Factors affecting the survival of sclerotia of Sclerotium cepivorum in the Fraser Valley of British Columbia

Sclerotia of Sclerotium cepivorum buried in muck soil in the Fraser Valley decayed with time. The rate of decay of sclerotia was influenced by local environmental conditions. A mixture of soil with sclerotia increased their survival but there was no difference in the rates of decay in three different soils. The decay was greatest during winter when Fraser Valley fields are often flooded. Sclerotial decay was also affected by pretreatment of the sclerotia. Dried sclerotia decayed significantly (P < 0.05) faster than sclerotia which had not been dried, a phenomenon which is apparently due to changes in micro-organisms on the sclerotia. Dried sclerotia which had been incubated in moist soil had fewer bacteria and more fungi than sclerotia which had been incubated in soil without being dried. The increase in fungi on the dried sclerotia was due to a dramatic increase in Trichoderma spp.     

Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia

Water availability is an important environmental factor which has major effects on fungal activity. The effects of osmotic (KCl amended agar) and matric Polyethylene glycol ((PEG) 8000 amended agar) potentials over the range -0.1 to -5.0MPa on mycelial growth and conidial germination of eight isolates of the sclerotial parasite Coniothyrium minitans was assessed. The influence of soil water potential on the ability of three selected isolates (LU112, LU545, and T5R42i) to parasitise sclerotia of the plant pathogen Sclerotinia sclerotiorum was determined. For all eight C. minitans isolates, decreasing osmotic and matric potentials caused a reduction in mycelial growth and conidial germination. Isolates were more sensitive to decreasing matric potential than osmotic potential. Across the isolates, growth at an osmotic potential of -5.0MPa was 30-70% of the growth seen in the control, whereas less than 20% of the control growth was seen at the corresponding matric potential. Across all isolates no conidial germination was seen at matric potential of -5.0MPa. The C. minitans isolates varied in their sensitivity to decreasing water potentials. Mycelial growth and conidial germination of three isolates (LU112, Conio, and CH1) were more tolerant of low osmotic potential and matric potential with respect to mycelial growth. Isolates T5R42i and LU430 were least tolerant. In contrast, conidial germination of isolates Conio, LU545, and T5R42i were less sensitive to decreasing matric potential. Soil water potential was seen to affect infection and viability of sclerotia by the three C. minitans isolates. Isolate LU545 reduced sclerotial viability over a wider water potential range (-0.01 to -1.5MPa) compared with LU112 (-0.01 to -1.0MPa), with isolate T5R42i being intermediate. Indigenous soil fungi (Trichoderma spp. and Clonostachys rosea) were recovered from sclerotia but did not result in reduction in sclerotial viability. The relevance of these results in relation to biocontrol activity of C. minitans in soil is discussed.     

How many fungi make sclerotia?

Most fungi produce some type of durable microscopic structure such as a spore that is important for dispersal and/or survival under adverse conditions, but many species also produce dense aggregations of tissue called sclerotia. These structures help fungi to survive challenging conditions such as freezing, desiccation, microbial attack, or the absence of a host. During studies of hypogeous fungi we encountered morphologically distinct sclerotia in nature that were not linked with a known fungus. These observations suggested that many unrelated fungi with diverse trophic modes may form sclerotia, but that these structures have been overlooked. To identify the phylogenetic affiliations and trophic modes of sclerotium-forming fungi, we conducted a literature review and sequenced DNA from fresh sclerotium collections. We found that sclerotium-forming fungi are ecologically diverse and phylogenetically dispersed among 85 genera in 20 orders of Dikarya, suggesting that the ability to form sclerotia probably evolved ≥14 different times in fungi.     

Antifungal effects of essential oils from oregano and fennel on Sclerotinia sclerotiorum

AIMS: The antifungal effects of essential oils of oregano (Origanum syriacum var. bevanii) and fennel (Foeniculum vulgare) were evaluated against Sclerotinia sclerotiorum. Effects of the essential oils on morphological structures of hyphae and sclerotia were studied under light and scanning electron microscopes (SEM). METHODS AND RESULTS: Inhibitory effects of volatile and contact phases of the essential oils used were determined on hyphae and sclerotia. Both essential oils have a marked antifungal effect against S. sclerotiorum. Soil amendment with essential oils has significant effect on reducing sclerotial viability. Both essential oils significantly inhibited the fungal growth in soil, thereby increasing the number of surviving tomato seedling by 69.8% and 53.3%, respectively. Light and SEM observations on pathogen hyphae and sclerotia revealed considerable morphological alterations in hyphae and sclerotia. CONCLUSIONS: The significant reduction in the mycelial growth and germination of sclerotia would greatly reduce the pathogen inoculum source. This may influence the rate of disease development in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the reduction in the number of diseased plants in infested soil amended with essential oils, we concluded that oregano and fennel essential oils could be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi.     

