We detected no directional selection on flowering time when averaged across all treatments , suggesting that treatment differences in selection intensity cancelled each other out. On average, western genotypes produced 0.75 more fruits than eastern—a 27% increase . In addition, genetic differences between populations within subspecies contributed 20.4% of the variance in fecundity . However, the lack of subspecies × treatment and genotype × treatment interactions shows that genetic effects on performance were not microbiota-dependent. Finally, fitness was consistent across microbial treatments , indicating no net effect of different microbiomes on fecundity . In contrast, we found no evidence that different sterilized field soils affect flowering time selection differentials or gradients . However, these treatments affected overall fecundity , indicating strong differences in soil quality. Western genotypes produced 69% more fruits than eastern genotypes on average , but this advantage was consistent across all soils . We detected no net selection gradient on flowering time across all field soils ; however, we did find a significant selection differential on flowering time .Sampled soil microbiomes differed between sites within year but not between years within site . These results are supported by principal coordinates analysis, in which samples clustered mainly by site .
The first three PCo cumulatively explained 69.8% of prokaryotic community variation . Because the MAH and JAM soils, square plastic pot which had the slowest and fastest flowering times respectively in our experiment, were separated primarily on the PCo2 axis , PCo2 became our candidate microbiome component to test for effects on flowering time. Because eastern genotypes appeared insensitive to microbes , we used flowering time residuals of western genotypes as the response variable. We did not find a significant relationship between mean flowering time in each treatment and the mean PCo2 score of soil samples from the corresponding site . Likewise, none of the individual OTUs with high PCo2 scores predicted flowering time ; for instance, abundance of OTU_96997 appeared to correlate with phenology but the relationship was non-significant after correction for multiple comparisons . The phyla Proteobacteria and Crenarchaeota were more abundant, and Acidobacteria were less abundant, in slow-flowering MAH compared to fast-flowering JAM soil communities . Within phyla, several families—including Koribacteraceae, Solibacteraceae, Opitutaceae, Verrucomicrobiaceae, Solirubrobacteraceae, and Mycobacteriaceae—differed in relative abundance between MAH and JAM soil communities . In addition, Verrucomicrobia and Gemmatimonadetes were enriched in the 5% of OTUs with the highest loadings on PCo2 compared to the full natural communities , indicating that these phyla contribute disproportionately to the microbiome variation summarized by PCo2.
These ‘candidate taxa’ are promising organisms for further study of microbial influences on flowering time.Our analysis of native plant genotypes and soil microbial communities from four undisturbed environments suggests that soil microbiomes contribute to the ecology and evolution of flowering phenology in Boechera stricta. First, we showed that soil microbiota influenced phenotypic plasticity of flowering time. Second, soil microbiota altered the strength and direction of selection on flowering time. Finally, we showed how this type of experiment could be combined with quantitative descriptions of soil community composition to search for microbial species that affect important phenotypes. Our results show that experimental dissection of complex environments can reveal the ecological interactions shaping phenotypic expression and natural selection. Our finding that both soil microbes and soil chemistry cause plasticity of flowering time agrees with previous reports, although to our knowledge only one other study has tested the effects of both biotic and abiotic components of the same soil. Lau & Lennon found that Brassica rapa flowered faster in dry conditions, and that a soil microbial community with a history of drought stress accelerated flowering compared to the wet-adapted replicate of the same community. Other soil properties reported to delay flowering include heavy metal concentration , nutrient depletion , high salinity , and a history of invasive plant growth . Notably, some species’ phenology may be more robust than others’ in response to soil heterogeneity . In general, such phenotypic plasticity has important ecological and evolutionary consequences . For B. stricta in particular, phenological plasticity affects the plants’ ability to time reproduction for optimal seed set . In fact, the exclusion of natural soil microbiomes from growth chamber replicates of that experiment might be partially responsible for the low genetic correlation of flowering time in the field and in the lab .
