Ice nucleation active bacteria in precipitation are genetically diverse and nucleate ice by employing different mechanisms.

A growing body of circumstantial evidence suggests that ice nucleation active (Ice+) bacteria contribute to the initiation of precipitation by heterologous freezing of super-cooled water in clouds. However, little is known about the concentration of Ice+ bacteria in precipitation, their genetic and phenotypic diversity, and their relationship to air mass trajectories and precipitation chemistry. In this study, 23 precipitation events were collected over 15 months in Virginia, USA. Air mass trajectories and water chemistry were determined and 33 134 isolates were screened for ice nucleation activity (INA) at -8 °C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at -8 °C in repeated tests. Concentrations of Ice+ strains in precipitation were found to range from 0 to 13 219 colony forming units per liter, with a mean of 384±147. Most Ice+ bacteria were identified as members of known and unknown Ice+ species in the Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae families, which nucleate ice employing the well-characterized membrane-bound INA protein. Two Ice+ strains, however, were identified as Lysinibacillus, a Gram-positive genus not previously known to include Ice+ bacteria. INA of the Lysinibacillus strains is due to a nanometer-sized molecule that is heat resistant, lysozyme and proteinase resistant, and secreted. Ice+ bacteria and the INA mechanisms they employ are thus more diverse than expected. We discuss to what extent the concentration of culturable Ice+ bacteria in precipitation and the identification of a new heat-resistant biological INA mechanism support a role for Ice+ bacteria in the initiation of precipitation.

First Report of Reduced Sensitivity to a QoI Fungicide in Apple Scab (Venturia inaequalis) in Virginia and Maryland.

Apple scab caused by Venturia inaequalis (Cooke) Winter continues to be a significant concern for apple growers in Virginia and Maryland. Management of scab has relied on foliar fungicides including strobilurins (QoIs) such as trifloxystrobin (TFX). In recent years, populations of V. inaequalis with reduced sensitivity to the QoIs have been reported in other apple-growing regions of the United States (1,2). Although QoIs generally remain effective in the mid-Atlantic, concerns about the development of resistance in some Virginia and Maryland orchards prompted this study. Twenty-five isolates of V. inaequalis were obtained from scabby leaves from commercial and experimental orchards in Virginia in 2010 (n = 6) and 2011 (n = 14) and from a commercial orchard in Maryland (n = 5) in 2011. Orchards had previously been treated with QoI or sterol-inhibiting (SI) fungicides. Isolates of V. inaequalis were grown on potato dextrose agar (PDA) amended with 0, 0.1, or 1.0 µg ml-1 TFX with 100 µg ml-1 salicylhydroxamic acid (SHAM) and incubated at 19°C. Colony growth was measured weekly for 4 weeks. To account for the SI use at some orchards, isolates of V. inaequalis were also evaluated on PDA amended with 0, 0.5, or 1.0 µg ml-1 myclobutanil. Fungicide sensitivities were expressed as a percentage of the difference in colony growth using a discriminatory dose of 1.0 µg ml-1 TFX with SHAM or 1.0 µg ml-1 myclobutanil at 28 days. Isolates with <25% growth suppression (GS) were classified as fully resistant, whereas those with >70% GS were classified as sensitive. Isolates with 25 to 70% GS were classified as partially resistant. Effective concentration (EC50) values (TFX concentration inhibiting colony growth by 50%) were also calculated for a subset of fully resistant and sensitive isolates. Of the 25 isolates tested, six were fully resistant to TFX (mean EC50 value greater than 10.0 µg ml-1) and 10 were sensitive (mean EC50 value of 0.04 µg ml-1 ± 0.05 µg ml-1). Nine isolates were classified as partially resistant. Some isolates showed more than a 200-fold increase in resistance to TFX, and one isolate grew almost as well on 10.0 µg ml-1 TFX as on the unamended control (GS of 3%). Current-season use of QoIs on isolate source trees was significantly associated with a lack of sensitivity Ç2 (1) = 3.72 (P < 0.06). All six fully resistant isolates originated from QoI-treated commercial orchards, which had shown control failures. Seven of 10 isolates sensitive to QoIs originated from trees that had been treated with SIs during the isolation year. Resistance to myclobutanil was not significantly associated with resistance to TFX Ç2 (1) = 1.220 (P < 0.5), and only one isolate was resistant (i.e. >25% GS) to both. Despite the long history of QoI use at the experimental orchards, no isolates fully resistant to TFX were identified there. To our knowledge, this is the first report of V. inaequalis isolates with resistance to TFX in Virginia and Maryland. Since SI resistance has been documented in Virginia (3) and resistance to both the SI and QoI chemical classes is a concern in the mid-Atlantic region (4), tank-mixing or alternating QoIs with broad-spectrum fungicides with different modes of action is recommended. References: (1). K. M. Cox et al. Phythopathology 99:S25, 2009. (2). K. E. Lesniak et al. Plant Dis. 95:927, 2011. (3) S. C. Marine et al. Plant Health Progress. doi:10.1094/PHP-2007-1113-01-RS, 2007. (4) E. E. Pfeufer and H. K. Ngugi. Phytopathology 102:272, 2012.

