Microcycle conidiation — A review

Microcycle conidiation is defined as the germination of spores by the direct formation of conidia without the intervention of mycelial growth, as occurs in most normal life cycles. It is a method of asexual spore formation in which the normal life cycle of the fungus is bypassed. Spores formed through sexual reproduction and species with unicellular thalli are not included in microcycle conidiation. The term secondary conidium or secondary spore is usually, but not always, synonymous with microcycle conidiation. In the laboratory various factors, but especially temperature, can induce the microcycle condition in such fungi asAspergillus niger, Penicillium andNeurospora crassa, providing a useful tool for research. Microcycle conidiation has also been reported in a broad range of species in nature, and comprises a normal part of the life cycle in several groups, including the Entomophthorales, Taphrinales, Clavicipitales, Uredinales, Ustilaginales, Tremellales and Exobasidiales. The presence of a microcycle in such fungi undoubtedly provides a survival mechanisn for spores that encounter unfavorable conditions.     

Microcycle Sporogenesis of Bacillus cereus in a Chemically Defined Medium

A chemically defined medium which allowed germination, outgrowth, and subsequent resporulation of Bacillus cereus T spores, without intervening cell division (microcycle sporogenesis), is described. No medium replacement was required. The second-stage spores were heat-stable and had similar germination characteristics and dipicolinic acid content to primary spores. Deoxyribonucleic acid (DNA) replication began soon after germination and there was a doubling in the DNA content of the cells within 2 hr.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

基于转录组的冬虫夏草菌微循环产孢研究

为研究冬虫夏草菌（Ophiocordyceps sinensis）微循环产孢过程中的分子机理,采用高通量测序技术,对微循环产孢前后冬虫夏草菌的转录组进行研究。筛选获得差异表达基因6 902个。Gene Ontology（GO）功能聚类分析表明,差异表达基因主要参与分子功能、胞内运输、核糖核蛋白复合体等生物学通路。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG)功能富集分析结果表明,差异表达基因参与了核糖体、嘧啶代谢、蛋白酶体、嘌呤代谢、甘氨酸代谢等过程。Mmc、Mcb、MaH1、MaPP5、MaAGA、Pyk等候选基因不同程度的参与了冬虫夏草菌微循环产孢过程,其中Mcb家族基因可能起到关键作用。通过qRT PCR验证差异表达基因,定量结果与转录组测序结果一致。本研究为进一步揭示冬虫夏草菌微循环产孢分子机制以及关键基因的调控提供了重要信息。     

Metabolic Requirements for Microcycle Sporogenesis of Bacillus megaterium

Spores of Bacillus megaterium QM B1551 germinated, elongated, and resporulated (microcycle sporogenesis) in simple chemically defined media which permitted no cell division. The second-stage spores thus produced were heat-stable and required heat activation for germination. The original amount of spore deoxyribonucleic acid tripled before completion of the cycle. Acetate and a small amount of a tricarboxylic acid cycle intermediate were the minimal organic metabolic requirements for microcycle sporogenesis. During this cycle, germinated cells oxidized acetate only after a delay, whether or not glucose was initially present. Spores that were germinated in the absence of a carbon source first oxidized an endogenous substrate, and then developed the ability to oxidize acetate.     

Achievement of complete Bacillus subtilis microcycle sporulation by the addition of S-adenosylmethionine and phospholipids.

In an attempt to find factors that may be responsible for the initiation of sporulation, a system in which the germination and outgrowth phases were separate was applied to Bacillus subtilis. Outgrowth of the germinated spores to only the primary singlet cells was followed in chemically defined medium. Addition of specific metabolites induced the primary singlet cells to sporulate via microcycle sporulation. Experiments are described that led to complete sporulation by the addition of diaminopimelic acid, S-adenosyl-L-methionine and phosphatidylethanolamine.     

Microcycle speculation in theClaviceps purpurea 244

The mutant strainClaviceps purpurea 244 forming hyphae composed mainly from sclerotiumlike cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 × 6.5 μm). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 μg/ml clavins, with elymoclavin predominating (82 %). The parent strainClaviceps purpurea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85 %) at a concentration of 4 mg/ml.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Light-independent conidiation in Trichoderma spp.: a novel approach to microcycle conidiation

Microcycle conidiation in Trichoderma hamatum and T. harzianum has been achieved in complete darkness for the first time. The time required for mass conidiation without intervening vegetative growth was decreased to 24 h instead of 6 to 7 days. The conidia produced by microcycle conidiation were viable and had pigmentation and antagonistic behaviour similar to those of the parental stock cultures.N. Khurana, R.K. Saxena, R. Gupta and R.C. Kuhad are with the Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India.     

Direct Transition of Outgrowing Bacterial Spores to New Sporangia Without Intermediate Cell Division.

Vinter, Vladimir (Syracuse University, Syracuse, N.Y.), and Ralph A. Slepecky. Direct transition of outgrowing bacterial spores to new sporangia without intermediate cell division. J. Bacteriol. 90:803-807. 1965.-A direct transition was observed of the primary cell developed after germination of Bacillus cereus spores into new sporangia without intermediate division stages. Two simple methods were used for replacement of outgrowing spores into diluted medium or saline. Elongated primary cells prevented from division by limitation of nutrients in the suspending medium were able to form new forespores in 8 hr and sporangia in 12 hr. These new sporangia were still marked by attached envelopes of the original spore. Under the same conditions, cells replaced during the first divisions quickly lysed. Spores formed in the elongated primary cell during "microcycle sporogenesis" possessed normal heat resistance and refractility and were later released from sporangia.     

