Molecular Characterization of the sdhB Gene and Baseline Sensitivity to Penthiopyrad, Fluopyram, and Benzovindiflupyr in Venturia inaequalis

The succinate dehydrogenase inhibiting (SDHI) fungicides are a class of single-site fungicides that are increasingly important in the management of Venturia inaequalis. In this study, the baseline sensitivity of V. inaequalis to penthiopyrad, fluopyram, and benzovindiflupyr was investigated. In all, 35 to 70 isolates with no prior exposure to single-site fungicides were used to determine the effective concentration at which growth was inhibited by 50% (EC50). Mean EC50 values for the conidial germ tube growth stage for penthiopyrad, fluopyram, and benzovindiflupyr were 0.086, 0.176, and 0.0016 μg ml−1, respectively. Linear correlation analysis revealed a significant and positive correlation between fluopyram and penthiopyrad (P ≤ 0.0001, r = 0.66) and fluopyram and benzovindiflupyr (P = 0.0014, r = 0.52). Baseline sensitivities of V. inaequalis during the mycelial growth stage were also determined for fluopyram and benzovindiflupyr. EC50 values were higher for fluopyram and benzovindiflupyr during this stage compared with the conidial germ tube growth stage, with means of 0.043 and 2.02 μg ml−1, respectively. In addition, the sdhB gene was characterized for three isolates of V. inaequalis collected from a research, baseline, and commercial orchard population. No common mutation sites associated with SDHI resistance in other phytopathogenic fungi were discovered in these isolates or isolates that were recovered following field applications of SDHI fungicides. The results of this study suggest that SDHI fungicides have a high level of activity during the conidial germ tube elongation stage in V. inaequalis and provide a basis for phenotypic and genotypic monitoring of shifts toward resistance of V. inaequalis populations to the SDHI fungicide class.

In vitro effect of substrate, temperature and photoperiod on Phakopsora pachyrhizi urediniospore germination and germ tube growth

In vitro experiments were conducted to assess the effects of substrate, temperature and time of exposure to temperature and photoperiod on P. pachyrhizi uredospore germination and germ tube growth. The following substrates were tested: water-agar and soybean leaf extract-agar at different leaf concentrations (0.5, 1.0, 2.0 and 4.0 g of leaves and 15g agar/L water), temperatures (10, 15, 20, 25, 30, and 35oC) and times of exposure (1, 2, 3, 4, 5, 6, 7, and 8 hours) to temperature and 12 different photoperiods. The highest germination and germ tube length was found for the soybean leaf extract agar. Maximum P. pachyrhizi uredospore germination was obtained at 21.8 and 22.3°C, and maximum germ tube growth at 21.4 and 22.1°C. The maximum uredospore germination was found at 6.4 hours exposure, while the maximum germ tube length was obtained at 7.7 h exposure. Regarding photoperiod, the maximum spore germination and the maximum uredospore germ tube length were found in the dark. Neither spore germination nor uredospore germ tube growth was completely inhibited by the exposure to continuous light.

Combinatorially Selected Peptides for Protection of Soybean Against Phakopsora pachyrhizi

Phakopsora pachyrhizi, the fungal pathogen that causes Asian soybean rust, has the potential to cause significant losses in soybean yield in many production regions of the United States. Germplasm with durable, single-gene resistance is lacking, and control of rust depends on timely application of fungicides. To assist the development of new modes of soybean resistance, we identified peptides from combinatorial phage-display peptide libraries that inhibit germ tube growth from urediniospores of P. pachyrhizi. Two peptides, Sp2 and Sp39, were identified that inhibit germ tube development when displayed as fusions with the coat protein of M13 phage or as fusions with maize cytokinin oxidase/dehydrogenase (ZmCKX1). In either display format, the inhibitory effect of the peptides on germ tube growth was concentration dependent. In addition, when peptides Sp2 or Sp39 in either format were mixed with urediniospores and inoculated to soybean leaves with an 8-h wetness period, rust lesion development was reduced. Peptides Sp2 and Sp39, displayed on ZmCKX1, were found to interact with a 20-kDa protein derived from germinated urediniospores. Incorporating peptides that inhibit pathogen development and pathogenesis into breeding programs may contribute to the development of soybean cultivars with improved, durable rust tolerance.

