Wheat genotypes differ in seed-borne ice nucleating bacterial (INB) population
Wheat genotypes differ in seed-borne ice nucleating bacterial (INB) population
Author: Amanuel Bekuma, Esther Walker, Brenton Leske, Sarah Jackson, Ben Biddulph, (Department of Primary Development and Regional Development (DPIRD)), Chaiyya Cooper, Rebecca Swift, (Curtin University). | Date: 03 Mar 2022
Key messages
- INB can be seed-borne and the level of infection varies according to wheat genotypes and the level of severity of the frost event.
- Wheat genotypes sown early and exposed to severe grain frost (>80%) host more than 100-fold INB compared to the late sown wheat genotypes exposed to very mild grain frost (<5%) in the same year.
- To minimise the risk of INB infection in subsequent seasons, it would be safer to avoid keeping frost affected grain for seeding purposes.
- qPCR-based testing is a promising method for quantifying INB from frost affected grains.
Aims
To develop methods for the detection and quantification of seed-borne INB and to compare wheat genotypes for the ability to host seed-borne INB
Introduction
Previous work has measured the reproductive frost damage of different wheat genotypes under Australian field conditions (March et al. 2016; Cocks et al. 2019). This data was used to generate a frost ranking system for wheat genotypes based on their frost susceptibility (https://nvt.grdc.com.au/grdc-nfi-frost-rankings). Further, physiological and metabolite analysis of wheat genotypes have also given an insight on how genotypes vary in response to frost events (Cheong at al. 2019; Livingston et al. 2021). However, breeding new varieties with improved frost tolerance is still a challenging task. This is partly because the molecular basis of frost tolerance in wheat during the reproductive stage is poorly understood (Reinheimer 2010). Additionally, it is difficult to measure frost damage under field conditions due to the sporadic and dynamic nature of frost events. However, recent field studies have demonstrated a role for ice nucleating bacteria in increasing frost risk during the reproductive stage in wheat grown in Western Australia (Biddulph et al. 2021; Bekuma et al. 2021). Understanding the interaction of different cereal genotypes with ice nucleating bacteria could contribute towards breeding programs for reproductive frost tolerance.
Ice nucleating bacteria, like Pseudomonas syringae, can be present in rainfall prior to frost events associated with weak frontal systems or in crop residue and dispersed by rainfall and/or actively growing on the plant canopy. These bacteria produce an ice nucleating protein that initiates the freezing of water at warmer temperature than ice would normally freeze at, even when compared to other ice nucleating agents like dust and spores (Lindow 1983). For instance, our previous work demonstrated that the presence of a bacterial ice nucleating protein significantly increased frost damage in field grown wheat at the DPIRD Dale Frost Nursery (WA) (Biddulph et al. 2021).
Understanding the link between INB and wheat genotypes will help inform breeders, industry and growers on the contribution of these bacteria to frost damage and inform strategies to manage the risk of frost damage. In this work, we assessed and developed methods to measure INB populations present on the grain of different wheat genotypes exposed to different levels of frost events during grain filling. We then used these methods to investigate variations in the wheat genotypes’ ability to host INB on the seed.
Materials and methods
Seed samples
Seed samples of four wheat genotypes (Young, Wyalkatchem, Scout and Cutlass) sown at the DPIRD-Dale Frost Nursery (WA) at different times of sowing (TOS) during the 2018 and 2019 field season were used in this study. For genotypes that differed in their flowering window, their developmental stage was matched by collecting seed samples from relevant TOS which flower at approximately the same period. This approach avoided environmental and developmental stage-related variation. For the 2018 samples, the flowering date of Cutlass sown on 10th April 2018 (TOS 1) was matched to Wyalkatchem and Scout sown on 19th April 2018 (TOS 2) and Young on 27th April 2018 (TOS 3). For the 2019 samples, the flowering date of Cutlass sown on 17th April 2019 (TOS 2) was matched to Wyalkatchem and Scout sown on 24th April 2019 (TOS 3) and Young on 1st May 2019 (TOS 4). To compare the results with late TOS which usually avoids frost damage, another set of seed samples of these genotypes planted on 10th May 2018 and 17th May 2019, respectively were also tested.
