Biochemical characteristics of Trichoderma atroviride associated with conidium fitness for biological control

Effects of abiotic factors during production (temperature, nutrients, water activity, pH) on conidium fitness (quantity and quality) of Trichoderma atroviride LU132 (a key biocontrol agent) were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture assays were selected to assess effects of storage conditions on conidial fitness over time. Further studies assessed interaction effects of temperatures (20°C or 30°C) and sugars (dextrose or sucrose) on conidium germination and bioactivity as fresh conidia, or after 6 months of storage. Biochemical analyses of sugars and fatty acids were carried out to determine relationships between quality variations and cellular characteristics for conidia produced in different culturing conditions. Low trehalose content in conidia (e.g. at 20°C) was associated with the least conidium fitness, although high trehalose content did not necessarily support conidium fitness. High proportions of total fatty acids in conidia were mostly associated with the least conidium fitness. When Trichoderma was grown at high carbon to nitrogen ratio (e.g. at C:N 160:1), the total conidium fatty acids content increased. This study also indicated that the monosaccharide dextrose is metabolically optimal for T. atroviride LU132 at 20°C while the disaccharide sucrose is optimal at 30°C. These studies indicate that physical growth conditions and nutritional requirements attribute in conidium fitness of T. atroviride LU132, and provide important knowledge supporting optimum production of biocontrol agents based on T. atroviride, and possibly other similar biocontrol agents.     

Materials for the fungus flora of Japan (56)Mariannaea camptospora andM. elegans var.punicea from Japan

One additional species and a variety ofMariannaea, M. camptospora andM. elegans var.punicea, were recorded for the first time in Japan.Mariannaea camptospora formed two types of conidiophores. One type was characterized by simple verticillate phialides sometimes with punctuate walls at the base, producing long oblique conidial chains, and symmetrical spindle-shaped conidia. The other type was characterized by more crowded and shorter phialides with small conidial droplets and hemispherical to concave smaller conidia.Mariannaea elegans var.punicea was characterized by distinct red purple pigmentation in agar media. (55): Udagawa, S. and Uchiyama, S., Mycoscience41: 263–267, 2000.     

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Spore dispersal in Alternaria porri (Ellis) Neerg. on onions in Nebraska

In spore-trapping studies in 1965, conidia of Alternaria porri (Ellis) Neerg. were detected in the atmosphere immediately above onions affected by purple blotch disease. On calm summer days, peak conidium concentration occurred between 08.00 and 14.00 hr., few conidia being trapped between 20.00 and 06.00 hr. On windy days, there were marked increases in concentration. Concentration tended to increase with increasing wind velocity. Increased conidium concentrations were also recorded after rainfall and irrigation, and during spraying operations. Laboratory observations indicate that in A. porri, the attachment of conidium to conidiophore is weakened, if not broken, by movements in the conidial apparatus which occur in response to a decrease in vapour pressure. Possible causes of fluctuations in atmospheric content of A. porri conidia are discussed.     

Temperature and Sporulation of Aquatic Hyphomycetes

Temperature appears to be an important factor affecting the occurrence and distribution of aquatic hyphomycetes, the dominant leaf litter-decomposing fungi in streams. We compared conidium production by eight species of aquatic hyphomycetes grown on yellow poplar leaves in stream-simulating microcosms at three temperatures (15, 20, and 25°C). The greatest conidium production occurred at 15°C for one species, 20°C for two species, and 25°C for two species. Two species produced similar numbers of conidia at 20 and 25°C, and one species produced similar numbers of conidia at all three temperatures. Linear growth rates were determined on malt extract agar. Six species had the same pattern of temperature responses for growth on malt extract agar as for sporulation on leaves, as shown by the positive correlations between the two parameters at the three temperatures. The species examined also exhibited differences in number of conidia produced from a similar amount of leaf material at a given temperature. These differences appeared to be due primarily to differences in individual conidium mass (determined by weighing conidia produced from cultures), as shown by the relationship of the type Y = k/X (r² = 0.96), where Y is the number of conidia produced, X is the individual conidium mass in milligrams, and k is a constant empirically determined to be 2.11. This finding supports the hypothesis that aquatic hyphomycetes allocate similar amounts of their resources to reproduction but vary with respect how these resources are partitioned into reproductive units (conidia).     

