did the vegetative cells.     

A new system using Solanum tuberosum for the co-cultivation of Glomus intraradices and its potential for mass producing spores of arbuscular mycorrhizal fungi

Freshly harvested potato tubers, Solanum tuberosum var ‘Pukhraj’, were inoculated for transformation with Agrobacterium rhizogenes strain Ri1600. Hairy roots were formed after 8 days of co-cultivation and the transformation efficiency was 40 %. The transformants were transferred from Murashige and Skoog medium (MS) to Modified White’s medium (MW) and finally on a hormone-free minimal medium (M). The putative transformants were confirmed using rolA and rolB gene specific primers for the polymerase chain reaction (PCR) analysis. The root inducing (Ri) T-DNA transformed potato roots were co-cultured with Glomus intraradices (CMCCROC7) to obtain arbuscular mycorrhizal root organ cultures (AM-ROC dual cultures), which were used for studying the symbiosis with Glomus intraradices and the potential for spore production in vitro. Sporulation was comparable with the existing in vitro carrot-dual culture system. Around 60,250 spores/jar could be harvested with around 38,314 extraradical spores/jar and around 21,936 intraradical spores/jar. The new method using potato is certainly promising for the mass production of mycorrhizal biofertilizers. The viability of the spores when tested on potato roots was nearly 100 % and more than half of the roots were colonized 12 weeks after inoculation.     

Spore production and mycorrhizal development in various tropical crop hosts infected with Glomus clarum

Plant growth, mycorrhizal development and vesicular arbuscular spore production were examined in five tropical crop host species inoculated with Glomus clarum and grown in a glasshouse. In one of the two experiments, sequential harvests of maize, sorghum and chickpea were made in order to study spore production in relation to plant growth and mycorrhizal development. Spore numbers in each of these hosts increased at a fairly constant rate until maximum plant dry weight, when spore production ceased. Sorghum and maize produced considerably more spores than chickpea, with spore numbers being closely correlated with mycorrhizal root length. In the second experiment, Glomus clarum was cultured on each of maize, millet, sorghum, groundnut and chickpea for three consecutive generations before cross-inoculation of the spores from each host onto all five hosts. Sporulation with respect to host size was generally greatest when the inoculum used to infect a host had been produced on that host. The growth-promoting effects of the fungus were not influenced by the source of the inoculum. More spores were produced on the cereals than the legumes. Differences in spore numbers amongst hosts and plant generations were apparently influenced mainly by infected root length and by the growth period.     

A method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica)

In experiments to develop a method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica) seed tubers were stab-inoculated near the stolon (attachment end), with a suspension of the bacterium, or with water, before planting. Disease symptoms were recorded in three years (1980–1982) and plant growth and yield in 1982. Estima and Maris Bard were the most susceptible cultivars with many plants failing to emerge and most of those that did showing disease symptoms. Pentland Crown was the most resistant: few plants failed to emerge and few showed blackleg. Nevertheless compared with water-inoculated plants bacterial inoculation of the seed tubers of this cultivar caused loss of yield and differences in tuber size distribution. Cara, Wilja and King Edward showed intermediate reactions.     

Relationship between basidiospore size,shape and life history characteristics: a comparison of polypores

Although spore dispersal is the predominant mode of spread in fungi, the functional ecology of offspring (spore) size and shape has received little attention. We investigated the relationship between spore size (volume) and shape and various life history parameters among 303 European polypore species. In an analysis of variance, basidiocarp size, nutritional mode (parasitic vs. saprotrophic) and host (conifer vs. deciduous) accounted for a significant part of the variation in spore size. Species producing large basidiocarps also produce large spores, parasites produce larger spores than saprotrophs, and species colonizing deciduous trees generally produce larger spores than those colonizing conifers. There was a correlation between spore size and shape, with larger spores being more spherical. The most important factors accounting for variation in spore shape were rot type (white rot vs. brown rot) and nutritional mode, with white rot species and parasites having more spherical spores compared to brown rotters and saprotrophs.     

The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.