Interestingly, our data hint that West genotypes may be more sensitive to soil microbiome than East genotypes , suggesting intraspecific genetic variation for microbe-induced flowering time plasticity. In contrast, the two subspecies show very similar sensitivities to abiotic soil properties , indicating that plasticity to these two stimuli may have different genetic bases. Typically, selection analyses allow us to infer and compare the adaptive value of particular traits in particular environments, but do not tell us why differential selection exists. Here we identified the soil microbiome as an agent of selection on flowering time in B. stricta . The evolutionary relevance of this finding is best illustrated by comparison with Anderson et al. , who measured a decrease in fecundity of 0.06 fruits/day to flowering in a typical B. stricta habitat. Our results suggest that all else remaining constant, a change in soil microbiome could increase or decrease that selection differential by up to 0.07 fruits/day, or >120% . The effects of the soil microbiome on both flowering time and its adaptive value may prove especially important in the context of conservation and adaptation to global change, given that bothmicrobial communities and plant phenology are sensitive to climate . Although we are not the first to report that soil biotas affect flowering time or selection on flowering time , our experiment adds to previous work in several unique ways. First, we used deep 16S rDNA sequencing methods to greatly enhance our resolution of the microbiome . Second, we described how this type of microbiome data can be combined with experimental phenotype data to determine which microbial taxa influence traits of interest. Third, including a diverse set of genotypes in the experiment allowed us to test for intraspecific genetic variation for flowering time plasticity in response to biotic and abiotic soil attributes, and link this ecological finding to the field of evolutionary biology. Finally, we are the first to use microbial communities from several undisturbed field sites to confirm that naturally occurring variation in soil microbes affects phenology and its adaptive value. We identified several taxa that are enriched or depleted in soils associated with fast flowering compared to slow flowering ; these groups are promising targets for future study of the microbe-flowering time relationship. As evolutionary biologists, a fundamental goal is to measure selection and trait expression in the field because the genotype-phenotype-fitness relationship is frequently contextdependent . In particular, the true relationships between plants and microbial communities may depend on neighboring plants or on other soil properties . For instance, Lau and Lennon found that the interaction between microbes and soil moisture synergistically affects fitness in Brassica rapa. Factorial application of a wider sampling of microbiomes and environmental variables—or, eventually, direct manipulation of these variables in the field —would be especially informative for understanding the ecological mechanisms of plantmicrobe interactions such as the flowering time effect we observed in B. stricta. Nonetheless, we demonstrate here that greenhouse experiments can reveal ecological interactions that may have been extremely difficult to detect directly in the field: all else remaining equal, natural differences in the soil microbiome can influence plant phenology and patterns of selection. This discovery required the isolation of soil microbiota from the larger, 25 liter pot more complex natural habitat. Further environmental simplification—the reduction of the microbiome into principal coordinates and then specific OTUs—potentially could reveal even more details of the relationship between genotype, phenotype, and the microbial environment . Although in this experiment we lacked power to fully utilize this method, it holds promise for future studies on the phenotypic effects of the environmental microbiome. This approach is generally applicable to search for microbial community members that alter biological characteristics of interest.Table grapes that meet minimum maturity standards, including sugar and acid content, and the ratio of sugar to acid, are harvested by hand and typically marketed as entire or partial clusters. The quality and value of the grapes are strongly affected by the size, texture, and color of the individual berries, and the overall appearance of the cluster . These quality attributes are commonly achieved, in part, through the use various plant growth regulators , agrochemicals with plant hormones or hormone-like compounds as active ingredients . For example, gibberellic acid is used to thin and size berries, forchlorfenuron is used to increase berry size, and firmness , and ethephon and abscisic acid may also be used to improve the color of red grapes .
Though the vast majority of table grapes are sold as entire or partial clusters, there is growing interest in marketing stemless fresh-cut grapes . However, destemming may damage grape berries, stimulating decay, and diminishing quality . Mechanical damage associated with destemming might be minimized through the use of abscission agents, PGRs which reduce fruit detachment force and promote the development of a dry stem scar, an abscission layer between the berry and pedicel . Research on the potential use of abscission agents as mechanical harvest aids for wine or raisin grapes have shown that 1-aminocyclopropane-1-carboxylic acid , coronatine, ethephon, and methyl jasmonate stimulate abscission of mature grape berries . Of those, ethephon is the only compound registered for use on grapes, though the registrations are for improving the color of red and black fruited grapes, or hastening grape maturity, both at considerably lower use rates than what is required to stimulate berry abscission. Ethephon is an ethylene-releasing molecule. Stable in a low pH solution, it hydrolyses in the higher pH of plant tissues releasing ethylene, a gaseous plant growth regulator . Ethephon’s chemical characteristics enable growers to apply it to grapes and other plants in the field with commercial spray equipment, and thereby stimulate ethylenedependent reactions. Ethephon absorption by plant tissues is influenced by temperature, relative humidity, and pH of the surface on which the spray droplets are deposited . Hedberg and Goodwin suggested that ethephon absorption by plant tissues is predominantly cuticular rather than stomatal and Nir and Lavee found that the thickness and composition of cuticle layers play an important role in penetration. How the molecule diffuses within the plant is not yet well understood. Studies conducted with the 2- chloroethylphosphoric acid marked with the 14C showed limited and mainly basipetal mobility . Ethylene regulates many aspects of fruit development including maturation, senescence, and abscission . Grape is considered non climacteric but an ethylene peak detected at veraison, the onset of ripening, may be higher than the physiological threshold for metabolic activities , and Giovannoni reported some aspects of non-climacteric ripening are probably associated with ethylene responses. Likewise, Chervin et al. reported that ethylene seems required for the increase in berry diameter, decrease in berry acidity and anthocyanins accumulation that occurs after veraison. Regardless of the endogenous role of ethylene in grape berry development, ethephon has well-established commercial uses in viticulture to promote fruit maturationrelated processes, including the synthesis and accumulation of anthocyanins in berries and the accumulation of soluble solids , and grape berries, which generally don’t abscise naturally, can be induced to abscise with exogenous application of ethephon or other compounds that stimulate ethylene production by grape berries . The potential for ethephon as an abscission agent for table grapes is a relatively new concept that has been little studied . If ethephon is to ever be registered for that use, the potential for excessive residues will have to be considered. This is especially important since relatively high rates of ethephon are needed to stimulate grape berry abscission, the process occurs quickly , and berries are consumed whole, without peeling. Therefore, the present study aimed to verify the effects of ethephon on the abscission of grape berries of two globally important seedless table grape Vitis vinifera cultivars, and on the residual concentration of ethephon in the berry in order to evaluate its potential for aiding in the production of fresh-cut fruit.Experiments were carried out in 2012 in Thompson Seedless and Crimson Seedless table grape vineyards located in the countryside of Adelfia and Francavilla Fontana , respectively. Both Thompson Seedless and Crimson Seedless were grafted onto 140 Ru and trained to an overhead trellis system , with the first spaced 2.8 m between rows and 2.5 m within rows and the latter 3.0 m between rows and 2.5 m within rows.