Resistance to gray leaf spot of maize: genetic architecture and mechanisms elucidated through nested association mapping and near-isogenic line analysis.

Gray leaf spot (GLS), caused by Cercospora zeae-maydis and Cercospora zeina, is one of the most important diseases of maize worldwide. The pathogen has a necrotrophic lifestyle and no major genes are known for GLS. Quantitative resistance, although poorly understood, is important for GLS management. We used genetic mapping to refine understanding of the genetic architecture of GLS resistance and to develop hypotheses regarding the mechanisms underlying quantitative disease resistance (QDR) loci. Nested association mapping (NAM) was used to identify 16 quantitative trait loci (QTL) for QDR to GLS, including seven novel QTL, each of which demonstrated allelic series with significant effects above and below the magnitude of the B73 reference allele. Alleles at three QTL, qGLS1.04, qGLS2.09, and qGLS4.05, conferred disease reductions of greater than 10%. Interactions between loci were detected for three pairs of loci, including an interaction between iqGLS4.05 and qGLS7.03. Near-isogenic lines (NILs) were developed to confirm and fine-map three of the 16 QTL, and to develop hypotheses regarding mechanisms of resistance. qGLS1.04 was fine-mapped from an interval of 27.0 Mb to two intervals of 6.5 Mb and 5.2 Mb, consistent with the hypothesis that multiple genes underlie highly significant QTL identified by NAM. qGLS2.09, which was also associated with maturity (days to anthesis) and with resistance to southern leaf blight, was narrowed to a 4-Mb interval. The distance between major leaf veins was strongly associated with resistance to GLS at qGLS4.05. NILs for qGLS1.04 were treated with the C. zeae-maydis toxin cercosporin to test the role of host-specific toxin in QDR. Cercosporin exposure increased expression of a putative flavin-monooxygenase (FMO) gene, a candidate detoxification-related gene underlying qGLS1.04. This integrated approach to confirming QTL and characterizing the potential underlying mechanisms advances the understanding of QDR and will facilitate the development of resistant varieties.

Genetic Characterization of Barley Net Blotch Resistance Genes.

Net blotch, caused by Pyrenophora teres f. teres, is one of the most devastating diseases of barley (Hordeum vulgare). Efficient utilization of available resistance sources is dependent upon successful characterization of genes conditioning resistance in diverse sources. Five net-blotch-resistant parents and one susceptible parent were intercrossed to identify novel resistance genes and postulate gene number and mode of inheritance. Seedling response to isolate ND89-19 was evaluated in a greenhouse test. Results indicate that the resistant spring barley lines CIho 2291 and CIho 5098 and the winter barley cv. Nomini each have single dominant genes for resistance. Resistance in CIho 5098 is governed by the same dominant gene conferring resistance in Nomini. Resistance in CIho 2291 is controlled by one dominant gene which, putatively, is the same gene conferring resistance in ND B112 but differs from the resistance genes carried by the other parents in this study. The resistance gene in Nomini or CIho 5098 could be pyramided with the resistance gene in CIho 2291 or ND B112 to enhance the durability of resistance against a wide spectrum of P. teres isolates.

Mefenoxam Sensitivity in Phytophthora cinnamomi Isolates.

Phytophthora cinnamomi is a destructive root pathogen of numerous woody plant species in the ornamental plant nursery. Sixty-five isolates of P. cinnamomi were evaluated for mefenoxam sensitivity on 20% clarified V8 agar amended with mefenoxam at 0 or 100 µg/ml. In the presence of mefenoxam at 100 µg/ml, eight isolates were intermediately sensitive, with mycelium growth ranging between 11 and 18% of the nonamended control, and 57 isolates were highly sensitive, with little or no mycelium growth. Five intermediately sensitive and five sensitive isolates were chosen to characterize their responses to mefenoxam at 0, 0.1, 1, 10, and 100 µg/ml. For intermediately sensitive isolates, the mefenoxam concentration causing 50% inhibition of mycelium growth (EC50 values) ranged between 0.03 and 0.08 µg/ml; EC50 values for sensitive isolates varied from 0.01 to 0.02 µg/ml. Five intermediately sensitive and seven sensitive isolates were selected further to assess in vivo sensitivity to mefenoxam using Lupinus angustifolius 'Russell Hybrids'. Lupine seedlings were treated with distilled water or mefenoxam at label rate (Subdue MAXX, 1 fl. oz. of product per 100 gal.) and then, 2 days later, inoculated with a 5-mm-diameter mycelial plug of P. cinnamomi on each cotyledon. Mefenoxam-treated plants averaged more than 96% less disease than water-treated plants. Mefenoxam provided adequate protection of lupines from infection by all 12 isolates regardless of their in vitro levels of sensitivity to mefenoxam. The ability to develop mefenoxam resistance was assessed in P. cinnamomi isolates with different mefenoxam sensitivity by UV mutagenesis and adapting mycelium to increasing concentrations of mefenoxam. Both UV mutagenesis and mycelium adaptation generated isolates with reduced sensitivity to mefenoxam. These isolates, however, did not grow as quickly as their corresponding parent. This study suggests that P. cinnamomi populations from ornamental nurseries in Virginia are sensitive to mefenoxam.

