Standard Area Diagrams for Pecan Leaf Scab: Effect of Rater Experience, Location, and Leaf Size on Reliability and Accuracy of Visual Estimates.

Assessments of the severity of scab (Venturia effusa), an economically significant disease of pecan, are critical for determining pecan cultivar susceptibility, disease epidemiology, and integrated disease management approaches. We developed a standard area diagram (SAD) set to aid in assessments of pecan leaflet scab. Leaflets with scab lesions were harvested and scanned using a flatbed scanner at 600 dpi, and Fiji (ImageJ) was used to determine the actual percent disease severity. The SADs had 10 leaflets ranging in severity from 0.2 to 48.9%. Forty "small" (1.34 to 7.43 cm2) and 40 "large" (7.67 to 25.9 cm2) leaflet images were randomized for rater assessments. The images were assessed twice by 36 raters, first without and then with the SADs as a guide. Data were subjected to analysis using Lin's concordance correlation coefficient (LCC, pc) to determine the accuracy of ratings and by intraclass correlation coefficient (ICC) analysis to determine interrater reliability. The effects of rater experience, rater location, and leaflet size were also determined. The SADs significantly improved the agreement between raters and the actual values (LCC, pc = 0.70 and 0.84 without and with the SADs, respectively). The reliability of estimates was improved (ICC = 0.54 and 0.82 without and with the SADs, respectively). The effect of rater location on overall concordance was significant without and with the SADs based on an analysis of variance using a generalized linear model and lsmeans separation (P < 0.05). A generalized linear mixed model analysis revealed that there was a significant interaction between rater location, experience, and the use of the SADs, with some raters having greater improvement in generalized bias and concordance. Raters had a significantly better accuracy when rating "small" leaves (LCC, pc = 0.86) compared with "large" leaves (LCC, pc = 0.82) when using the SADs, highlighting the impact of psychophysics on field evaluations of plant disease severity. The proposed SADs will serve as an improved tool for performing pecan leaflet scab assessments by the pecan research community.

Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans.

Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

Evidence for Seed Transmission of Xylella fastidiosa in Pecan (Carya illinoinensis).

Pecan bacterial leaf scorch, caused by Xylella fastidiosa subsp. multiplex, is an economically significant disease of pecan with known detrimental effects on the yield of susceptible cultivars. In this study, endosperm was harvested from developing pecan seeds, and direct qPCR and sequencing were used to detect and confirm the presence of X. fastidiosa. DNA was isolated from mature seeds originating from seven trees, revealing a positivity rate up to 90%, and transmission of X. fastidiosa from infected seed to the germinated seedlings was found to be over 80%. Further epidemiological analyses were performed to determine where X. fastidiosa localizes in mature seed and seedlings. The highest concentrations of X. fastidiosa DNA were found in the hilum and outer integument of the seeds and the petioles, respectively. High-, medium-, and low-density seeds were harvested to determine the impact of the bacterium on seed density and seedling growth rate. The growth rate of seedlings originating from low-density seeds was significantly reduced compared to the medium- and high-density seeds. Despite the increased growth and germination rates, the high-density seed group had a greater proportion of samples that tested positive for the presence of X. fastidiosa by qPCR. The results demonstrate the ability of X. fastidiosa to colonize developing seeds and be efficiently transmitted from well-developed seeds to germinated seedlings. Continued research is needed to understand the plant-microbe interactions involved in the colonization of pecan seeds by X. fastidiosa and to develop effective phytosanitary approaches to reduce the risks posed by seed transmission.

Thermal treatment using microwave irradiation for the phytosanitation of Xylella fastidiosa in pecan graftwood.

Pecan bacterial leaf scorch caused by Xylella fastidiosa is an emerging disease for the U.S. and international pecan industries and can be transmitted from scion to rootstock via grafting. With the expanse of global transportation and trade networks, phytosanitation is critical for reducing the spread of economically significant pathogens, such as X. fastidiosa. We developed and evaluated thermal treatments using microwave irradiation and microwave absorbers [sterile deionized water (dH2O) and carbon nanotubes (CNTs)] as novel disinfectant methods for remediating X. fastidiosa in pecan scions. Partial submergence of scions in dH2O or CNT dispersions resulted in the transport of microwave absorbers in the xylem tissue via transpiration but did not compromise plant health. The microwave absorbers effectively transferred heat to the scion wood to reach an average temperature range of 55-65°C. Microwave radiation exposure for 6 sec (3 sec for two iterations) of CNT- or dH2O-treated scions reduced the frequency of X. fastidiosa-positive in pecan scions without negatively affecting plant viability when compared to the control group (dH2O-treated with no microwave). The efficacy of the new thermal treatments based on microwave irradiation was comparable to the conventional hot-water treatment (HWT) method, in which scions were submerged in 46°C water for 30 min. Microwave irradiation can be employed to treat X. fastidiosa-infected scions where the conventional HWT treatment is not feasible. This study is the first report to demonstrate novel thermal treatment methods based on the microwave irradiation and microwave absorbers of dH2O and CNT as an application for the phytosanitation of xylem-inhabiting bacteria in graftwood.

Identification and Characterization of Pathogenic and Endophytic Fungal Species Associated with Pokkah Boeng Disease of Sugarcane.

Pokkah Boeng is a serious disease of sugarcane, which can lead to devastating yield losses in crop-producing regions, including southern China. However, there is still uncertainty about the causal agent of the disease. Our aim was to isolate and characterize the pathogen through morphological, physiological, and molecular analyses. We isolated sugarcane-colonizing fungi in Fujian, China. Isolated fungi were first assessed for their cell wall degrading enzyme capabilities, and five isolates were identified for further analysis. Internal transcribed spacer sequencing revealed that these five strains are Fusarium, Alternaria, Phoma, Phomopsis, and Epicoccum. The Fusarium isolate was further identified as F. verticillioides after Calmodulin and EF-1α gene sequencing and microscopic morphology study. Pathogenicity assay confirmed that F. verticillioides was directly responsible for disease on sugarcane. Co-inoculation of F. verticillioides with other isolated fungi did not lead to a significant difference in disease severity, refuting the idea that other cellulolytic fungi can increase disease severity as an endophyte. This is the first report characterizing pathogenic F. verticillioides on sugarcane in southern China.