Cloning of the Cytochrome b Gene From the Tomato Powdery Mildew Fungus Leveillula taurica Reveals High Levels of Allelic Variation and Heteroplasmy for the G143A Mutation.

Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (QoIs) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica (Lt cytb) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to QoIs. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against QoIs in several fungi. The other four amino acid substitutions were novel mutations, whose effect on QoI resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that QoI resistance in this fungus is likely to be quantitative rather than qualitative.

Comparative Genomics of the Sigatoka Disease Complex on Banana Suggests a Link between Parallel Evolutionary Changes in Pseudocercospora fijiensis and Pseudocercospora eumusae and Increased Virulence on the Banana Host.

The Sigatoka disease complex, caused by the closely-related Dothideomycete fungi Pseudocercospora musae (yellow sigatoka), Pseudocercospora eumusae (eumusae leaf spot), and Pseudocercospora fijiensis (black sigatoka), is currently the most devastating disease on banana worldwide. The three species emerged on bananas from a recent common ancestor and show clear differences in virulence, with P. eumusae and P. fijiensis considered the most aggressive. In order to understand the genomic modifications associated with shifts in the species virulence spectra after speciation, and to identify their pathogenic core that can be exploited in disease management programs, we have sequenced and analyzed the genomes of P. eumusae and P. musae and compared them with the available genome sequence of P. fijiensis. Comparative analysis of genome architectures revealed significant differences in genome size, mainly due to different rates of LTR retrotransposon proliferation. Still, gene counts remained relatively equal and in the range of other Dothideomycetes. Phylogenetic reconstruction based on a set of 46 conserved single-copy genes strongly supported an earlier evolutionary radiation of P. fijiensis from P. musae and P. eumusae. However, pairwise analyses of gene content indicated that the more virulent P. eumusae and P. fijiensis share complementary patterns of expansions and contractions in core gene families related to metabolism and enzymatic degradation of plant cell walls, suggesting that the evolution of virulence in these two pathogens has, to some extent, been facilitated by convergent changes in metabolic pathways associated with nutrient acquisition and assimilation. In spite of their common ancestry and shared host-specificity, the three species retain fairly dissimilar repertoires of effector proteins, suggesting that they likely evolved different strategies for manipulating the host immune system. Finally, 234 gene families, including seven putative effectors, were exclusively present in the three Sigatoka species, and could thus be related to adaptation to the banana host.

Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector.

Effects of manure-application practices on curli production by Escherichia coli transported through soil.

The release of Escherichia coli into the environment from untreated manure can pose a threat to human health. Environmental survival of E. coli has been linked to extracellular fibers called curli. We investigated the effect of manure management (surface application followed by incorporation versus immediate incorporation) on the relative abundance of curli-producing E. coli in subsurface drainage effluent. Samples were collected from three dairy farms. The proportion of curli-producing E. coli in the manure storage facilities was uniform across the farms. However, the abundance of curli-producing E. coli was much greater (P < 0.05) in the tile drains of farms performing surface application of manure than in the tile drain of the farm that incorporated manure. This field observation was tested with controlled soil column experiments; the abundance of curli-producing E. coli in soil column effluents was greater (P < 0.05) when manure was surface-applied than when it was incorporated. Our findings suggest selection pressures resulting from the different manure application methods affected curli production by E. coli isolates transported through soil. Given the importance of curli production in pathogenesis, this work highlights the effect that manure management strategies may have on pathogenesis-associated phenotypes of bacteria in agricultural subsurface runoff.

Colloid transport and retention in unsaturated porous media: effect of colloid input concentration.

Colloids play an important role in facilitating transport of adsorbed contaminants in soils. Recent studies showed that under saturated conditions colloid retention was a function of its concentration. It is unknown if this is the case under unsaturated conditions. In this study, the effect of colloid concentration on colloid retention was investigated in unsaturated columns by increasing concentrations of colloid influents with varying ionic strength. Colloid retention was observed in situ by bright field microscopy and quantified by measuring colloid breakthrough curves. In our unsaturated experiments, greater input concentrations resulted in increased colloid retention at ionic strength above 0.1 mM, but not in deionized water (i.e., 0 mM ionic strength). Bright field microscope images showed that colloid retention mainly occurred at the solid-water interface and wedge-shaped air-water-solid interfaces, whereas the retention at the grain-grain contacts was minor. Some colloids at the air-water-solid interfaces were rotating and oscillating and thus trapped. Computational hydrodynamic simulation confirmed that the wedge-shaped air-water-solid interface could form a "hydrodynamic trap" by retaining colloids in its low velocity vortices. Direct visualization also revealed that colloids once retained acted as new retention sites for other suspended colloids at ionic strength greater than 0.1 mM and thereby could explain the greater retention with increased input concentrations. Derjaguin-Landau-Verwey-Overbeek (DLVO) energy calculations support this concept. Finally, the results of unsaturated experiments were in agreement with limited saturated experiments under otherwise the same conditions.