Fusarium fractiflexum sp. nov. and two other species within theGibberella fujikuroi species complex recently discovered in Japan that form aerial conidia in false heads

Morphological and molecular phylogenetic analyses were conducted on 12 strains ofFusarium, deposited in MAFF asF. subglutinans (≡F. moniliforme var.subglutinans≡F. sacchari var.subglutinans) orFusarium sp. because they formed aerial conidia in false heads in the dark. These strains were resolved as three distinct species within theGibberella fujikuroi species complex. A new species,F. fractiflexum, and two species new to Japan,F. circinatum andF. concentricum, are described and illustrated and their morphological features are discussed.Fusarium fractiflexum, isolated from diseased yellow leaf spots ofCymbidium spp., is differentiated from other fusaria based on its yellowish colonies and aerial conidia formed in false heads in the dark and in zigzag-like conidial chains under black light. Japanese strains ofF. circinatum also formed elongate, coiled sterile hyphae. Phialidic aerial conidia with a pointed apex and a wedgeshaped base were found inF. concentricum cultured under black light and represent a new diagnostic character of the species, in addition to colonies with alternating concentric rings when cultured on PDA. Based on DNA sequences of the β-tubulin gene and two other loci, strains ofF. fractiflexum were resolved phylogenetically as members of the Asian clade of theG. fujikuroi species complex. In addition, Japanese strains ofF. circinatum andF. concentricum were phylogenetically identical to the ex-type strains.     

Biological fitness of Trichoderma atroviride during long-term storage,after production in different culture conditions

Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C?:?N 5?:?1 and C?:?N 160?:?1, indicating that C?:?N ratio in culture medium is not important for conidium survival of T. atroviride.     

Evolutionary relationships among aquatic anamorphs and teleomorphs: Lemonniera, Margaritispora, and Goniopila

The hypothesis that similar conidial morphologies in aquatic hyphomycetes are a result of convergent evolution was tested using molecular sequence data. Cladistic analyses were performed on partial sequences of 28S rDNA of seven species of Lemonniera, one species of Margaritispora and one species of Goniopila. Lemonniera has tetraradiate conidia with long arms, whereas Margaritispora and Goniopila have typically globose (isodiametric) conidia, with short conical protuberances in a stellate or quadrangular arrangement. Lemonniera and Margaritispora have phialidic conidiogenesis and both produce dark, minute sclerotia in culture whereas Goniopila has holoblastic conidiogenesis and does not produce sclerotia in culture. Goniopila produces a microconidial phialidic synanamorph in culture. All three genera have schizolytic conidial secession. Molecular analyses demonstrate that Lemonniera species are placed in two distinct clades: one within Leotiomycetes; the other within Pleosporales, Dothideomycetes. Margaritispora is placed with Lemonniera species within Leotiomycetes. Goniopila and Lemonniera pseudofloscula are placed within Dothideomycetes. No morphological character was entirely congruent with the molecular derived phylogeny. This suggests that for the group of species studied, conidial shape is not a reliable indicator of phylogeny but more likely the result of convergent evolution in response to the aquatic environment.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

Nonspecific Factors Involved in Attachment of Entomopathogenic Deuteromycetes to Host Insect Cuticle

The attachment of the conidia of the insect-pathogenic fungi Nomuraea rileyi, Beauveria bassiana, and Metarrhizium anisopliae to insect cuticle was mediated by strong binding forces. The attachment was passive and nonspecific in that the conidia adhered readily to both host and nonhost cuticle preparations. The hydrophobicity of the conidial wall and the insect epicuticle appeared to mediate the adhesion process. Detergents, solvents, and high-molecular-weight proteins known to neutralize hydrophobicity reduced conidial binding when added to conidium-cuticle preparations. However, these chemicals did not remove the hydrophobic components from the epicuticle or from conidial preparations. The outer surface of the conidium consists of a resilient layer of well-organized fascicles of rodlets. Intact rodlets extracted from B. bassiana conidia bound to insect cuticle and exhibited the hydrophobicity expressed by intact conidia. Both electrostatic charges and various hemagglutinin activities were also present on the conidial surface. However, competitive-inhibition studies indicated that these forces played little, if any, role in the adhesion process.     

Conidial water affinity is an important characteristic for thermotolerance in entomopathogenic fungi

The interspecific thermotolerance of several species of entomopathogenic fungi was evaluated based on the conidial water affinity. The species were divided between hydrophilic and hydrophobic conidia. The species with hydrophobic conidia were Beauveria bassiana (ARSEF 252), Metarhizium brunneum (ARSEF 1187), Metarhizium robertsii (ARSEF 2575), Isaria fumosorosea (ARSEF 3889) and Metarhizium anisopliae s.l. (ARSEF 5749). The species with hydrophilic conidia were Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), Simplicillium lanosoniveum (ARSEF 6430), Lecanicillium aphanocladii (ARSEF 6433), S. lanosoniveum (ARSEF 6651), Aschersonia placenta (ARSEF 7637) and Aschersonia aleyrodis (ARSEF 10276). The conidial surface tension of each isolate was also studied. Conidial suspensions were exposed to 38, 41 or 45 °C. After exposure, the suspensions were inoculated on media and conidial germination was evaluated. Considerable differences in thermotolerance were found among the 12 entomopathogenic fungal species. Species with hydrophobic conidia were generally more thermotolerant than species with hydrophilic conidia. All isolates with hydrophobic conidia showed higher conidial surface tension than the isolates with hydrophilic conidia.     

