Exploring the life cycles of three South American rusts that have potential as biocontrol agents of the stipoid grass Nassella neesiana in Australasia

Three rusts, Puccinia nassellae, Uromyces pencanus, and Puccinia graminella, are being studied as potential biological control agents for Nassella neesiana (Chilean needle grass) in Australia and New Zealand. An understanding of the life cycle of a pathogen is desirable before its release as a biocontrol agent is considered, to enable the assessment of the risks involved in such a release. Field observations and experiments have been carried out to elucidate the life cycles of these three pathogens. Puccinia nassellae cycles as urediniospores and produces dormant teliospores. Dormancy of teliospores has been broken through manipulation in the laboratory, but resulting basidiospores have failed to infect N. neesiana plants under the conditions tested. Uromyces pencanus cycles as urediniospores and its telia appear to have lost the capacity to produce basidiospores. Aecia have been reported for this rust in the literature. However, evidence is provided that these aecia in fact belong to the life cycle of P. graminella. Puccinia graminella produces only aecia and telia. The aeciospores have been shown to be repetitive (aecidioid urediniospores). Teliospores germinate directly without a dormant phase, but resulting basidiospores failed to infect N. neesiana plants under the conditions tested. The role of teliospores in the life cycle of all three rusts remains unknown. Although the autoecious nature of their life cycles has not been proven experimentally, neither is there any evidence that they are heteroecious. Practical and theoretical implications of these findings are discussed.     

Detection of fungal spores using a generic surface plasmon resonance immunoassay

This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.     

28份人工合成小麦对禾谷孢囊线虫、纹枯病、条锈病和叶锈病的抗性

为了发现具有兼抗多种病害的小麦种质,本研究采用田间病圃法和人工接种法,对28份人工合成小麦的禾谷孢囊线虫、纹枯病、条锈病和叶锈病进行了抗性鉴定。人工合成小麦对这些病害表现不同程度的抗性反应。C2和C20对鉴定的4种病害都具有抗性,C5、C10和C25对这些病害都表现感病。8份材料对Heterodera avenae和H. filipjevi两种病原线虫都表现抗性反应型,也有的材料只抗一种线虫。供试材料对纹枯病的抗性表现较好,其中19份材料表现抗性反应型。9份材料对接种的条锈菌小种CY30、CY31、CY32和CY33均表现抗性反应型,5份材料对叶锈菌小种THT和PHT都具抗性。     

Spore Liberation and Dispersal in Smut Fungi*

Teliospores are the most important diaspores of smut fungi, albeit not the only ones. The role of basidla, basidiospores, secondary spores, yeast cells, and infected parts of the host for dispersal has often been neglected. Many smut species have soral structures like galls, peridia, and elaters, which cause teliospores to be liberated over prolonged periods. This increases the chance that at least some spores are released under favourable wet climatic conditions and while host plants are susceptible. In this review, the diversity of dispersal units as well as vectors of smut fungi are presented. The importance of timing of diaspore liberation, flexibility in dispersal strategies, and the genetic and evolutionary implications of dispersal strategies of smut fungi are discussed. The general considerations are complemented by examples based on original field and laboratory observations: peridia of Farysia corniculata and certain species of Sporisorium expose the spore mass by hygroscopic movement under wet conditions (hygrochasy) favourable for teliospore germination and infection of a host plant. Basidia with firmly attached basidiospores liberated from spore balls of Doassansiopsis deformans, branched basidiospores of Rhamphospora nymphaeae, needle-shaped basidiospores of species of Entyloma, folded basidiospores of Mycosyrinx cissi, and stellate groups of yeast cells of Trichocintractia utriculicola show enlarged surfaces, which are advantageous for dispersal in water. Galls filled with spore balls of Doassansiopsis limnocharidis and witches' brooms formed by spikelets infected by Cintractia standleyana separate from the host and fall into water where they are dispersed.     