Fungal species residing in the sclerotia of <Emphasis Type="Italic">Polyporus umbellatus</Emphasis>

Sclerotia of Polyporus umbellatus is a traditional Chinese herb. The sclerotia can survive in soils for long time and their growth depends upon a symbiotic association with Armillariella mellea. But it is unclear whether other fungi reside or play a role in the sclerotia. In this study, wild sclerotial samples were collected from seven provinces, which span southwest to northeast China. A total of 148 fungal isolates were recovered from the sclerotia of P. umbellatus and classified into 19 morphological taxa. Seventeen belonged to five genera: Fusarium, Eurotium, Penicillium, Geomyces and Mucor. The fungi found within the sclerotia varied depending on the province from which they were collected. The possible role of these fungi is discussed.     

植物病原真菌的菌核研究进展

真菌菌核是相关真菌在特殊环境下由营养体菌丝交织和聚集形成的具有抵御恶劣环境能力的休眠结构,在真菌生活史及病原真菌的病害循环中具有重要的生物学和生态学意义。许多引起严重植物病害的病原真菌能够形成菌核,这类真菌通过菌核度过逆境。菌核在适宜条件下萌发形成子囊盘和孢子或菌丝,造成植物的侵染。本文从形成菌核的真菌种类、生物学特性、黑色素及分子生物学等方面进行综述,旨在为深入研究真菌菌核及其在真菌学、植物病理学及药用真菌学等领域应用提供研究思路和参考依据。     

Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage.

Mycelial cultures of 64 isolates of 14 species of ectomycorrhizal fungi and 27 isolates of 15 species of plant pathogenic fungi were grown on agar medium in Petri dishes. Mycelial discs, 8 mm in diameter, were removed from the cultures and stored in sterile distilled water in test tubes at 5 degrees C. Sixty-four, 61, and 41 isolates of the symbiotic fungi were viable after 1, 2, and 3 years storage respectively. Only 19, 10, and 8 isolates of the pathogenic fungi were viable after 1, 2, and 3 years storage, respectively. Time in pure culture before water storage did not affect viability of any fungal species following water storage. After 3 years storage, four fungi (three symbionts and one pathogen) were tested and found to have retained their original growth rates and root-infecting abilities on pine seedlings. The same four isolates, however, maintained on agar slants at 5 degrees C and subcultured every 4 to 6 months, grew slower and did not infect as many feeder roots of pine as the water-stored isolates.     

Survival of Sclerotia of Rhizoctonia solani AG3PT and Effect of Soil‐Borne Inoculum Density on Disease Development on Potato

Sclerotia produced by a single isolate of Rhizoctonia solani AG3PT were buried in small plot experiments to investigate the effects of sclerotial production method, soil type and burial depth on sclerotial viability in field soil. The factor with the greatest effect on sclerotial viability, defined as the percentage of sclerotia germinating on agar following retrieval, in all experiments was the duration of burial. After 18 months, on average across all experiments, 20% of retrieved sclerotia were viable. A comparison between sclerotia produced in vitro on malt yeast extract agar and in vivo using micropropagated tubers in field soil found no significant differences between the two production methods on sclerotial viability. Burial in field soil at 20‐cm depth was found to significantly reduce sclerotial viability to 50% compared to 60% at 5 cm. In two pot experiments, amending the growing medium and soil with increasing inoculum densities of R. solani was found to increase stem number, stem canker and black scurf severity regardless of whether this soil‐borne inoculum was derived from mycelium or sclerotia. Black scurf incidence and severity were assessed 30–32 days posthaulm destruction and found to be similar for a range of sclerotial soil‐borne inoculum densities (1.0 × 10^?1 g/kg d.w. soil to 6 × 10^?3 g/kg d.w. soil). The significance of these findings in relation to pathogen survival, detection in soil and disease development is discussed.     

<Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> from rice husks forming sclerotia in agar culture

Sclerotia were formed in agar culture by a fungus with clamp connections isolated from rice husks at Tsukuba, Japan. The sclerotia were brown, globose to ellipsoidal, small, up to 200 μm in diameter, and composed of external rind tissue and internal medulla tissue. Such tiny sclerotia have not been commonly reported among basidiomycetous fungi in the literature. The fungus was identified as Coprinopsis cinerea on the basis of morphological characteristics together with molecular analyses. Three reference strains of C. cinerea formed sclerotia similarly under identical cultural conditions.     

