Gary C. Bergstrom

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

BackgroundThe discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.ResultsLarge-scale screening identified a range of hydrolytic activities among 348 unique isolates representing 156 species of plant pathogenic and non-pathogenic fungi. Hierarchical clustering was used to identify groups of species with similar hydrolytic profiles. Among moderately and highly active species, plant pathogenic species were found to be more active than non-pathogens on six of eight substrates tested, with no significant difference seen on the other two substrates. Among the pathogenic fungi, greater hydrolysis was seen when they were tested on biomass and hemicellulose derived from their host plants (commelinoid monocot or dicot). Although T. reesei has a hydrolytic profile that is highly active on cellulose and pretreated biomass, it was less active than some natural isolates of fungi when tested on xylans and untreated biomass.ConclusionsSeveral highly active isolates of plant pathogenic fungi were identified, particularly when tested on xylans and untreated biomass. There were statistically significant preferences for biomass type reflecting the monocot or dicot host preference of the pathogen tested. These highly active fungi are promising targets for identification and characterization of novel cell wall degrading enzymes for industrial applications.

An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts.

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose‐ or switchgrass‐containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5‐dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high‐throughput bioprospecting for new sources of novel enzymes for biofuel production. Biotechnol. Bioeng. 2009;102: 1033–1044.

Genetic Structure of Atmospheric Populations of Gibberella zeae

ABSTRACT Gibberella zeae, causal agent of Fusarium head blight (FHB) of wheat and barley and Gibberella ear rot (GER) of corn, may be transported over long distances in the atmosphere. Epidemics of FHB and GER may be initiated by regional atmospheric sources of inoculum of G. zeae; however, little is known about the origin of inoculum for these epidemics. We tested the hypothesis that atmospheric populations of G. zeae are genetically diverse by determining the genetic structure of New York atmospheric populations (NYAPs) of G. zeae, and comparing them with populations of G. zeae collected from seven different states in the northern United States. Viable, airborne spores of G. zeae were collected in rotational (lacking any apparent within-field inoculum sources of G. zeae) wheat and corn fields in Aurora, NY in May through August over 3 years (2002 to 2004). We evaluated 23 amplified fragment length polymorphism (AFLP) loci in 780 isolates of G. zeae. Normalized genotypic diversity was high (ranging from 0.91 to 1.0) in NYAPs of G. zeae, and nearly all of the isolates in each of the populations represented unique AFLP haplotypes. Pairwise calculations of Neis unbiased genetic identity were uniformly high (>0.99) for all of the possible NYAP comparisons. Although the NYAPs were genotypically diverse, they were genetically similar and potentially part of a large, interbreeding population of G. zeae in North America. Estimates of the fixation index (G(ST)) and the effective migration rate (Nm) for the NYAPs indicated significant genetic exchange among populations. Relatively low levels of linkage disequilibrium in the NYAPs suggest that outcrossing is common and that the populations are not a result of a recent bottleneck or invasion. When NYAPs were compared with those collected across the United States, the observed genetic identities between the populations ranged from 0.92 to 0.99. However, there was a significant negative correlation (R = -0.59, P < 0.001) between genetic identity and geographic distance, suggesting that some genetic isolation may occur on a continental scale. The contribution of long-distance transport of G. zeae to regional epidemics of FHB and GER remains unclear, but the diverse atmospheric populations of G. zeae suggest that inoculum may originate from multiple locations over large geographic distances. Practically, the long-distance transport of G. zeae suggests that management of inoculum sources on a local scale, unless performed over extensive production areas, will not be completely effective for the management of FHB and GER.

Daily inoculum levels of Gibberella zeae on wheat spikes

The inoculum level of Gibberella zeae on wheat spikes was measured during 1995 and 1996 in nine locations of Canada and the United States prone to Fusarium head blight of wheat. Spikes were exposed after exsertion and until kernel milk or soft dough stage in fields with wheat or corn residue as a source of inoculum; other spikes were exposed in a location remote from any obvious inoculum source; and in 1995 only, control plants remained in a greenhouse. After 24 h, spikes were excised and vigorously shaken in water to remove inoculum. Propagules were enumerated on selective medium and identified as G. zeae from subcultures. Significantly more inoculum was detected from fields in epidemic areas than from remote sites in an epidemic and from fields in nonepidemic areas. The median inoculum level was 20 CFU of G. zeae per spike per day in fields experiencing an epidemic, 4 CFU in locations remote from epidemic fields, 2 CFU in nonepidemic fields, and 1 CFU in locations remote from a source of inoculum in non-epidemic areas. In an epidemic region, inoculum levels near corn stubble reached up to 587 CFU of G. zeae per spike per day, and the median inoculum level of 126 CFU was significantly higher than the median of 13 CFU found near wheat residue. Inoculum was not detected or occurred sporadically during extended dry periods. While inoculum increased during rainy periods, timing of increased levels was variable. Fusarium head blight epidemics were associated with multiple inoculation episodes and coincident wet periods.

Fusarium head blight.

The first symptoms of Fusarium head blight include a tan or brown discoloration at the base of a floret within the spikelets of the head. As the infection progresses, the diseased spikelets become light tan or bleached in appearance (Figure 1). The infection may be limited to one spikelet, but if the fungus invades the rachis the entire head may develop symptoms of the disease. The base of the infected spikelets and portions of the rachis often develop a dark brown color. When weather conditions have been favorable for pathogen reproduction, the fungus may produce small orange clusters of spores or black reproductive structures called perithecia on the surface of the glumes. Infected kernels are often shriveled, white, and chalky in appearance (Figure 2). In some cases, the diseased kernels may develop a red or pink discoloration.

