A detached leaf assay to rapidly screen for resistance of maize to Bipolaris maydis, the causal agent of southern corn leaf blight

Southern corn leaf blight (SCLB), caused by the fungus Bipolaris maydis, is a disease that significantly affects maize productivity across the globe. A detached leaf assay (DLA) was developed to rapidly assess maize resistance to SCLB. Several experiments were conducted to: (i) identify a highly virulent B. maydis isolate; and to determine the most appropriate (ii) phytohormone to maintain viability of maize leaf tissue, (iii) leaf age for the assay, and (iv) inoculum concentration. Once optimized, the DLA was compared with screenhouse and field experiments. Use of DLA required a maximum of 28 days for resistance assessment, in contrast to screenhouse and field tests at a minimum of 33 and 72 days, respectively. DLA positively correlated with screenhouse (r = 0.48, P = 0.08) and field experiments (r = 0.68, P = 0.008). Assessments of diverse B. maydis strains and host genotypes indicated that the DLA could be used to detect both highly virulent SCLB strains and highly resistant maize genotypes. Here we report that DLA is a rapid, reliable technique to screen maize resistance to SCLB. Use of this tool in maize breeding programs can speed up the process of identification of sources of resistance to multiple variants of SCLB.

Quantifying the Effects of a G137S Substitution in the Cytochrome bc1 of Venturia effusa on Azoxystrobin Sensitivity Using a Detached Leaf Assay

The quinone outside inhibitor (QoI) fungicides are known for their inherently high resistance risk owing to substitutions in amino acid residues 129, 137, or 143 of the cytochrome b gene of phytopathogens. In Venturia effusa, cause of pecan scab, an intron adjacent to position 143 likely reduces this risk; however, the effects of a recently discovered substitution at position 137 (G137S) are unknown. Traditional in vitro assays are not useful for determining sensitivity of isolates of V. effusa to the QoI fungicides, owing to the fungitoxic effects of required alternative oxidase inhibitors. A detached leaf assay was developed to quantify the sensitivity of 59 isolates to azoxystrobin: 45 wild-type isolates and 14 carrying G137S. Isolate EC50 values ranged from <0.0001 to 0.3047 µg/ml; EC50 values for wild-type isolates ranged from <0.0001 to 0.2007 µg/ml (median 0.0023 µg/ml), whereas EC50 values for G137S isolates ranged from 0.0033 to 0.3047 µg/ml (median 0.0178 µg/ml). The median EC50 value for G137S isolates was significantly greater than that of the wild-type isolates; however, there was overlap between the two groups. This is the first report of sensitivity of V. effusa isolates to a QoI fungicide and evidence of G137S as a potential mechanism of partial resistance. However, although a complete control failure is unlikely, the impact of this substitution on QoI efficacy in Georgia pecan orchards remains to be determined.

Bioactivity-driven high throughput screening of microbiomes of medicinal plants for discovering new biological control agents

ABSTRACTIn a preliminary DNA-based microbiome studies, diverse culturable and unculturable bacterial taxa were identified in the roots and rhizospheres of different medicinal plants. In this report, culturable endophytic bacteria were isolated from four economically important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata and Calendula arvensis. On the basis of initial antimicrobial screening, nine bacterial species in seven different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Pantoea, Pseudarthrobacter and Delftia, were selected for further analyses. These bacteria were identified using 16S rRNA gene sequencing. Antimicrobial assays of these selected bacteria revealed that Pseudomonas taiwanensis has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis inhibited growth of P. parasitica more than 80%. Ethyl acetate extracts of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1 and B. tequilensis MOSEL-FLS3 and D. lacustris MB322 also displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4 µg/mL – 400 µg/mL). Bacterial extracts showing higher bioactivity (>80% inhibition in vitro) were selected for detached-leaf assay against P. parasitica on tobacco. In detached-leaf assay, application of 1% ethyl acetate bacterial extract of MOSE L-RD3, MOSEL-RD23, MOSEL-FLS1, MOSEL-FLS3 and MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Over all P. taiwenensis MOSEL-RD23 showed positive activities for all the assays. Analysing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for sustainable production of crops.

Resistance Identification of Tree Peony Cultivars of Different Flowering Time to Gray Mold Pathogen Botrytis cinerea

Tree peonies are valuable ornamental plants and are widely cultivated in China and many other countries. Gray mold caused by Botrytis cinerea is an increasingly severe disease in Luoyang of China and seriously affects the ornamental value of tree peonies both in the open air and in greenhouses. However, the resistance of different tree peony cultivars to B. cinerea remains unknown. In this study, 15 tree peony cultivars belonging to three different flowering times were evaluated for resistance to B. cinerea by detached leaf assay measure. Results showed that the resistance of early-flowering peonies was stronger than that of later flowering peonies. Moreover, the correlation between flowering time and resistance of tree peonies was extremely significant (P < 0.01). The information obtained in this study can provide theoretical basis both for further exploring the resistance genes of tree peony to B. cinerea and for the prevention and controlling of the gray mold.

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.

Screening Ornamental Cherry (Prunus) Taxa for Resistance to Infection by Blumeriella jaapii

Ornamental flowering cherry trees are important landscape plants in the United States but are susceptible to several serious pests and disease problems. Cherry leaf spot, incited by the fungus Blumeriella jaapii, is characterized by defoliating susceptible trees in late summer, leading to weakening or even death of the tree. To identify resistant plants for use in landscape plantings and in our breeding program, we used a detached leaf assay to screen 69 diverse ornamental flowering cherry taxa for resistance to cherry leaf spot. We found clear differences in susceptibility among the accessions, with seven accessions developing essentially no symptoms at all. A variance decomposition showed that most of the variance (59%) occurred among accessions, indicating that genotype, even more than species, determined susceptibility. The detached leaf assay used in this study is an effective method for screening large numbers of plants for relative resistance to cherry leaf spot. These methods will be particularly useful to characterize germplasm and screen hybrids in breeding and selection programs.