Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

Background - Monilinia blight caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) is a major disease of wild blueberry that can result in severe crop losses in the absence of an integrated disease management programme. The fungus causes blight in the emerging floral and vegetative buds, but the degree of susceptibility varies among the different wild blueberry phenotypes, ranging from the highly susceptible V. a. f. nigrum to the moderately susceptible V. angustifolium and the least susceptible V. myrtilloides. Results - The present study evaluated the defense responses of these major phenotypes during their primary infection (floral buds) with M.vc. The temporal expression profiles of PR genes (PR3 and PR4) and the flavonoid pathway structural genes (CHS, ANS, ANR, DFR and FLS) were analysed. The PR3 and PR4 gene expression profiles revealed that V. myrtilloides responded to M.vc infection by activating the expression of both PR genes. V. a. f. nigrum, on the other hand, failed to activate these genes, while V. angustifolium, exhibited an intermediate response. Our study with the flavonoid pathway genes indicated variability in activation of the genes during post-infection time points with ANS and ANR in V. myrtilloides, FLS in V. angustifolium and no response observed in V. a. f. nigrum. Conclusions - Altogether, this study highlights that the degree of phenotype susceptibility is associated with the timely activation of host defense responsive genes. Data obtained in this study provided a starting point for a better understanding of the wild blueberry- M. vaccinii-corymbosi pathosystem

Susceptibility to Botrytis blight at different floral stages of wild blueberry phenotypes

Botrytis blossom blight is an important disease of wild blueberries with yield losses in excess of 20% frequently occurring. Two field experiments were conducted in 2016 and 2017 to determine the susceptibility of four phenotypes (Vaccinium angustifolium, V. angustifolium f. nigrum, V. myrtilloides and V. angustifolium var. Fundy] at different floral stages [(Bud break (F5); bud prebloom; (F6), corolla fully open (F7), and senescent corolla (F8)] to Botrytis infection. Specific flower clusters on tagged stems from different phenotypes were inoculated with Botrytis cinerea conidial suspension (106 conidia·ml-1). Disease development were assessed eight days after inoculation. Disease incidence and severity in phenotype ranged from 14.1 to 22.6% and 37.4 to 42.3% in 2016, respectively, and 39.8 to 44.1% and 9.70 to 19.1% in 2017, respectively. Results indicated that V. angustifolium was the most susceptible followed by V. angustifolium f. nigrum and V. angustifolium var. Fundy. Vaccinium myrtilloides was found to be least susceptible. Incidence and severity on floral stages ranged from 2.95 to 36.4% and 7.81 to 75.5% in 2016, respectively, and 7.28 to 66.9% and 11.1 to 27.1% in 2017, respectively. Floral stage F7 was the most susceptible with incidence up to 66.9% and severity up to 75.5% followed by F6, F5 and F8. Therefore, results from this study indicated that V. myrtilloides was less susceptible to B. cinerea than V. angustifolium phenotypes, and F6 and F7 stages were the most susceptible to Botrytis blight. These results will assist producers in making more informed decisions on Botrytis blight control and as its management practices shift from blanket to precise delivery of disease control products

Relative roles of dispersal dynamics and competition in determining the isotopic niche breadth of a wetland fish

1. The niche variation hypothesis predicts that among-individual variation in niche use will increase in the presence of intraspecific competition and decrease in the presence of interspecific competition. We sought to determine whether the local isotopic niche breadth of fish inhabiting a wetland was best explained by competition for resources and the niche variation hypothesis, by dispersal of individuals from locations with different prey resources or by a combination of the two. We analysed stable isotopes of carbon and nitrogen as indices of feeding niche and compared metrics of within-site spread to characterise site-level isotopic niche breadth. We then evaluated the explanatory power of competing models of the direct and indirect effects of several environmental variables spanning gradients of disturbance, competition strength and food availability on among-individual variation of the eastern mosquitofish (Gambusia holbrooki). 2. The Dispersal model posits that only the direct effect of disturbance (i.e. changes in water level known to induce fish movement) influences among-individual variation in isotopic niche. The Partitioning model allows for only direct effects of local food availability on among-individual variation. The Combined model allows for both hypotheses by including the direct effects of disturbance and food availability. 3. A linear regression of the Combined model described more variance than models limited to the variables of either the Dispersal or Partitioning models. Of the independent variables considered, the food availability variable (per cent edible periphyton) explained the most variation in isotopic niche breadth, followed closely by the disturbance variable (days since last drying event). 4. Structural equation modelling provided further evidence that the Combined model was best supported by the data, with the Partitioning and the Dispersal models only modestly less informative. Again, the per cent edible periphyton was the variable with the largest direct effect on niche variability, with other food availability variables and the disturbance variable only slightly less important. Indirect effects of heterospecific and conspecific competitor densities were also important, through their effects on prey density. 5. Our results support the Combined hypotheses, although partitioning mechanisms appear to explain the most diet variation among individuals in the eastern mosquitofish. The results also support some predictions of the niche variation hypothesis, although both conspecific and interspecific competition appeared to increase isotopic niche breadth in contrast to predictions that interspecific competition would decrease it. We think this resulted from high diet overlap of co-occurring species, most of which consume similar macroinvertebrates

Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds

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Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes

Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions. Molecular mechanisms regulating defence responses of wild blueberry phenotypes towards this causal fungus are not yet fully known. A reliable quantification of gene expression using quantitative real time PCR (qPCR) is fundamental for measuring changes in target gene expression. A crucial aspect of accurate normalisation is the choice of appropriate reference genes. This study evaluated the expression stability of seven candidate reference genes (GAPDH, UBC9, UBC28, TIP41, CaCSa, PPR and RH8) in floral tissues of diploid and tetraploid wild blueberry phenotypes challenged with M.vc. The expression stability was calculated using five algorithms: geNorm, NormFinder, BestKeeper, deltaCt and RefFinder. The results indicated that UBC9 and GAPDH were the most stable reference genes, while RH8 and PPR were the least stable ones. To further validate the suitability of the analyzed reference genes, the expression level of a pathogenesis related protein gene (i.e., PR3) was analysed for both phenotypes at four time points of infection. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in wild blueberry species.publishedVersio

Modulation of defense genes and phenolic compounds in wild blueberry in response to Botrytis cinerea under field conditions

Botrytis blight is an important disease of wild blueberry [(Vaccinium angustifolium (Va) and V. myrtilloides (Vm))] with variable symptoms in the field due to differences in susceptibility among blueberry phenotypes. Representative blueberry plants of varying phenotypes were inoculated with spores of B. cinerea . The relative expression of pathogenesis-related genes (PR3, PR4), flavonoid biosynthesis genes, and estimation of the concentration of ten phenolic compounds between uninoculated and inoculated samples at different time points were analyzed. Representative plants of six phenotypes (brown stem Va , green stem Va , Va f. nigrum, tall, medium, and short stems of Vm ) were collected and studied using qRT-PCR. The expression of targeted genes indicated a response of inoculated plants to B. cinerea at either 12, 24, 48 or 96 h post inoculation (hpi). The maximum expression of PR3 occurred at 24 hpi in all the phenotypes except Va f. nigrum and tall stem Va . Maximum expression of both PR genes occurred at 12 hpi in Va f. nigrum. Chalcone synthase, flavonol synthase and anthocyanin synthase were suppressed at 12 hpi followed by an upregulation at 24 hpi. The expression of flavonoid pathway genes was phenotype-specific with their regulation patterns showing temporal differences among the phenotypes. Phenolic compound accumulation was temporally regulated at different post-inoculation time points. M-coumaric acid and kaempferol-3-glucoside are the compounds that were increased with B. cinerea inoculation. Results from this study suggest that the expression of PR and flavonoid genes, and the accumulation of phenolic compounds associated with B. cinerea infection could be phenotype specific. This study may provide a starting point for understanding and determining the mechanisms governing the wild blueberry- B. cinerea pathosystem

Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes

Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions. Molecular mechanisms regulating defence responses of wild blueberry phenotypes towards this causal fungus are not yet fully known. A reliable quantification of gene expression using quantitative real time PCR (qPCR) is fundamental for measuring changes in target gene expression. A crucial aspect of accurate normalisation is the choice of appropriate reference genes. This study evaluated the expression stability of seven candidate reference genes (GAPDH, UBC9, UBC28, TIP41, CaCSa, PPR and RH8) in floral tissues of diploid and tetraploid wild blueberry phenotypes challenged with M.vc. The expression stability was calculated using five algorithms: geNorm, NormFinder, BestKeeper, deltaCt and RefFinder. The results indicated that UBC9 and GAPDH were the most stable reference genes, while RH8 and PPR were the least stable ones. To further validate the suitability of the analyzed reference genes, the expression level of a pathogenesis related protein gene (i.e., PR3) was analysed for both phenotypes at four time points of infection. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in wild blueberry species

Potential use of biofungicides and conventional fungicide for the management of Botrytis blossom blight in lowbush blueberries

Botrytis blight is an economically important disease of lowbush blueberry that causes significant yield loss annually. In this study, the biofungicides, Diplomat 5SC® (polyoxin D), Timorex Gold® (tea tree oil), Fracture® (BLAD) and Serenade MAX® (Bacillus subtilis) were evaluated for their disease suppression potential against B. cinerea individually and in rotation with Switch® (cyprodinil and fludioxonil) under field conditions. Three applications of each biofungicide were made for the stand-alone treatments at 7–10 day intervals, with Switch® replacing the biofungicides during the second applications in the combined treatment. Results indicated that the products have the potential for use as a biofungicide in lowbush blueberries. All the stand-alone and rotational applications brought about a significant reduction in disease development, especially in 2019. The application of Diplomat 5SC® and Fracture®-Switch® rotation decreased disease development by over 63% in 2018. In 2019, all stand-alone treatments reduced disease development by more than 42%, whereas their rotation with Switch® reduced disease by over 69% at one site and at least 30% at another site. Stand-alone Diplomat 5SC® and Timorex Gold® along with Fracture®, Timorex Gold® and Serenade MAX® rotation resulted in over 20% more berries. This study suggests that the biofungicides and their integration with chemical fungicides have the potential as an alternative management strategy against Botrytis blossom blight to reduce the use of conventional fungicides and produce fruit with no detectable fungicide residues