Effects of Temperature on Urediniospore Germination, Germ Tube Growth, and Initiation of Infection in Soybean by Phakopsora Isolates

Temperature is a critical factor in plant disease development. As part of a research program to determine how specific environmental variables affect soybean rust, we determined temperature effects on urediniospore germination and germ tube growth of four isolates of Phakopsora pachyrhizi, one each from Brazil, Hawaii, Taiwan, and Zimbabwe, and an isolate of P. meibomiae from Puerto Rico, collected over a 25-year period. Also compared were the effects of temperature during a night dew period on initiation of disease by the P. pachyrhizi isolates. All variables were fit to a nonlinear beta function with temperature as the independent variable. Minimum, maximum, and optimum temperatures, along with shape parameters of the beta function for each variable, were statistically analyzed. All Phakopsora isolates behaved similarly as to how temperature affected urediniospore germination, germ tube growth, and initiation of disease. The results suggest that P. pachyrhizi has changed little in the past few decades with respect to how it responds to temperature and that previously collected research data continues to be valid, simplifying the development of soybean rust disease models.

Effect of Temperature on Thekopsora minima Urediniospores and Uredinia

Thekopsora minima is a heteroecious rust, with spermogonia and aecia occurring on the needles of hemlock (Tsuga spp.) and uredinia, telia, and basidia occurring on the leaves of ericaceous genera, including species of Rhododendron. The effect of temperature was determined for urediniospore germination, germ tube growth, and infection efficiency on Rhododendron ‘White Lights’. Percent germination and germ tube growth were assessed at 10, 15, 20, 25, and 30°C after 3 h of incubation on 1.5% water agar in the dark. Polynomial regression analyses revealed a significant effect of temperature on both germination (P < 0.001, R2adj = 0.936) and germ tube growth (P < 0.001, R2adj = 0.933), with predicted optimum temperatures of 21.5 and 22.0°C, respectively. Germination and germ tube growth were reduced greatly at 30°C and below 15°C. Temperature also was found to have a significant effect on infection efficiency, as measured by incubation period (P < 0.001, R2adj = 0.808) and uredinia produced (P < 0.001, R2adj = 0.866). On excised leaf disks of Rhododendron ‘White Lights’ maintained under a 14-h photoperiod, the shortest mean incubation periods of 10.7 and 10.0 days were at 20 and 25°C, respectively, with a predicted optimum of 23°C. The mean incubation period at 15 and 30°C was approximately 4 and 3 days longer, respectively, than at the predicted optimum temperature. The number of uredinia produced was similar at 15, 20, and 25°C, but was reduced sixfold at 30°C. The predicted optimum temperature for uredinial production was 19.5°C, with a 5% variation in uredinia production between 17.5 and 22°C.

Osmotin and Thaumatin from Grape: A Putative General Defense Mechanism Against Pathogenic Fungi

Little information is available concerning the expression of pathogenesis-related (PR) proteins in grapevine (Vitis vinifera) and their effect properties on the major fungal pathogens of grape. A systematic study was performed on the effect of total or individual grape proteins on mycelial growth, spore germination, and germ tube growth of Uncinula necator, Phomopsis viticola, and Botrytis cinerea. Two proteins, identified as PR proteins by immunological methods and by N-terminal sequencing as osmotin and thaumatin-like protein, exhibited strong antifungal activities in vitro, blocking the growth of Phomopsis viticola and Botrytis cinerea mycelia. In addition, they inhibited spore germination and germ tube growth of U. necator, Phomopsis viticola, and Botrytis cinerea. The presence of both proteins displayed a synergistic effect. The expression of osmotin and thaumatin-like protein was induced in grapevine leaves and berries infected with U. necator and Phomopsis viticola. Thaumatin previously was thought to occur exclusively in berries. Immunoblot analyses revealed the accumulation of the two PR proteins in infected leaves and berries, supporting a role in vivo in increasing the resistance of grapevine to fungal attack.