Changes in the Content of Phytyl and Geranylgeranyl Esters of Germinating Polytrichum commune Spores

Polytrichum commune spores contained esterified phytol and geranylgeraniol, 706 and 114 μg, respectively, per 100 mg dry weight of freshly collected spores. After storage for 9 months the level of esterified phytol of the spores was decreased by c. 600 μg, whereas the level of esterified geranylgeraniol was more or less unchanged. The changes in the level of esterified prenols during germination follow the same pattern in freshly collected and in 9 month-old spore material. An immediate steep decrease between 0 and 3 h was followed by an increase in the level of esterified phytol between 3 and 12 h and by a constant value for esterified geranylgeraniol during the same period. Between 12 and 48 h the level of both types of esterified prenols decreased. In the freshly collected spores the amount of esterified prenols increased after 48 h of germination, in the older spores after 72 h. Free phytol was found in trace amounts in dry and germinating spores and in the protonema.     

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines

Background

Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR.

Materials and Methods

We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori.

Results

Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log.

Conclusions

This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.     

Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs

Aims: To determine if orally ingested Bacillus spores used as probiotics or direct‐fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. Methods and Results: Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore‐feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4–6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid‐colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2·14‐fold compared to time zero. Conclusions: The experiments showed that 70–90% of dietary‐supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. Significance and Impact of the Study: A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.     

The occurrence and effect of a protozoan parasite, Ophryocystis elektroscirrha (Neogregarinida: Ophryocystidae) on overwintering monarch butterflies, Danaus plexippus (Lepidoptera: Danaidae) from two California winter sites

Abstract.

• 1 Monarch butterflies, Danaus plexippus, from two overwintering populations, were found to have Ophryocystis elektroscirrha spores on their scales at rates between 53% and 68%. The frequency of butterflies with O.elektroscirrha spores remained about the same between sites and throughout the winter.
• 2 The spores, recovered from all parts of the body of the butterfly, were most numerous on the abdomen, particularly near the posterior third.
• 3 Butterflies with spores survived as long as those without detectable spores at 10.1°C ±0.4 SE and 78.3% r.h. ±0.6SE. Insects with spores held at 19.4°C ±0.4SE and 44.9% r.h. ±1.5SE showed a significantly higher rate of moisture loss and survived a shorter period than monarch butterflies without detectable spores.

     

Daily and seasonal variations ofAlternaria andCladosporium airborne spores in León (North-West, Spain)

The spores ofAlternaria andCladosporium are present throughout the year in the atmosphere of León (NW Spain), although they show an important seasonal variation. To understand the relationship between the number of spores and climatic factors,Alternaria andCladosporium spores counts for January 1994 to December 1995 were examined by means of correlation analyses. The results of weekly samples of both years showed that the spores concentration of two taxa are significantly and positively correlated with maximum and minimum temperature and sunshine hours and negatively with relative humidity. The statistical analysis of daily samples generally showed the same results. In the hourly distribution of spore concentrations we can see a similar behaviour ofAlternaria andCladosporium, with most spores collected in the 12–14 h period.     

UV protectants for the biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary toxins from UV radiation

The UV protectant properties of 26 natural and synthetic compounds were investigated for a biopesticide based on an indigenously isolated strain (ISPC-8) of Bacillus sphaericus Neide. In initial screening, spores of ISPC-8 with 0.1% (w/w for solid and v/w for liquid materials) concentration of different compounds were exposed to UV-B radiation (4.9 × 10⁵ J/m²) for 6 h and their spore viability and larvicidal activity were studied. The larvicidal activity was evaluated against third-instar larvae of Culex quinquefasciatus Say. There was a complete loss of spore viability (1.4% viable spores) and partial reduction in larvicidal activity (57.7% of original activity) after exposure of spores to UV-B for 6 h. However, spore viability as well as larvicidal activity protected significantly when spores were mixed with different compounds before exposing them to UV-B. Among the different compounds tested benzaldehyde, congo red, para-aminobenzoic acid (PABA) and cinnamaldehyde were found to be promising in protecting the spores from UV-B radiation. The presence of binary toxins (41.9 kDa and 51.4 kDa) in protected and unprotected samples were examined by SDS–PAGE. The binary toxin bands disappeared in unprotected spores after 24 h of exposure to UV-B, whereas toxin bands were distinctly visible when spores with benzaldehyde and cinnamaldehyde were exposed to UV-B for 96 h and 120 h, respectively. Congo red and PABA were found to be most effective in protecting binary toxins even after 168 h of exposure to UV-B. Incorporation of these promising UV protectant compounds in biopesticides would help in protecting the spores from the adverse effects of UV radiation and prolong the persistence of biopesticides under field conditions.     

Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Induction of microcycle sporulation in Bacillus brevis spp. AG4 by amethopterin

Addition of amethopterin to medium before inoculation inhibited DNA synthesis and induced microcycle sporogenesis in Bacillus brevis spp. AG4. Synthesis of RNA and proteins occurred at a considerably reduced rate.Abbreviations TVC total viable counts - HSC heat stable counts - CDGS chemically defined medium for growth and sporulation - TCA trichloro acetic acid