An evaluation of two methods used for microscopic analysis of airborne fungal spore concentrations from the Burkard Spore Trap

The Burkard Volumetric Spore Trap is a common and efficient instrument used to collect outdoor air samples. In North America, two slide counting methods have been widely used by aerobiologists: the single longitudinal traverse method and the twelve transverse traverse method. The purpose of this study was to compare the two counting methods by assessing fungal spore concentrations of ascospores, basidiospores, smut teliospores, Cladosporium, Alternaria, Epicoccum, Curvularia, Drechslera, Pithomyces, other spores, and total spores at two metropolitan Tulsa, Oklahoma sites (Tulsa and Hectorville) during September 1996. Results showed that both methods were sensing parallel fluctuations in average daily spore concentration, although the twelve transverse traverse method usually resulted in higher concentrations. At the Tulsa site, the twelve transverse traverse method gave statistically higher concentrations than the single longitudinal traverse method except for Epicoccum, Pithomyces, smut teliospores, and other spores. At the Hectorville site, however, only Cladosporium and basidiospores showed that the twelve transverse traverse method was statistically higher than the single longitudinal traverse method. Comparison with concentrations obtained by counting the total slide surface of two slides indicated that neither method was equivalent to the total slide spore count, although the twelve transverse traverse method gave a lower absolute percent difference from the total slide surface concentration. While the twelve transverse traverse method gave slightly better approximations of the spore concentration, the increase in accuracy may not justify the extra effort required to analyze with this method.     

Effects of fixation, dehydration and staining on dimensions of myxosporidan

The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores.     

Seasonal and Diurnal Variation of Airborne Basidiomycete Spore Concentrations in Mexico City

Seasonal and diurnal changes in concentrations of airborne basidiomycete spores (basidiospores, rusts, smuts) were studied, using Burkard volumetric spore traps, in two areas of Mexico City with different degrees of urbanization and related to changes in climatic variables through 1991. Basidiomycete spores formed a large component of the total airborne fungal spore load in the atmosphere of Mexico City. They were the second most abundant spore type after Deuteromycotina (Hyphomycetes), forming 32% of the total fungal spores trapped in an urban-residential area and 28% in an urban-commercial area. The most abundant basidiomycete spores were basidiospores although smut-type spores were trapped on more days than basidiospores and rusts on fewer. Basidiospores occurred in concentrations up to 2,000 spores m-3 in the urban-residential area. Basidiospores showed a marked seasonal distribution, especially in the southern area, with their greatest abundance during the wet season. The correlation coefficients associated with regressions between basidiospore concentration and some environmental factors were increased when a lag period of 2 to 4 days was used between environmental measurements and the day of spore collection. Basidiospore concentrations exceeded the 75 percentile concentration (>400 spores m^-3) most often when rainfall was up to 6 mm and relative humidity was >70%. Basidiospores showed a diurnal periodicity with greatest concentrations in the early morning. The most common basidiospore type was Coprinus which formed 67% of basidiospores trapped in the southern area and 63% in the central area. Smut spores were trapped on 87% of days through the year while rust spores occurred in only 35%. Both rusts and smuts were present in only small concentrations.     

Effect of light exposure on in vitro germination and germ tube growth of eight species of rust fungi

The effects of light on urediniospore germination and germ tube elongation was studied with eight species of rust fungi that infect ornamental plants or row crops. Exposure of six species of fungi to cool white fluorescent light at 400 or 600 micromol s(-1) m(-2) for 24 h significantly reduced germination with largest decreases typically observed at 600 micromol s(-1) m(-2). Germination and germ tube elongation did not recover during 24 h dark incubation after 18 h exposure to fluorescent light at 600 micromol s(-1) m(-2), indicating the effects were not reversible. Germ tube elongation of all fungi was negatively affected by increased length of exposure to fluorescent light. Increased exposure to fluorescent light differentially affected germination of the fungi with Puccinia hemerocallidis, Phakopsora pachyrhizi, Pucciniastrum vaccinii and Puccinia menthae negatively affected and Puccinia sorghi, Puccinia triticina, Puccinia pelargonii-zonalis and Puccinia iridis relatively unaffected in 10 h incubation. Exposure of Ph. pachyrhizi and P. triticina urediniospores to sunlight rapidly reduced germination and germ tube elongation with no germination observed for Ph. pachyrhizi after 2.5 h. Germ tube elongation but not germination of hydrated urediniospores of Ph. pachyrhizi and P. triticina was significantly reduced compared to dry urediniospores exposed to 10 h fluorescent light followed by 24 h dark incubation. Exposure to fluorescent light (all fungi) or sunlight (two fungi) negatively affected urediniospore germ tube elongation. Differences observed in urediniospore germination between fungi suggest some species have co-evolved with their host for differing light conditions. Our data suggests exposure of urediniospores to strong light could inactivate rust fungi on plant surfaces or in the atmosphere.     