猪苓菌核结构性质的研究

利用光学显微镜和电子显微镜对猪苓菌核的结构及其发育进行了研究。组成猪苓菌核的菌丝与平板培养或发酵培养的猪苓菌丝比较,具有多分枝、融合频率高、菌丝形态不规则等特点。猪苓菌核表现了高度的结构分化,有表皮、表皮下层、疏松菌丝层和结构菌丝之分,结构菌丝是组成菌核的主要成分,疏松菌丝层类似一般菌核的髓,但大小、位置在菌核中变化较大。菌核的个体繁殖是由母体菌核的一束或几柬菌丝突破表皮而萌发产生新的菌核;较系统的观察了猪苓菌核细胞分裂及其菌丝内部结构的变化。     

Sclerotia formed by citric acid producing strains of Aspergillus niger: Induction and morphological analysis

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea

Effect of tricyclazole on production and melanin contents of sclerotia of Botrytis cinerea Tricyclazole retarded production of sclerotia of Botrytis cinerea on agar medium more severely than mycelium growth. At a concentration range (50–200 mg/l) that did not control Botrytis on grape leaves, sclerotia production was significantly reduced. There was a negative relation between the bleaching duration of sclerotia and the tricyclazole concentrations in the medium on which they were formed. Light microscopical studies showed that sclerotia from tricyclazole-containing medium contained a significantly poorer developed outer melanin layer than that from the control medium. Ultrastructural studies with 5 days old untreated sclerotia revealed intense electron impermeable deposits in the intercellular spaces and a small electron dense layer in the outer cell walls, on the other hand treated sclerotia of the same age showed only sporadic small pigmented deposits between the cells and the pigmentation of the outer cell wall was absent.     

Potential for biocontrol of sclerotinia rot of carrot with foliar sprays of Contans® WG (Coniothyrium minitans)

A glasshouse and field trial were conducted to evaluate foliar sprays of Contans® WG (Coniothyrium minitans) conidial suspensions for control of sclerotinia rot of carrot and infection of Sclerotinia sclerotiorum sclerotia by C. minitans. In the glasshouse trial, foliar sprays (1×10⁴–10⁸ conidia mL^?1) decreased the viability of sclerotia recovered from diseased plants and increased infection by C. minitans. In the field trial, three successive foliar sprays applied at 14-day intervals failed to reduce foliage disease severity, but significantly reduced viability of sclerotia recovered from diseased plants/crop debris and increased infection by C. minitans. No significant differences in sclerotial viability or infection were observed between two conidial concentrations (2.4 and 4.8×10⁶ conidia mL^?1). Foliar sprays of Contans® WG have potential for reducing viability of sclerotia produced on diseased foliage.     

猪苓菌丝形成菌核伴生菌的发现及应用

在野生猪苓(Grifola umbellata (Pers.) Pilát)菌核生长穴中首次分离到猪苓菌丝形成菌核所必需的伴生菌(Grifola sp.).实验证明:纯培养的猪苓菌丝不能形成菌核,但其与伴生菌共培养,无论在实验室培养基上或用树棒栽培,猪苓菌核形成很快且发育正常;伴生菌是猪苓菌丝形成菌核的关键生物因子.另外,伴生菌菌丝和猪苓菌菌丝二者形态差别较大,前者多为细长的薄壁菌丝,后者多为细胞直径较大的薄壁和厚壁菌丝.     

Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum

Biological control agents (BCAs) Bacillus subtilis QST 713, Coniothyrium minitans CON/M/91-08, Streptomyces lydicus WYEC 108, and Trichoderma harzianum T-22 were evaluated for their efficacy in the reduction of survival of sclerotia and production of apothecia of Sclerotinia sclerotiorum under controlled environments. A growth chamber assay was conducted where 25 sclerotia were buried in pots containing potting soil, and BCAs were drenched into the soil at various concentrations, and five soybean seeds were planted in each pot. The presence and number of S. sclerotiorum apothecia were recorded daily. Sclerotinia sclerotiorum sclerotia were retrieved six weeks after seeding and viability was assessed on water agar plates. All BCAs were effective in reducing S. sclerotiorum inoculum at various efficacies. In general, efficacy was positively correlated with the rate of application. At the rate of application when the efficacy did not change significantly by increasing the rate, the BCAs had various reductions of apothecia and sclerotia. B. subtilis reduced apothecia and sclerotia by 91.2 and 29.6%, respectively; C. minitans reduced apothecia and sclerotia by 81.2 and 50%, respectively; Streptomyces lydicus reduced apothecia and sclerotia by 100 and 29.6%, respectively; Trichoderma harzianum reduced apothecia and sclerotia by 80.5 and 31.7%, respectively. In addition, the commercial strain of C. minitans CON/M/91-08, and a wild Michigan strain of C. minitans W09 were compared for their growth and sclerotial reduction. W09 had faster growth rate than the commercial strain, indicating potential diversities of biological control strains to be studied.     