Lagrangian coherent structures are associated with fluctuations in airborne microbial populations.

Many microorganisms are advected in the lower atmosphere from one habitat to another with scales of motion being hundreds to thousands of kilometers. The concentration of these microbes in the lower atmosphere at a single geographic location can show rapid temporal changes. We used autonomous unmanned aerial vehicles equipped with microbe-sampling devices to collect fungi in the genus Fusarium 100 m above ground level at a single sampling location in Blacksburg, Virginia, USA. Some Fusarium species are important plant and animal pathogens, others saprophytes, and still others are producers of dangerous toxins. We correlated punctuated changes in the concentration of Fusarium to the movement of atmospheric transport barriers identified as finite-time Lyapunov exponent-based Lagrangian coherent structures (LCSs). An analysis of the finite-time Lyapunov exponent field for periods surrounding 73 individual flight collections of Fusarium showed a relationship between punctuated changes in concentrations of Fusarium and the passage times of LCSs, particularly repelling LCSs. This work has implications for understanding the atmospheric transport of invasive microbial species into previously unexposed regions and may contribute to information systems for pest management and disease control in the future.

First Report of Cephalosporium gramineum, Causal Agent of Cephalosporium Stripe of Wheat, in a Commercial Winter Wheat Field in Virginia.

Cephalosporium stripe (CS) (2) was identified in a commercial field of winter wheat (Triticum aestivum) near Riner, Montgomery County, Virginia in May 2006. Nearly 15% of the field was severely affected. Broad, yellow-brown stripes were observed on the leaf blades of affected plants, and many plants were stunted and had ripened prematurely. Symptomatic plants were associated with low acidic (pH 5.2), wet spots of the field. Leaves and nodes of affected plants were surface disinfested for 1 min in 5% sodium hypochlorite, plated on corn meal agar (CMA), and incubated at 20°C for 5 days. Cephalosporium gramineum was isolated from numerous plants. Cultures of the fungus produced hyaline conidiophores approximately 5 µm long and unicellular conidia 3 to 7 µm long. Aqueous suspensions of mycelia and conidia were prepared from pure cultures. Several spring wheat cultivars were wounded by severing the root mass and were inoculated when the fifth stem node was detectable (35 on Zadoks scale). Noninoculated plants were wounded as controls. Plants were kept in the greenhouse at temperatures of 22 to 27°C. After 14 days, inoculated plants produced symptoms of CS, and the fungus was reisolated from the leaves of these plants. No symptoms were observed on noninoculated control plants. Though CS had been observed in Virginia in research nurseries (1), to our knowledge, this is the first confirmed report of the disease in a commercial wheat field in Virginia. References: (1) J. B. Jones et al. Plant Dis. 64:325, 1980. (2) C. M. Stiles and T. D. Murray. Phytopathology 86:177, 1996.