Cloning and characterization of the actin gene from Puccinia striiformis f. sp. tritici

The fungus Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Urediniospores are the most common spore type involved in the epidemiology of this disease. Tip growth of germ tubes of germinated urediniospores is a key step during infection of wheat, but few studies have investigated it so far. Recent research has found that actin is closely associated with hyphal tip growth. In this study, we have cloned and obtained the full-length actin cDNA from P. striiformis f. sp. tritici and characterized its expression. Furthermore, actin filament (F-actin) patterns were visualized microscopically during germ tube formation. The most conspicuous actin-containing structures were actin patches. They were mainly concentrated near the hyphal tip and scattered throughout the cortex. By using cytochalasin B, we observed that depolymerization of F-actin greatly reduced the germination rate of urediniospores and disrupted the transport of vesicles to the germ tube tip, indicating that F-actin played a key role in the tip growth of P. striiformis f. sp. tritici. This work helps us to understand the tip growth mechanism of P. striiformis f. sp. tritici, and may provide a theoretical framework for designing novel pesticides.     

The high-resolution architecture and structural dynamics of Bacillus spores

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.     

RUST FUNGI AND HOST PLANT COEVOLUTION: DO PRIMITIVE HOSTS HARBOR PRIMITIVE PARASITES?

Abstract— The classical view of rust phylogeny is that rusts found on ferns and conifers are primitive, while rusts that parasitize angiosperms are advanced. This belief was based on the theory that primitive hosts harbor primitive parasites; that is, it assumed coevolution (co-speciation) of hosts and parasites. A cladistic analysis of 30 genera and 28 characters representative of the major patterns of rust fungi diversity is presented. The results of this analysis suggest that tropical short-cycle rusts on angiosperms form the cladistically basal group of rusts, while the rusts on conifers and ferns (Melampsoraceae sensu lato ) form a nested terminal clade. These results suggest that rusts and their hosts have not undergone a long period of parallel cladogenesis (co-speciation); host transfer has probably been at least as frequent as co-speciation. The cladograms indicate evolutionary trends of spore stages and life history: urediniospores evidently preceded the evolution of aeciospores and pycniospores within Uredinales, and heteroecism is a derived condition which evolved at least several times. This study stresses the importance of making use of independent cladistic analyses of both host and parasite in order to test assumptions of coevolution and host transfer.     

Quest to elucidate the life cycle of Puccinia psidii sensu lato

There is controversy surrounding the described life cycle of the rust fungus Puccinia psidii sensu lato, which causes disease on several plant species in the family Myrtaceae. The objective of this study was to determine whether P. psidii s.l. is autoecious by performing basidiospore inoculations, and microscopically examining the fate of basidiospores on the leaf surface and nuclear condition at different stages of rust development. No spermogonia developed on leaves of Agonis flexuosa inoculated either with a teliospore suspension or basidiospores naturally discharged from telia. Uredinial sori that developed in all three inoculations with teliospore suspensions and in one of the five inoculations with naturally-discharged basidiospores from telia were most likely the result of urediniospore infections. Microsatellite analysis revealed that isolates made from these uredinial sori had the same multilocus genotype as that of the original isolate. No signs of penetration of plant cells by basidiospores were observed on A. flexuosa and Syzygium jambos. The nuclear condition of mycelia of uredinial sori, urediniospores, teliospores, and four-celled metabasidia was typical of that in many rust fungi. Our study could not provide unequivocal proof that P. psidii s.l. is autoecious. While it is possible that it could be heteroecious, with an unknown alternate aecial host, it is also possible that basidiospores have lost the ability to infect Myrtaceae or are infrequently operational.     

Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores

Botrytis cinerea and Puccinia recondita spores adhere strongly to polystyrene microtiter plates coincident with germination. We developed assays for inhibition of spore adhesion in 96-well microtiter plates by using sulforhodamine B staining to quantify the adherent spores. In both organisms, fungicides that inhibited germination strongly inhibited spore adhesion, with 50% effective concentrations (EC(50)s) comparable to those for inhibition of germination. In contrast, fungicides that acted after germination in B. cinerea inhibited spore adhesion to microtiter plates only at concentrations much higher than their EC(50)s for inhibition of mycelial growth. Similarly, in P. recondita the ergosterol biosynthesis inhibitors myclobutanil and fenbuconazole acted after germination and did not inhibit spore adhesion. The assays provide a rapid, high-throughput alternative to traditional spore germination assays and may be applicable to other fungi.     

How far and how fast can mushroom spores fly? Physical limits on ballistospore size and discharge distance in the Basidiomycota

Active discharge of basidiospores in most species of Basidiomycota is powered by the rapid movement of a droplet of fluid, called Buller’s drop, over the spore surface. This paper is concerned with the operation of the launch mechanism in species with the largest and smallest ballistospores. Aleurodiscus gigasporus (Russulales) produces the largest basidiospores on record. The maximum dimensions of the spores, 34 × 28 μm, correspond to a volume of 14 pL and to an estimated mass of 17 ng. The smallest recorded basidiospores are produced by Hyphodontia latitans (Hymenochaetales). Minimum spore dimensions in this species, 3.5 × 0.5 μm, correspond to a volume of 0.5 fL and mass of 0.6 pg. Neither species has been studied using high-speed video microscopy, but this technique was used to examine ballistospore discharge in species with spores of similar sizes (slightly smaller than A. gigasporus and slightly larger than those of H. latitans). Extrapolation of velocity measurements from these fungi provided estimates of discharge distances ranging from a maximum of almost 2 mm in A. gigasporus to a minimum of 4 μm in H. latitans. These are, respectively, the longest and shortest predicted discharge distances for ballistospores. Limitations to the distances traveled by basidiospores are discussed in relation to the mechanics of the discharge process and the types of fruit-bodies from which the spores are released.     

Study of airborne fungal spores in Madrid, Spain

The concentration of fungal spores in the atmosphere of Madrid was recorded and analyzed for the year 2003. Airborne spores were sampled continuously with a Hirst-type spore trap located on the roof of a building of the School of Pharmacy, at about 8 m above ground level. Correlation between the mean daily spore concentrations and meteorological variables were explored by means of Spearman’s correlation analyses. Seventy spore types were identified, of which the most numerous were Cladosporium, Aspergillaceae (conidia), Coprinus, Agaricales (basidiospores), Ustilago (teliospores) and Pleospora (ascospores). These six types of spores represented more than 70% of the total. Cladosporium represented 41% of the total fungal spores, while Ustilago spores, the concentrations of which in May and June exceeded 47% of the monthly total spore count, constituted the second most important group. Spores reached their highest concentrations in the spring months, and in the autumn, mainly in October. A␣positive significant correlation was found between airborne spore counts and temperature and relative humidity. The results provide a picture of the spectrum of airborne fungal spores present in the atmosphere of Madrid and of the `peak' periods of their presence. Future studies will provide more detailed information on the seasonal dynamics of the spores most frequently found in the air as well as on the extent to which atmospheric conditions influence their release, dispersion and sedimentation processes.     

