Iron Sulfate and Phosphorous Acid Affect Turfgrass Surface pH and Microdochium Patch Severity on Annual Bluegrass.

Microdochium patch is a turfgrass disease caused by the fungal pathogen Microdochium nivale. Iron sulfate heptahydrate (FeSO4•7H2O) and phosphorous acid (H3PO3) applications have previously been shown to suppress Microdochium patch on annual bluegrass putting greens when applied alone, although either disease suppression was inadequate or turfgrass quality was reduced from the applications. A field experiment was conducted in Corvallis, Oregon, U.S.A., to evaluate the combined effects of FeSO4•7H2O and H3PO3 on Microdochium patch suppression and annual bluegrass quality. The results of this work suggest that the addition of 3.7 kg H3PO3 ha-1 with 24 or 49 kg FeSO4•7H2O ha-1 applied every 2 weeks improved the suppression of Microdochium patch without substantially compromising turf quality, which occurred when 98 kg FeSO4•7H2O ha-1 was applied with or without H3PO3. Spray suspensions reduced the pH of the water carrier, therefore two additional growth chamber experiments were conducted to better understand the effects of these treatments on leaf surface pH and Microdochium patch suppression. On the application date in the first growth chamber experiment, at least a 19% leaf surface pH reduction was observed compared with the well water control when FeSO4•7H2O was applied alone. When 3.7 kg H3PO3 ha-1 was combined with FeSO4•7H2O, regardless of the rate, the leaf surface pH was reduced by at least 34%. The second growth chamber experiment determined that sulfuric acid (H2SO4) at a 0.5% spray solution rate was always in the group that produced the lowest annual bluegrass leaf surface pH, but did not suppress Microdochium patch. Together, these results suggest that while treatments decrease leaf surface pH, this decrease in pH is not responsible for the suppression of Microdochium patch.

A new leaf blight disease of turfgrasses caused by Microdochium poae, sp. nov.

A novel species of Microdochium was identified as the causal agent of a leaf blight of Poa pratensis (Kentucky blue grass) and Agrostis stolonifera (Creeping bentgrasses), two cold-season turfgrasses widely grown on golf courses in northern China. This disease first appears as small, water-soaked, and scattered leaf spots. Under conditions of high temperatures and successive days of rain, the infected leaves rapidly lose their integrity and large diseased patches appear. Fungal strains were isolated from blighted leaf spots. A phylogenetic analysis based on the nuc rDNA internal transcribed spacer regions and 5.8S rRNA gene (ITS1-5.8S-ITS2 = ITS) and parts of the ß-tubulin (TUB2) and RNA polymerase II second largest subunit (RPB2) genes strongly supported that these isolates are a distinct evolutionary lineage in Microdochium (Microdochiaceae, Xylariales) that represents a new taxonomic species, herein named as M. poae. Microscopic characters confirmed that these strains were morphologically distinct from known Microdochium species. The pathogenicity of M. poae was confirmed by inoculating spore suspension on both grasses and reisolation of the pathogen from symptomatic tissues. The optimal growth temperature suggests that the occurrence of the new leaf blight disease caused by M. poae was significantly different from the microdochium patch disease caused by M. nivale.

Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR.

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = -0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = -0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = -0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

Soil microarthropods alter the outcome of plant-soil feedback experiments.

Plant-soil feedback (PSF) effects are studied as plant growth responses to soil previously conditioned by another plant. These studies usually exclude effects of soil fauna, such as nematodes, soil arthropods, and earthworms, although these organisms are known to influence plant performance. Here, we aimed to explore effects of a model microarthropod community on PSFs. We performed a PSF experiment in microcosms with two plant species, Phleum pratense and Poa pratensis. We added a model microarthropod community consisting of three fungivorous springtail species (Proisotoma minuta, Folsomia candida, and Sinella curviseta) and a predatory mite (Hypoaspis aculeifer) to half of the microcosms. We measured seedling establishment and plant biomass, nematode and microbial community composition, microbial biomass, and mycorrhizal colonization of roots. Microarthropods caused changes in the composition of nematode and microbial communities. Their effect was particularly strong in Phleum plants where they altered the composition of bacterial communities. Microarthropods also generally influenced plant performance, and their effects depended on previous soil conditioning and the identity of plant species. Microarthropods did not affect soil microbial biomass and mycorrhizal colonization of roots. We conclude that the role of soil microarthropods should be considered in future PSF experiments, especially as their effects are plant species-specific.

Level of contamination with mycobiota and contents of mycotoxins from the group of trichothecenes in grain of wheat , oats, barley, rye and triticale harvested in Poland in 2006- 2008.

