An Improved Detached-Leaf Assay for Phenotyping Net Blotch of Barley Caused by Pyrenophora teres

Net blotch, caused by Pyrenophora teres, is a major barley (Hordeum vulgare) leaf disease worldwide. P. teres occurs as two forms—P. teres f. teres, and P. teres f. maculata—inducing net and spot-like symptoms, respectively. An intact-seedling assay, where entire seedlings are inoculated by spraying with a conidial suspension, is frequently used for phenotyping net blotch. However, this presents a biosecurity risk in the glasshouse when nonlocal isolates are being screened. Alternatively, a detached-leaf assay (DLA-droplet method) can be used in which leaf segments laid out in a covered tray are inoculated with droplets of a conidial suspension, confining the inoculum. However, using this method, net and spot form symptoms cannot be distinguished from each other. We have developed an improved DLA (DLA-spray method) in which detached whole leaves are sprayed with the inoculum to produce distinct lesions. We compare the results for the three phenotyping methods above using four isolates from both net and spot forms of the disease to inoculate a standard set of eight barley genotypes. Results indicate that the DLA-spray method is a functional, informative and rapid test that readily differentiates the two forms of the pathogen in a biosecure environment.

A comparison of analytical approaches for assessing freezing damage in black spruce using electrolyte leakage methods

To compare different methods of quantifying shoot frost damage during controlled plant freezing tests, frost hardening of black spruce (Picea mariana (Mill.) BSP) seedlings exposed to three temperature hardening regimes over 16 weeks was assessed using electrolyte leakage and intact seedling methods. Electrolyte leakage was expressed as index of injury and was quantified either as the temperature needed to induce an index of injury of 5% (DT5) or as the critical temperature (CT), the mildest temperature at which damage was first detected statistically. Damage to intact shoots was expressed as percent shoot browning and was quantified as the temperature at which 50% of needle tissue on the shoots was damaged (sLT50) or as the temperature at which 50% of terminal buds were killed (bLT50). Seedling response to hardening temperature varied, depending on the method used to quantify frost hardiness. When expressed as critical temperature, hardening continued over the 16 weeks at a constant rate with no differences detected between treatments. Intact seedling shoot damage, sLT50 and bLT50, described a hardening process in which there was a large initial increase in hardening in the first 8 weeks, with less hardening occurring during the subsequent 8 weeks. Also, significant temperature effects were detected, with the greatest hardening occurring in a cool temperature (4 °C), the least in a warm temperature (20 °C), and an intermediate amount in a moderate temperature (10 °C). When quantified as DT5, the pattern of hardening was somewhat intermediate to the other two. Methods of determining frost hardiness were highly correlated, with the strongest correlation being between sLT50 and bLT50 (r2 = 0.903). Both electrolyte leakage methods, DT5 and CT, were linearly related to one another (r2 = 0.666) and were more sensitive than the intact seedling methods, since they both detected damage at warmer temperatures. DT5 was better correlated to intact measures of hardiness than was CT. Keywords: black spruce, index of injury, frost hardiness, critical temperature, damaging temperature, LT50.