铁线莲上隐匿柄锈菌性孢子器和锈孢子器发育的组织学和细胞学观察

通过光学显微镜技术和电镜技术对铁线莲上隐匿柄锈菌Puccinia recondita性孢子器和锈孢子器发育进行了组织学和超微结构的观察。观察表明,铁线莲上隐匿柄锈菌性孢子器呈瓶状,内生有大量单核的性孢子梗和侧丝。性孢子梗顶端产生大量的性孢子,每形成一个性孢子后,都在性孢子梗上留下一环痕。性孢子长椭圆形,表面无特殊纹饰。铁线莲上隐匿柄锈菌锈孢子器呈杯状,其内生长大量平行排列的长形双核锈孢子梗,每个锈孢子梗由上向下连续产生多个锈孢子。锈孢子梗通过产生隔膜先形成锈孢子原体,再分隔形成锈孢子和间生细胞。随着锈孢子的进一步发育,间生细胞解体,锈孢子彼此分开。成熟的锈孢子表面产生有密集的瘤状结构。     

Vertical Concentrations of Fungal Spores above Wheat Fields

Kramer-Collins volumetric spore samplers were used to measure concentrations of Puccinia graminis, P. recondita, Erysiphe graminis, Cladosporium, and Alternaria spores and fungal hyphal fragments within the canopy and at 1, 3, 6 and 14 m above ground level over wheat fields near Manhattan, Kansas, USA. The largest numbers of spores of each of the named fungi and hyphal fragments were trapped during hours when free moisture was not present on host leaf surfaces. As wind velocity increased, the number of spores and hyphal fragments trapped at all heights increased. Airspora trapped at the various sampling heights under all combinations of biometeorological conditions were calculated as ratios. Location and severity of infection or frequency of occurrence of the parent fungi greatly affected the percentage of propagules released in the canopy and escaped into the atmosphere. Less than 40% of P. recondita urediniospores released from tillering plants in the fall and trapped at 20–25 cm were trapped at 1 m, while only 6% of those trapped at 10 cm were trapped at 1 m. Ratios for the average number of spores trapped at 3 m: 1 m during the entire sampling period were similar for P. recondita (0.54), Alternaria (0.57), and E. graminis (0.48). However, the higher ratios that occurred with spores of P. graminis (0.72) and Cladosporium (0.76), and hyphal fragments (0.77) indicate considerable mixing of locally released airspora with airspora from exogenous sources.     

Phragmidium satoanum,a new rust pathogen of Rosa hirtula in Japan

The causal fungus of a rust disease of Rosa hirtula, endemic to mountainous areas of Fuji-Hakone-Izu National Park, Japan, was thought to be a common species Phragmidium rosae-multiflorae. Continued field observations, morphological examination, and experimental inoculations proved that the fungus produced laterally three-angled aeciospores and urediniospores together with multi-cellular teliospores on the same R. hirtula trees. These morphological features were different from those of P. rosae-multiflorae. The fungus parasitized only R. hirtula. Experimental inoculations and field observations did not prove that R. banksiae, R. laevigata, and R. multiflora supported infection and sporulation of the fungus. Under the field observations, R. multiflora, the most common host of P. rosae-multiflorae, was not proven to harbor the R. hirtula fungus. Therefore, the fungus was concluded to be a species distinct from P. rosae-multiflorae; and a new name, P. satoanum, was proposed for it.     

金针菇担孢子核相及遗传属性的研究

以3个不同的金针菇菌株为材料,研究了其担孢子的核相及遗传属性。荧光染色观察显示,担孢子核相以双核为主,双核孢子、单核孢子和无核孢子分别占80.2%、7.5%和12.3%。源于单孢分离物的菌丝为有隔膜、无锁状联合的多核菌丝。在交配试验中,源于不同菌株单孢分离物的菌丝原生质体的配对形成具锁状联合的菌落,而源于同一单孢分离物的菌丝原生质体的配对则形成无锁状联合的菌落,暗示担孢子中的两个核具有相同的交配型。RAPD分析显示,源于同一单孢分离物的菌丝原生质体为10个随机引物所扩增的图谱彼此完全相同,印证了担孢子中的双核是同质的。此外,观察表明,一个担子上着生有4个担孢子。因此,金针菇是一种具4个含同质双核担孢子的四极性蕈菌。