蝉棒束孢内菌核、菌膜及其生境土壤真菌群落结构及生态功能分析

通过分析蝉棒束孢Isaria cicadae内菌核、菌膜及其生境土壤的真菌群落结构与核心真菌组的生态功能,初步探索蝉棒束孢自然生长过程中内环境真菌与其周围环境中真菌的关系。本研究利用高通量测序技术检测6株采自贵阳大将山和贵阳森林公园的蝉棒束孢及土壤样本,结果表明,蝉棒束孢内菌核共检测到43个真菌属,菌膜检测到58个真菌属,菌际土检测到260个真菌属,且内菌核和菌膜真菌群落结构相似。而两地生境土的真菌群落结构有差异,大将山以棒束孢属Isaria、被孢霉属Mortierella和一分类地位未知属占优势,而贵阳森林公园以被孢霉属、隐球酵母属Cryptococcus、红菇属Russula、棒束孢属占优势,且两地菌际土中棒束孢属的丰度均显著高于其他土样。核心真菌组检测分析显示,内菌核的核心真菌组有10个OTUs,菌膜的核心真菌组有5个OTUs,菌际土的核心真菌组有20个OTUs,3种样本的核心真菌组的生态功能群均检测到有与植物相关的功能群,内菌核中检测到外生菌根真菌群,菌膜中检测到植物病原真菌群,菌际土中检测到丛枝菌根真菌群和植物病原菌群。     

Symbiosis of a termite and a sclerotium-forming fungus: Sclerotia mimic termite eggs

Brown balls, of a similar size but different shape to termite eggs, were found frequently in the piles of eggs of the subterranean termite Reticulitermes speratus. rDNA analysis identified the ball as the sclerotia of the fungus, Fibularhizoctonia sp. nov, which is phylogenetically closest to decay fungi, Athelia spp. Laboratory observation showed that the workers gathered the eggs and the sclerotia indiscriminately, even if they were widely scattered in a Petri dish, and piled them up in a specific place for egg care. We compared the morphology of the eggs with that of sclerotia of Fibularhizoctonia spp. and Athelia spp. in relation to egg carrying behaviour, and found that the workers could only carry the Fibularhizoctonia spp. sclerotia whose diameters were similar to the short diameter of the eggs. We also conducted a bioassay using termite eggs and dummy eggs (glass beads and sea sand) of two diameter-classes, coated with or without the egg-derived chemicals. The workers recognized the eggs based on a combination of the size, shape, and chemical cues. All the results suggested that the sclerotia mimic the eggs both morphologically and chemically. Finally, we found that the workers suppressed germination of sclerotia, and termite egg survival increased in the presence of sclerotia only if they were tended by the workers. If the workers were removed experimentally, the sclerotia germinated and grew by exploiting termite eggs. These results suggest that the sclerotia protect termite eggs from putative pathogens.     

Viability of VA-mycorrhizal fungi following soil solarization and fumigation

Two field experiments were conducted to examine the effect of soil solarization on the survival of arbuscular mycorrhizal (AM) fungi and root colonization of three crops. The experiments were carried out in a loamy sand soil (Rehovot) and a silty soil (Bet She'an Valley). For both experiments, assessment of indigenous AM fungal populations by the most probable number (MPN) method indicated that populations were reduced to zero after 2 or 4 weeks of solarization treatment. However, Glomus intraradices inoculum applied to the soil prior to solarization remained viable even after 8 weeks of solarization. After soil fumigation with methyl bromide both indigenous and applied AM fungi were nondetectable. Percentage root colonization by the indigenous AM fungal populations, together with plant-growth parameters, were assessed for three crops: onion and wheat (Rehovot), and carrot (Bet She'an). When sown on solarized field plots, onion and carrot seedlings showed a plant growth retardation, whereas wheat showed an increased growth response. Root colonization by indigenous AM fungi was not evident until 6 weeks after seedling emergence. Fumigation with methyl bromide reduced root colonization by indigenous AM populations, and reduced onion and wheat plant development at early growth stages. In a laboratory experiment, a temperature of 45° C for up to 24 h did not affect AM spore viability, indicating that temperatures reached during the solarization treatment cannot solely account for the reduced AM fungi viability in the field. Apparently, soil solarization temporarily delays root colonization by indigenous AM fungi until 6-8 weeks after plant emergence.     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.