Effects of Propamocarb Hydrochloride on Mycelial Growth, Sporulation, and Infection by Phytophthora nicotianae Isolates from Virginia Nurseries.

Propamocarb hydrochloride is a systemic fungicide commonly used for control of Phytophthora diseases of nursery crops. Here we report on the effect of this compound on different growth stages of Phytophthora nicotianae, a major pathogen of numerous herbaceous and some woody ornamental plants. A total of 71 isolates were assayed for sensitivity to propamocarb at two concentrations of 1.8 mg/ml (label rate) and 10 mg/ml using clarified V8 agar as a base medium. All isolates grew at 10 mg/ml with the most sensitive isolate having 34.8% relative growth compared with growth on nonamended medium. Nine isolates were selected and further tested for mycelial growth at 0, 1, 10, 25, 50, and 100 mg/ml, and for sporangium production, zoospore motility, and germination at 0, 5, 50, 500, 5,000, and 50,000 µg/ml. EC50 values ranged from 2.2 to 90.1 mg/ml for mycelial growth, 133.8 to 481.3 µg/ml for sporangium production, 88.1 to 249.8 µg/ml for zoospore motility, and 1.9 to 184.6 µg/ml for zoospore germination, respectively. In addition, 17 selected isolates were evaluated for propamocarb sensitivity on Pelargonium × hortorum cv. White Orbit. Two days after seedlings were treated with propamocarb at 3.6 mg/ml, they were inoculated by either inverting one 5-mm-diameter plug of a 3-day-old culture or applying a 10-µl drop containing 20 zoospores onto each cotyledon. Propamocarb hydrochloride provided good protection of geranium seedlings from zoospore infections but not from mycelial infections. These results suggest that this fungicide must be used preventively for Phytophthora disease management and that mycelial growth may not be the most reliable measurement to determine the development of fungicide resistance to this compound in Phytophthora species at production facilities and in the landscape.

Fluctuations of Phytophthora and Pythium spp. in Components of a Recycling Irrigation System.

Stringent standards of water quality have prompted many horticultural enterprises to limit pollutant discharge associated with nutrient and pesticide applications. Collecting and recycling effluent is a method that has been implemented by many operations to contain pollutants; however, plant pathogens may be spread through recycled effluent. In this study, Phytophthora and Pythium spp. present in a water-recycling irrigation system at a perennial container nursery in southwestern Virginia were characterized using filtering and baiting techniques with two selective media. Members of Phytophthora were identified to species, whereas Pythium spp. were identified to genus only. Pythium spp. were recovered more frequently and in greater numbers than Phytophthora spp. Phytophthora capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri, and P. nicotianae were recovered in filtering assays. Only P. cryptogea and P. drechsleri were identified from baits placed on the surface of the irrigation reservoir, whereas P. cactorum, P. capsici, P. citricola, P. citrophthora, P. cryptogea, and P. drechsleri were recovered at depths, specifically at 1 and 1.5 m. This research provides data for development of detection technology and management practices for plant pathogens in irrigation water and may lead to improvements in conventional assay protocols.

Gaeumannomyces graminis vars. avenae, graminis, and tritici Identified Using PCR Amplification of Avenacinase-like Genes.

Identifying take-all pathogens, Gaeumannomyces graminis varieties avenae (Gga), graminis (Ggg), and tritici (Ggt), is difficult. Rapid identification is important for development of disease thresholds. We developed a single-tube, polymerase chain reaction (PCR) method differentiating among Gga, Ggg, and Ggt. Nucleotide base sequence analyses of avenacinase-like genes from Gga, Ggg, and Ggt isolates provided the basis for designing variety-specific primers. Sequences from Ggg and Ggt were highly related (99% identity), but Gga sequences were <95% identical to Ggg and Ggt sequences. Three 5' primers specific for Gga, Ggt, and Ggg and a single 3' common primer allowed amplification of variety-specific fragments of 617, 870, and 1,086 bp, respectively. Each 5' primer was specific in mixed populations of primers and templates. No PCR products were amplified from related fungi including Gaeumannomyces cylindrosporus and Phialophora spp. We surveyed 16 putative Ggt isolates using our assay; nine produced Ggt-specific fragments and seven produced Ggg-specific fragments. Five Gga isolates produced Gga-specific fragments. However, Gga- and Ggt-specific fragments were observed from a sixth Gga isolate, RB-W, which indicates a mixed culture or a heterokaryon. Our single-tube, PCR method rapidly differentiates among the important take-all pathogens commonly encountered together in cereal fields.