Phase-change-mediated transport and agglomeration of fungal spores on wheat awns.

Wheat and other staple crops are devastated by fungal diseases. Many fungal plant pathogens are spread via active or passive discharge of microscopic spores. Here, we described the unique transport of spores of the fungal pathogen Epicoccum tritici, causal agent of black sooty mould, on wheat awns. The unique multi-scale architecture of wheat awns, coupled with condensation and evaporation of dew droplets, facilitated the transport and agglomeration of spores of the fungus. First, dew droplets spontaneously transported spores from the tips of awn hairs to the neighbouring stomatal ridges, driven by gradients in Laplace pressure and surface wettability. Subsequently, spores agglomerated into dry clusters due to the Cheerios effect and evaporation, increasing the likelihood of passive spore removal via wind shear and/or rainsplash. Future plant breeding approaches should consider the development of modified spike structures, such as those without awns or awn hairs, to reduce the potential for spread of fungal plant pathogens.

Synergistic dispersal of plant pathogen spores by jumping-droplet condensation and wind.

Plant pathogens are responsible for the annual yield loss of crops worldwide and pose a significant threat to global food security. A necessary prelude to many plant disease epidemics is the short-range dispersal of spores, which may generate several disease foci within a field. New information is needed on the mechanisms of plant pathogen spread within and among susceptible plants. Here, we show that self-propelled jumping dew droplets, working synergistically with low wind flow, can propel spores of a fungal plant pathogen (wheat leaf rust) beyond the quiescent boundary layer and disperse them onto neighboring leaves downwind. An array of horizontal water-sensitive papers was used to mimic healthy wheat leaves and showed that up to 25 spores/h may be deposited on a single leaf downwind of the infected leaf during a single dew cycle. These findings reveal that a single dew cycle can disperse copious numbers of fungal spores to other wheat plants, even in the absence of rain splash or strong gusts of wind.

Dynamics of splashed droplets impacting wheat leaves treated with a fungicide.

Wheat is threatened by diseases such as leaf rust. One significant mechanism of disease spread is the liberation and dispersal of rust spores due to rainsplash. However, it is unclear to what extent the spore-laden splashed droplets can transmit the disease to neighbouring leaves. Here, we show that splashed droplets either bounce or stick, depending on the orientation of the leaf and whether the surface of the leaf has been treated with a fungicide. A scaling model revealed that bouncing was enabled when the droplet's kinetic energy exceeded its pinning energy to the surface. Our findings indicate that, ironically, the application of fungicide to protect a wheat plant may also facilitate pathogen spread and infection by making leaves sticky to spore-laden droplets.

'Sneezing' plants: pathogen transport via jumping-droplet condensation.

We show that condensation growing on wheat leaves infected with the leaf rust fungus, Puccinia triticina, is capable of spontaneously launching urediniospores off the plant. This surprising liberation mechanism is enabled by the superhydrophobicity of wheat leaves, which promotes a jumping-droplet mode of condensation powered by the surface energy released from coalescence events. We found that urediniospores often adhere to the self-propelled condensate, resulting in liberation rates of approximately 10 cm-2 h-1 for leaves infected with rust. Urediniospores were catapulted up to 5 mm from the leaf's surface, a distance sufficient to clear the laminar boundary layer for subsequent dispersal even in gentle winds.

Vortex-induced dispersal of a plant pathogen by raindrop impact.

Raindrop impact on infected plants can disperse micron-sized propagules of plant pathogens (e.g., spores of fungi). Little is known about the mechanism of how plant pathogens are liberated and transported due to raindrop impact. We used high-speed photography to observe thousands of dry-dispersed spores of the rust fungus Puccinia triticina being liberated from infected wheat plants following the impact of a single raindrop. We revealed that an air vortex ring was formed during the raindrop impact and carried the dry-dispersed spores away from the surface of the host plant. The maximum height and travel distance of the airborne spores increased with the aid of the air vortex. This unique mechanism of vortex-induced dispersal dynamics was characterized to predict trajectories of spores. Finally, we found that the spores transported by the air vortex can reach beyond the laminar boundary layer of leaves, which would enable the long-distance transport of plant pathogens through the atmosphere.