INTRODUCTION AND OBJECTIVE: The risk of cereal exposure to microbial contamination is high and possible at any time, starting from the period of plant vegetation, through harvest, up to the processing, storage and transport of the final product. Contents of mycotoxins in grain are inseparably connected with the presence of fungal biomass, the presence of which may indicate the occurrence of a fungus, and indirectly also products of its metabolism. MATERIALS AND METHOD: Analyses were conducted on 378 grain samples of wheat, triticale, barley, rye and oats collected from grain silos located at grain purchase stations and at mills in Poland in 2006, 2007 and 2008. The concentrations of ERG and mycotoxins from the group of trichothecenes, as well as CFU numbers were analysed. RESULTS: The tested cereals were characterised by similarly low concentrations of both the investigated fungal metabolites and the level of microscopic fungi. However, conducted statistical analyses showed significant variation between tested treatments. Oat and rye grain contained the highest amounts of ERG, total toxins and CFU. In turn, the lowest values of investigated parameters were found in grain of wheat and triticale. CONCLUSIONS: Chemometric analyses, based on the results of chemical and microbiological tests, showed slight differences between contents of analysed metabolites between the years of the study, and do not confirm the observations on the significance of the effect of weather conditions on the development of mycobiota and production of mycotoxins; however, it does pertain to treatments showing no significant infestation. Highly significant correlations between contents of trichothecenes and ERG concentration (higher than in the case of the correlation of the total toxin concentrations/log cfu/g), indicate that the level of this metabolite is inseparably connected with mycotoxin contents in grain.

Communities of endophytic sebacinales associated with roots of herbaceous plants in agricultural and grassland ecosystems are dominated by Serendipita herbamans sp. nov.

Endophytic fungi are known to be commonly associated with herbaceous plants, however, there are few studies focusing on their occurrence and distribution in plant roots from ecosystems with different land uses. To explore the phylogenetic diversity and community structure of Sebacinales endophytes from agricultural and grassland habitats under different land uses, we analysed the roots of herbaceous plants using strain isolation, polymerase chain reaction (PCR), transmission electron microscopy (TEM) and co-cultivation experiments. A new sebacinoid strain named Serendipita herbamans belonging to Sebacinales group B was isolated from the roots of Bistorta vivipara, which is characterized by colourless monilioid cells (chlamydospores) that become yellow with age. This species was very common and widely distributed in association with a broad spectrum of herbaceous plant families in diverse habitats, independent of land use type. Ultrastructurally, the presence of S. herbamans was detected in the cortical cells of Plantago media, Potentilla anserina and Triticum aestivum. In addition, 13 few frequent molecular operational taxonomic units (MOTUs) or species were found across agricultural and grassland habitats, which did not exhibit a distinctive phylogenetic structure. Laboratory-based assays indicate that S. herbamans has the ability to colonize fine roots and stimulate plant growth. Although endophytic Sebacinales are widely distributed across agricultural and grassland habitats, TEM and nested PCR analyses reinforce the observation that these microorganisms are present in low quantity in plant roots, with no evidence of host specificity.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P. aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E. amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P. fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E. amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae.

In North America, one of the most important root diseases of Poa and Festuca turf is summer patch, caused by Magnaporthe poae. Detection and identification of M. poae in infected roots by conventional culture-based methods is difficult and time consuming, typically taking 3 wk or longer to accomplish. In this study, a culture-independent, TaqMan real-time PCR assay was developed for the detection of M. poae from the roots of fungicide treated and non-treated Kentucky bluegrass (Poa pratensis) turf. The assay was validated with the target pathogen, closely related fungal species and a number of other microorganisms that inhabit the same host and soil environment. This assay was more sensitive (could detect as little as 3.88 pg genomic DNA of M. poae), rapid and accurate compared to direct microscopic observation and isolation on a selective medium. The real-time PCR detection results corresponded closely to visual assessments of disease severity in the field. Utilization of this assay in diagnostic laboratories will enable turfgrass managers to more quickly and effectively detect and potentially reduce fungicide usage through early and accurate identification of the pathogen.

A new Epichloe species with interspecific hybrid origins from Poa pratensis ssp. pratensis in Liyang, China.