<Emphasis Type="Italic">Cylindrosporella</Emphasis> on betulaceous trees in Japan

Three Cylindrosporella species on the leaves of betulaceous trees – C. carpini, C. coryli, and C. microsperma – were first reported from Japan. The genus Cylindrosporella is sometimes treated as congeneric with Asteroma; however, we considered these to differ based on conidial morphology following the concept of Arx. The genus Cylindrosporella is characterized by one-celled, filiform or fusiform, small conidia that are often curved, have hyaline, thin walls, and are produced from phialidic conidiogenous cells in subcuticular, fiat acervuli. The three species are distinguished from each other on the basis of conidium size. Received: September 27, 2001 / Accepted: November 5, 2001     

The nature of the secondary conidia of Sporothrix schenckii

Observations of secondary conidium production in an African strain ofS. schenckii revealed that secondary conidia are produced (1) acropetally on short sterigmata, (2) sympodially on short conidial appendages, and/or (3) sympodially or acropetally on short sporogenous cells which arise directly from the primary conidium. The secondary conidia therefore appear also to be sympodulospores.

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Zusammenfassung Beobachtungen von sekundärer Konidienbildung in einer afrikanischen Kultur vonS. schenckii zeigten, daß sekundäre Konidien gebildet werden:1) acropetal an kurzen Sterigmata; 2) sympodial oder acropetal an kurzen, sporentragenden Zellen, und 3) sympodial oder acropetal an kurzen sporentragenden Zellen, die direkt vom primären Konidium entspringen. Die sekundären Konidien erscheinen deshalb auch Sympodulosporen zu sein.

     

哈氏木霉分生孢子发育的超微结构和细胞化学

通过电子显微镜和细胞化学标记研究了哈氏木霉分生孢子发育的超微结构和细胞化学。分生孢子发育的超微结构研究表明,分生孢子壁的发育是有个由薄而光滑到厚而有疣的过程;期间脂肪体在分生孢子和产孢细胞中不断累积,最后脂肪体沿着内壁排列成一层。免疫金标记结果显示,幼嫩的分生孢子壁中缺乏几丁质和纤维素,只有在成熟的分生孢子壁中含有几丁质;出乎意料的是在成熟分生孢子中发现有少量纤维素的存在。     

FINE STRUCTURE OF ANNELLOPHORES. I. SCOPULARIOPSIS BREVICAULIS AND S. KONINGII

Fine-structure observations of annelloconidium production in filamentous Hyphomycetes are reported for the first time. The difference in conidium morphology between Scopulariopsis brevicaulis and S. koningii was quite distinct. In S. brevicaulis, verrucosities appeared early in conidium ontogeny and formed an integral part of the primary wall layer of mature conidia. In S. koningii, verrucosities were absent. In S. brevicaulis, annellations did not invariably result on conidiophore necks with the production of each conidium in the basipetal sequence, but alternatively could be left on subapical regions of subsequently formed conidia. In S. koningii, annellations were more distinct, and the position of a conidium-delimiting septum was variable. If a septum were formed at a position proximal to previous septa, a portion of the annellophore neck remained attached to the base f the seceding conidium. In both species, a spherical electron-dense body, perhaps analogous to septal pore plugs in vegetative hyphae, plugged the pore between conidia and conidiophores and remained embedded in the base of seceded conidia.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Shoot and leaf demography of Carex curvula ssp. Curvula and Carex curvula ssp. rosae in the Central Alps

Abstract. Populations of Carex curvula ssp. curvula, C. curvula ssp. rosae and their intermediate form were investigated in the Central Alps over a three-year period. The closely related taxa showed a different dominance behaviour in their respective communities. This may be caused by different growth strategies and a different reproduction biology. Therefore, the main aim of the study was to compare the demography of the three taxa. Shoot and leaf turnover, flower and seed production and population half-lives were determined. Differences in growth dynamics were less pronounced between the two species of Carex curvula than between these taxa and their intermediate form which showed the highest shoot turnover, highest number of fertile inflorescences and highest number of seeds per inflorescence. Carex curvula ssp. rosae showed a slightly higher shoot and leaf production but a lower reproduction capacity than Carex curvula ssp. curvula. Recruitment of populations of the three Carex-forms was only by vegetative shoots. Flowering had a striking effect on the shoot dynamics of the species grown in the grassland sites: up to about 70 % of all dead shoots could be identified as dead flowering shoots from the previous year. The different dominance behaviour of the three taxa could not be explained by their demographical features. Interspecific interactions and the occurrence of microniches might affect the growth and reproduction processes of the taxa.