Differential detection of a surrogate biological threat agent (Bacillus globigii) with a portable surface plasmon resonance biosensor.

New methods and technology are needed to quickly and accurately detect potential biological warfare agents, such as Bacillus anthracis, causal agent of anthrax in humans and animals. Here, we report the detection of a simulant of B. anthracis (B. globigii) alone and in a mixture with a different species of Bacillus to test non-specific interference using a portable surface plasmon resonance (SPR) biosensor (SPIRIT 4.0, Seattle Sensor Systems). Both direct capture and antibody amplification were used to determine the limit of detection for spores of B. globigii, and to detect spores of B. globigii in a mixed sample containing another Bacillus spp. Spores of B. globigii were detected by anti-B. globigii (anti-Bg) coated sensors by direct capture at a concentration of 10(7)spores/mL, and with a secondary antibody amplification at a concentration of 10(5)spores/mL. Spores of B. globigii were differentially detected in a 1:1 mixture with B. pumilus spores from equal concentrations (10(7)spores/mL) with a secondary antibody amplification. To our knowledge, this is the first report of the differential detection of B. globigii with SPR in a mixed sample containing at least one additional Bacillus spp., highlighting the potential for SPR to detect any target bacterium in a mixed sample of closely related species. With the availability of portable instrumentation to accurately detect biological warfare agents such as B. anthracis, emergency responders can implement protocols in a timely fashion, limiting the amount of exposed individuals.

AMR1, an Arabidopsis gene that coordinately and negatively regulates the mannose/l-galactose ascorbic acid biosynthetic pathway.

Ascorbic acid (AsA) biosynthesis in plants occurs through a complex, interconnected network with mannose (Man), myoinositol, and galacturonic acid as principal entry points. Regulation within and between pathways in the network is largely uncharacterized. A gene that regulates the Man/l-galactose (l-Gal) AsA pathway, AMR1 (for ascorbic acid mannose pathway regulator 1), was identified in an activation-tagged Arabidopsis (Arabidopsis thaliana) ozone-sensitive mutant that had 60% less leaf AsA than wild-type plants. In contrast, two independent T-DNA knockout lines disrupting AMR1 accumulated 2- to 3-fold greater foliar AsA and were more ozone tolerant than wild-type controls. Real-time reverse transcription-polymerase chain reaction analysis of steady-state transcripts of genes involved in AsA biosynthesis showed that AMR1 negatively affected the expression of GDP-Man pyrophosphorylase, GDP-l-Gal phosphorylase, l-Gal-1-phosphate phosphatase, GDP-Man-3',5'-epimerase, l-Gal dehydrogenase, and l-galactono-1,4-lactone dehydrogenase, early and late enzymes of the Man/l-Gal pathway to AsA. AMR1 expression appears to be developmentally and environmentally controlled. As leaves aged, AMR1 transcripts accumulated with a concomitant decrease in AsA. AMR1 transcripts also decreased with increased light intensity. Thus, AMR1 appears to play an important role in modulating AsA levels in Arabidopsis by regulating the expression of major pathway genes in response to developmental and environmental cues.

An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate.

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.

High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics.

Fragaria vesca L., a diploid (2n = 2x = 14) relative of the commercial octoploid strawberry, is an attractive model for functional genomics research in Rosaceae. Its small genome size, short reproductive cycle, and facile vegetative and seed propagation make F. vesca a promising candidate for forward and reverse genetics experiments. However, the lack of a high-efficiency transformation protocol required for systematic production of thousands of T-DNA insertional mutant lines and high-throughput gene validation is a major bottleneck. We describe a new transformation procedure that uses leaf explants from newly unfolded trifoliate leaves obtained from stock plants 6-7 weeks after seed germination, co-cultivation with Agrobacterium strain GV3101, and stringent selection on MS medium containing 4 mg l(-1) hygromycin. Using this protocol we achieved 100% transformation efficiency for 6 of 14 F. vesca accessions tested. Accession PI 551572 was determined to be the best candidate for a model in F. vesca functional genomics research, as it showed the greatest propensity for callus formation, transformation, shoot regeneration, ex vitro establishment, and plant growth, requiring only 14-15 weeks to complete its life cycle in different seasons in the greenhouse.

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.