Phytophthora Species Detected in Two Ozark Forests with Unusual Patterns of White Oak Mortality.

Widespread decline and mortality of white oaks (Quercus alba) occurred in Missouri Ozark forests between 2011 and 2017. Symptoms included rapid crown death with bronzing of leaves, retention of dead leaves, crown dieback and thinning, and loss of large limbs within one year of death. Decline and mortality were associated with hillside drainages and fit descriptions of European oak forests predisposed to decline by pathogenic Phytophthora species. A survey was performed at two locations in 2014 and 2015 to assess the distribution of dead and declining white oaks, and the occurrence and distribution of Phytophthora species. Multiple Phytophthora species were detected, including P. cinnamomi, P. cactorum, P. europaea, and P. pini. P. cinnamomi was the most common and widely distributed species among plots at both locations. The detection of P. cinnamomi at the base of white oaks was not associated with poor crown vigor. However, more quantitative survey techniques are necessary to clearly evaluate this relationship. P. cinnamomi kills fine roots of white and red oaks in North America and has been associated with the decline of white oaks in the United States (Ohio) and other countries. Further studies are needed to determine the importance of P. cinnamomi in oak decline within the Ozark highlands.

Colonization of Artificially Stressed Black Walnut Trees by Ambrosia Beetle, Bark Beetle, and Other Weevil Species (Coleoptera: Curculionidae) in Indiana and Missouri.

Thousand cankers disease (TCD) is a new disease of black walnut (Juglans nigra L.) in the eastern United States. The disease is caused by the interaction of the aggressive bark beetle Pityophthorus juglandis Blackman and the canker-forming fungus, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley & Tisserat, carried by the beetle. Other insects also colonize TCD-symptomatic trees and may also carry pathogens. A trap tree survey was conducted in Indiana and Missouri to characterize the assemblage of ambrosia beetles, bark beetles, and other weevils attracted to the main stems and crowns of stressed black walnut. More than 100 trees were girdled and treated with glyphosate (Riverdale Razor Pro, Burr Ridge, Illinois) at 27 locations. Nearly 17,000 insects were collected from logs harvested from girdled walnut trees. These insects represented 15 ambrosia beetle, four bark beetle, and seven other weevil species. The most abundant species included Xyleborinus saxeseni Ratzburg, Xylosandrus crassiusculus Motschulsky, Xylosandrus germanus Blandford, Xyleborus affinis Eichhoff, and Stenomimus pallidus Boheman. These species differed in their association with the stems or crowns of stressed trees. Multiple species of insects were collected from individual trees and likely colonized tissues near each other. At least three of the abundant species found (S. pallidus, X. crassiusculus, and X. germanus) are known to carry propagules of canker-causing fungi of black walnut. In summary, a large number of ambrosia beetles, bark beetles, and other weevils are attracted to stressed walnut trees in Indiana and Missouri. Several of these species have the potential to introduce walnut canker pathogens during colonization.

Combinatorially selected peptides for protection of soybean against Phakopsora pachyrhizi.

Phakopsora pachyrhizi, the fungal pathogen that causes Asian soybean rust, has the potential to cause significant losses in soybean yield in many production regions of the United States. Germplasm with durable, single-gene resistance is lacking, and control of rust depends on timely application of fungicides. To assist the development of new modes of soybean resistance, we identified peptides from combinatorial phage-display peptide libraries that inhibit germ tube growth from urediniospores of P. pachyrhizi. Two peptides, Sp2 and Sp39, were identified that inhibit germ tube development when displayed as fusions with the coat protein of M13 phage or as fusions with maize cytokinin oxidase/dehydrogenase (ZmCKX1). In either display format, the inhibitory effect of the peptides on germ tube growth was concentration dependent. In addition, when peptides Sp2 or Sp39 in either format were mixed with urediniospores and inoculated to soybean leaves with an 8-h wetness period, rust lesion development was reduced. Peptides Sp2 and Sp39, displayed on ZmCKX1, were found to interact with a 20-kDa protein derived from germinated urediniospores. Incorporating peptides that inhibit pathogen development and pathogenesis into breeding programs may contribute to the development of soybean cultivars with improved, durable rust tolerance.

Subcellular localization and biochemical comparison of cytosolic and secreted cytokinin dehydrogenase enzymes from maize.