We describe a new Epichloë species found in symbiosis with Poa pratensis ssp. pratensis in Liyang, China. Stromata characteristic of Epichloë spp. were present on some of the reproductive tillers of individual host grasses. Only three of the 98 stromata observed on field plants became orange and produced perithecia. Phylogenetic analyses based on sequences of tubB and tefA indicated that this Epichloë sp. was an interspecific hybrid related to both E. yangzii and members in the E. typhina complex clade (ETC). Allele-1 of tefA and tubB grouped in the E. bromicola/E. yangzii clade; allele-2 of these two genes clustered in a distinct subclade in the ETC. This is the first report of an Epichloë species that has interspecific hybrid origins. We propose the name Epichloë liyangensis Z. Wang, Y. Kang et H. Miao, sp. nov. for this species.

Cold-induced responses in annual bluegrass genotypes with differential resistance to pink snow mold (Microdochium nivale).

Greens-type annual bluegrass (Poa annua L.) is susceptible to winter stresses including subfreezing temperatures and pink snow mold (SM). To better understand the mechanisms of SM resistance in annual bluegrass, four SM-resistant and four SM-sensitive genotypes were incubated at low temperature with Microdochium nivale (Fries) Samuels & Hallett, the causal agent of pink snow mold. We assessed the impact of a 6-week incubation period with SM at 2 °C under high humidity (≥ 98%) on the accumulation of cold-induced metabolites and on freezing tolerance. Incubation of annual bluegrass inoculated with SM lead to a major decrease in concentration of cryoprotective sugars such as sucrose and HDP (high degree of polymerization) fructans. Conversely, major amino acids linked to stress resistance such as glutamine and arginine increased in crowns of annual bluegrass in response to SM inoculation. One of the major differences between resistant and sensitive genotypes was found in the concentration of HDP fructans, which remained higher in SM-resistant genotypes throughout the incubation period. HDP fructans were also more abundant in freeze-tolerant genotypes, reinforcing their positive impact on winter survival of annual bluegrass. The identification of genotypes that are resistant to both SM and freezing shows the possibility of being able to improve both traits concomitantly.

Forage as a primary source of mycotoxins in animal diets.

The issue of moulds and, thus, contamination with mycotoxins is very topical, particularly in connexion with forages from grass stands used at the end of the growing season. Deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM) and aflatoxins (AFL) are among the most common mycotoxins. The aim of the paper was to determine concentrations of mycotoxins in selected grasses (Lolium perenne, Festulolium pabulare, Festulolium braunii) and their mixtures with Festuca rubra an/or Poa pratensis during the growing season as a marker of grass safety, which was assessed according to content of the aforementioned mycotoxins. During the growing season grass forage was contaminated with mycotoxins, most of all by DON and ZEA. The contents of AFL and FUM were zero or below the limit of quantification. Moreover, the level of the occurrence of mould was quantified as ergosterol content, which was higher at the specific date of cut. All results were statistically processed and significant changes were discussed.

Evidence for functional divergence in arbuscular mycorrhizal fungi from contrasting climatic origins.

A considerable amount of phenotypic, genetic and symbiotic functional variability has been documented in arbuscular mycorrhizal fungi (AMF). However, little is known about whether distinct AMF ecotypes have evolved within their geographic range. We tested the hypothesis that AMF growing at temperatures closer to those prevalent within their origin would benefit their host and grow more than isolates distant from their native conditions. For each of six AMF species, we chose pairs of isolates that originated from distant areas with contrasting climates. Each isolate was grown in association with two grass species of different thermal optima at two temperature settings. Thus, we also tested whether AMF from different climatic origins were dependent on the thermal adaptation of the host plant species or to temperature per se. Although fungal growth was not directly affected by temperature, we found that AMF isolates originating from contrasting climates consistently and differentially altered plant growth. Our results suggest that AMF from contrasting climates have altered symbiotic function, thus linking an abiotic factor to ecotypic differentiation of putatively important symbionts.

Phylogenetic trait conservatism and the evolution of functional trade-offs in arbuscular mycorrhizal fungi.

The diversity of functional and life-history traits of organisms depends on adaptation as well as the legacy of shared ancestry. Although the evolution of traits in macro-organisms is well studied, relatively little is known about character evolution in micro-organisms. Here, we surveyed an ancient and ecologically important group of microbial plant symbionts, the arbuscular mycorrhizal (AM) fungi, and tested hypotheses about the evolution of functional and life-history traits. Variation in the extent of root and soil colonization by AM fungi is constrained to a few nodes basal to the most diverse groups within the phylum, with relatively little variation associated with recent divergences. We found no evidence for a trade-off in biomass allocated to root versus soil colonization in three published glasshouse experiments; rather these traits were positively correlated. Partial support was observed for correlated evolution between fungal colonization strategies and functional benefits of the symbiosis to host plants. The evolution of increased soil colonization was positively correlated with total plant biomass and shoot phosphorus content. Although the effect of AM fungi on infection by root pathogens was phylogenetically conserved, there was no evidence for correlated evolution between the extent of AM fungal root colonization and pathogen infection. Variability in colonization strategies evolved early in the diversification of AM fungi, and we propose that these strategies were influenced by functional interactions with host plants, resulting in an evolutionary stasis resembling trait conservatism.

Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland.

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 x 10(5) to 8.9 x 10(5) copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 x 10(3) to 2.6 x 10(4), 7.4 x 10(2) to 1.4 x 10(3), 2.5 x 10(2) to 6.4 x 10(3), and 1.2 x 10(3) to 5.5 x 10(3), respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.

Microbial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central Alps.

Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.

The role of clp-regulated factors in antagonism against Magnaporthe poae and biological control of summer patch disease of Kentucky bluegrass by Lysobacter enzymogenes C3.

A global regulator was previously identified in Lysobacter enzymogenes C3, which when mutated, resulted in strains that were greatly reduced in the expression of traits associated with fungal antagonism and devoid of biocontrol activity towards bipolaris leaf-spot of tall fescue and pythium damping-off of sugarbeet. A clp gene homologue belonging to the crp gene family was found to globally regulate enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes C3 (Kobayashi et al. 2005). Here, we report on the expansion of the biocontrol range of L. enzymogenes C3 to summer patch disease caused by Magnaporthe poae. The clp- mutant strain 5E4 was reduced in its ability to suppress summer patch disease compared with the wild-type strain C3 and was completely devoid of antifungal activity towards M. poae. Furthermore, cell suspensions of 5E4 were incapable of colonizing M. poae mycelium in a manner that was distinct for C3. Strain C3 demonstrated biosurfactant activity in cell suspensions and culture filtrates that was associated with absorption into the mycelium during the colonization process, whereas 5E4 did not. These results describe a novel interaction between bacteria and fungi that intimates a pathogenic relationship.

Labyrinthula terrestris sp. nov., a new pathogen of turf grass.

A species of Labyrinthula that causes 'rapid blight' and death of turfgrass has been isolated and studied. We name this new species Labyrinthula terrestris and briefly summarize morphological characteristics and growth patterns of this pathogen of turfgrass.

The infection process of Colletotrichum graminicola and relative aggressiveness on four turfgrass species.

Detached 3-week-old leaves of Agrostis palustris, Lolium perenne, Poa annua, and Poa pratensis were inoculated with conidial suspensions of two isolates of Colletotrichum graminicola obtained from A. palustris. Inoculated leaves were incubated at 23 degrees C under high relative humidity (>95%). The infection process was investigated by light microscopy from 2 to 168 h after inoculation (AI). Spore germination was observed within 2 h AI, appressoria within 6 h AI, and penetration pores within 8 h AI on all four hosts. Infection hyphae were observed inside epidermal cells within 24 h AI on all four hosts, but significantly greater infection was observed in A. palustris and P. annua than in L. perenne or P. pratensis at both 96 and 120 h AI. Acervuli appeared on leaves of A. palustris at 72 h AI and on L. perenne at 96 h AI but were not found on either P. annua or P. pratensis during the first 168 h AI. The infection process was similar to that reported for C. graminicola from other hosts; however, disease development of the two isolates of C. graminicola from A. palustris was faster or fungal growth more extensive on detached leaf tissue of A. palustris than on other turfgrass species tested.

Soil community composition and the regulation of grazed temperate grassland.

The effect of the community composition of soil microbes on ecosystem processes has received relatively little attention. Here we examined the variation in soil microbial composition in a Yellowstone National Park grassland and the effect of that variation on the growth, in a greenhouse, of the dominant grass in the community. Plants and their rhizospheric soil were collected from paired, Poa pratensis-dominated grassland plots located inside and outside a 40-year-old exclosure. P. pratensis aboveground, belowground, and whole plant growth were greater in pots with soil communities from grazed grassland compared to fenced grassland, indicating (1) soil microbial communities differed, and (2) this difference influenced the growth of the plant that dominated both grasslands. Treating pots with fungicide (benomyl) suppressed the soil community influence, indicating that different fungal communities caused the soil microbe effect. In addition, two lines of evidence are consistent with the hypothesis that arbuscular mycorrhizal fungal (AMF) species composition affected P. pratensis: (1) a divergence in AMF spore communities in the two field soils, and (2) little evidence of pathogenic fungi. These findings emphasize the need to examine the role that the composition of the soil microbial community plays in controlling terrestrial ecosystems.