Efficacy and Stability of Integrating Fungicide and Cultivar Resistance to Manage Fusarium Head Blight and Deoxynivalenol in Wheat

Integration of host resistance and prothioconazole + tebuconazole fungicide application at anthesis to manage Fusarium head blight (FHB) and deoxynivalenol (DON) in wheat was evaluated using data from over 40 trials in 12 U.S. states. Means of FHB index (index) and DON from up to six resistance class-fungicide management combinations per trial (susceptible treated [S_TR] and untreated [S_UT]; moderately susceptible treated [MS_TR] and untreated [MS_UT]; moderately resistant treated [MR_TR] and untreated [MR_UT]) were used in multivariate meta-analyses, and mean log response ratios across trials were estimated and transformed to estimate mean percent control ( ) due to the management combinations relative to S_UT. All combinations led to a significant reduction in index and DON (P < 0.001). MR_TR was the most effective combination, with a of 76% for index and 71% for DON, followed by MS_TR (71 and 58%, respectively), MR_UT (54 and 51%, respectively), S_TR (53 and 39%, respectively), and MS_UT (43 and 30%, respectively). Calculations based on the principle of treatment independence showed that the combination of fungicide application and resistance was additive in terms of percent control for index and DON. Management combinations were ranked based on percent control relative to S_UT within each trial, and nonparametric analyses were performed to determine management combination stability across environments (trials) using the Kendall coefficient of concordance (W). There was a significant concordance of management combinations for both index and DON (P < 0.001), indicating a nonrandom ranking across environments and relatively low variability in the within-environment ranking of management combinations. MR_TR had the highest mean rank (best control relative to S_UT) and was one of the most stable management combinations across environments, with low rank stability variance (0.99 for index and 0.67 for DON). MS_UT had the lowest mean rank (poorest control) but was also one of the most stable management combinations. Based on Piephos nonparametric rank-based variance homogeneity U test, there was an interaction of management combination and environment for index (P = 0.011) but not for DON (P = 0.147), indicating that the rank ordering for index depended somewhat on environment. In conclusion, although the magnitude of percent control will likely vary among environments, integrating a single tebuconazole + prothioconazole application at anthesis with cultivar resistance will be a more effective and stable management practice for both index and DON than either approach used alone.

Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum.

Mating type idiomorphs (MAT1-1 and MAT1-2) were identified from the heterothallic loculoascomycete Phaeosphaeria nodorum (wheat biotype) using DNA from a pair of isolates from Poland and Georgia, USA that are known to mate. MAT predicted proteins of P. nodorum are similar in sequence and in phylogenetic relationship to those described for other loculoascomycetes such as Cochliobolus spp., Alternaria alternata, and Didymella zeae-maydis. The organization of the MAT locus of the P. nodorum differs from these species in that its idiomorph begins within an adjacent upstream conserved ORF of unknown function. MAT-specific primers were used to identify isolates of both mating types in field populations, demonstrating that an absence of either mating type is not the reason that the teleomorph has not been found in New York. Portions of MAT1-1 and MAT1-2 were sequenced from geographically diverse isolates, including those from regions where the teleomorph has been reported. MAT was highly conserved and no significant differences in sequence were found.

Plant pathogens as a source of diverse enzymes for lignocellulose digestion

The plant cell wall is a major barrier that many plant pathogens must surmount for successful invasion of their plant hosts. Full genome sequencing of a number of plant pathogens has revealed often large, complex, and redundant enzyme systems for degradation of plant cell walls. Recent surveys have noted that plant pathogenic fungi are highly competent producers of lignocellulolytic enzymes, and their enzyme activity patterns reflect host specificity. We propose that plant pathogens may contribute to biofuel production as diverse sources of accessory enzymes for more efficient conversion of lignocellulose into fermentable sugars.

Spatial patterns of viable spore deposition of Gibberella zeae in wheat fields

ABSTRACT An increased understanding of the epidemiology of Gibberella zeae will contribute to a rational and informed approach to the management of Fusarium head blight (FHB). An integral phase of the FHB cycle is the deposition of airborne spores, yet there is no information available on the spatial pattern of spore deposition of G. zeae above wheat canopies. We examined spatial patterns of viable spore deposition of G. zeae over rotational (lacking cereal debris) wheat fields in New York in 2002 and 2004. Viable, airborne spores (ascospores and macroconidia) of G. zeae were collected above wheat spikes on petri plates containing a selective medium and the resulting colonies were counted. Spores of G. zeae were collected over a total of 68 field environments (three wheat fields during 54 day and night sample periods over 2 years) from spike emergence to kernel milk stages of local wheat. Spatial patterns of spore deposition were visualized by contour plots of spore counts over entire fields. The spatial pattern of spore deposition was unique for each field environment during each day and night sample period. Spore deposition patterns during individual sample periods were classified by spatial analysis by distance indices (SADIE) statistics and Mantel tests. Both analyses indicated that the majority (93%) of the spore deposition events were random, with the remainder being aggregated. All of the aggregated patterns were observed during the night. Observed patterns of spore deposition were independent of the mean number of viable spores deposited during individual sample periods. The spatial pattern for cumulative spore deposition during anthesis in both years became aggregated over time. Contour maps of daily and cumulative spore deposition could be compared with contour maps of FHB incidence to gain insights into inoculum thresholds and the timing of effective inoculum for infection.

Inheritance of partial resistance to powdery mildew in spring wheat

SummaryFour spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.