Cytokinin dehydrogenase (CKX; EC 1.5.99.12) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in plant cells. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the subcellular localization of ZmCKX10, and compare its biochemical properties with those of ZmCKX1. Expression profiling of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine their transcript abundance and organ-specific expression. For determination of the subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, the ZmCKX1-GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10-GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1-GFP formed significantly more mass in comparison with the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor and trans-zeatin, N(6)-(Delta(2)-isopentenyl)adenine (iP) and N(6)-(Delta(2)-isopentenyl)adenosine (iPR) as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates.

Combinatorially selected defense peptides protect plant roots from pathogen infection.

Agricultural productivity and sustainability are continually challenged by emerging and indigenous pathogens. Currently, many pathogens can be combatted only with biocides or environmentally dangerous fumigants. Here, we report a rapid and pathogen-specific strategy to reduce infection by organisms that target plant roots. Combinatorially selected defense peptides, previously shown to effect premature encystment of Phytophthora capsici zoospores, were fused to maize cytokinin oxidase/dehydrogenase as a display scaffold. When expressed in tomato roots, the peptide-scaffold constructs were secreted and accumulated to sufficient concentrations in the rhizosphere to induce zoospore encystment and thereby deter taxis to the root surface. Pathogen infection was significantly inhibited in roots expressing bioactive peptides fused to the maize cytokinin oxidase/dehydrogenase scaffold. This peptide-delivery technology is broadly applicable for rapid development of plant defense attributes against plant pathogens.

Tissue localization of cytokinin dehydrogenase in maize: possible involvement of quinone species generated from plant phenolics by other enzymatic systems in the catalytic reaction.

The degradation of cytokinins in plants is controlled by the flavoprotein cytokinin dehydrogenase (EC 1.5.99.12). Cytokinin dehydrogenase from maize showed the ability to use oxidation products of guaiacol, 4-methylcatechol, acetosyringone and several other compounds as electron acceptors. These results led us to explore the cability for indirect production of suitable electron acceptors by different quinone-generating enzymes. The results reported here revealed that the electron acceptors may be generated in vivo from plant phenolics by other enzymatic systems such as peroxidase and tyrosinase/laccase/catechol oxidase. Histochemical localization of cytokinin dehydrogenase by activity staining and immunochemistry using optical and confocal microscopy showed that cytokinin dehydrogenase is most abundant in the aleurone layer of maize kernels and in phloem cells of the seedling shoots. Cytokinin dehydrogenase was confirmed to be present in the apoplast of cells. Co-staining of enzyme activity for laccase, an enzyme poised to function on the cell wall in the apoplast, in those tissues suggests a possible cooperation of the enzymes in cytokinin degradation. Additionally, the presence of precursors for electron acceptors of cytokinin dehydrogenase was detected in phloem exudates collected from maize seedlings, suggestive of an enzymatic capacity to control cytokinin flux through the vasculature. A putative metabolic connection between cytokinin degradation and conversion of plant phenolics by oxidases was proposed.

Catalytic reaction of cytokinin dehydrogenase: preference for quinones as electron acceptors.

The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors.

Genetic diversity and virulence of Rhizoctonia species associated with plantings of Lotus corniculatus.

Species of Rhizoctonia cause a blight of Lotus corniculatus, a perennial forage legume. We characterized genetic variation and virulence in populations of R. solani and binucleate Rhizoctonia's associated with diseased L. corniculatus in field plantings over several years. Isolates of anastomosis groups AG-1 and AG-4 accounted for the R. solani recovered from diseased leaf and shoot tissues. Isolates of binucleate Rhizoctonia were recovered predominantly from soil and associated plant debris. Isolates of R. solani were more virulent on leaves and shoots of L. corniculatus than were binucleate Rhizoctonia isolates. Numerous unique DNA restriction patterns were observed among binucleate isolates and anastomosis groups of R. solani. Variation in restriction patterns was greater among isolates of AG-1 from the lower plant canopy than from the upper canopy. No restriction pattern was shared by any isolate from AG-1 and AG-4. Allelic and genotypic heterogeneity of AG-1 isolates were also greater in the lower plant canopy. Binucleate isolates exhibited greater heterogeneity than AG-1 isolates from either canopy region. L. corniculatus offers significant opportunities for investigating temporal and spatial dynamics of genetic structure of Rhizoctonia populations in perennial plant systems.

Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores.

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.