Isolation of bacteria from seed
Seeds were harvested in 2018 and 2019 at physiological maturity, threshed, cleaned, and stored in a cold room at 4°C. Recovery of seed-borne INB from wheat grain samples was carried out following the procedure by Mohan and Schaad (1987). Briefly, 10 g of seed was soaked in a cold (5°C) sterile saline (0.85% NaCl) solution with 0.01% Tween 20 buffer in a ratio of 1:2.5 (1 g of seed in 2.5 ml of solution) in 50 ml plastic vials and refrigerated overnight at (5°C). The tubes were then shaken on a rotary shaker for one hour in a cold room (5oC). Ten-fold dilutions series with dilutions 10-1, 10-2,10-3 and 10-4 were made from the soaking solution and 100 µl of each dilution was plated in triplicate on King’s B medium (KB) (King et al. 1954) and incubated for four days at 20°C to allow maximum expression of ice nucleation activity (Sherman and Lindow 1995).
INB enumeration by replica plate freezing technique
The number of IN active bacteria was assayed by replica plating technique developed by Lederberg and Lederberg (1952) and modified by Lindow et al. (1978). A square of sterile velvet cloth stretched across a circular polystyrene block was first pressed against the surface of the dilution plate with colonies to be assayed and then pressed onto a sheet of paraffin-coated aluminium foil (coating mix 3% w/v paraffin dissolved in xylene). The edges (ca. 1 cm) of the sheet were then folded upward to form a "boat" to allow floating on the surface of a cooling bath held at -8°C. A fine mist of sterile distilled water (ice nucleus-free) was sprayed on the surface of the boat. After about 2 min, discrete areas of ice (having a frosty, white appearance) were counted visually for each dilution plate as a proxy for number of INB then, the total INA bacteria per ml was calculated. Ambiguous areas were tested physically for solidity with the tip of a sterile pipette tip. When only a small fraction or no INA were detected, plates bearing very large numbers of colonies were checked by the replica freezing procedure. On the higher dilution plates, it is possible to miss detection of active colonies due to inter-colony antagonism on our nonselective media.
qPCR-based quantification of the ice nucleation gene (inA)
Seed samples were prepared as indicated above and a 300µl aliquot of the soaking solution was placed in a clean tube containing 450 µl of extraction buffer (200 mM Tris-HCL pH 8.0, 250 mM NaCl, 25 mM EDTA pH 8.0). After appropriate dilution with 10mM Tris-HCL (pH 8.0), the extract was used directly as template in qPCR. Each qPCR reaction was carried out in a 10 µl volume containing 5 µl SensiFAST SYBR Lo-ROX mix (Meridian Bioscience, OH, USA), 500 nM each of forward and reverse primer (Macrogen Inc. Seoul, South Korea), 1% PVP (w/v) and 3 µl of template. Amplification was performed on a portable real time PCR cycler (Magnetic induction cycler, Bio Molecular Systems, QLD, Australia) under the following 2-step cycling conditions: 1 cycle of 95⁰C for 3 min, followed by 35 cycles of 95⁰C for 5 sec then 65⁰C for 10 sec. Data were analysed using the Absolute Quantification method within the micPCR v2.10.5 software. Results were first assessed as presence/absence of the inA fragment and then quantified against a standard curve. Inhibition was assessed by spiking duplicate samples with a known amount of the standard. The standard curve was prepared from Snomax (Snomax International, CO, USA), which is a commercial product containing strain P. syringae 31R1, to maximize the ice nucleation activity. One milligram of Snomax pellets was dissolved in 1 ml of sterile molecular grade water. One mg ml−1 of Snomax contains approximately 7X108 ice nucleating bacterial cells (Mohler et al. 2008). Assuming one inA copy per genome, the product was serially diluted 105 to obtain 101 copies/µl with 10mM Tris-HCL (pH 8.0).
Results and Discussion
qPCR-based detection of ice nucleation gene (inA) correlates with bacterial count
All qPCR runs showed efficiency between 90 and 100% and R2-value of 0.999 relative to the standard suggesting the high efficiency and reproducibility of the assay (data not shown). The assay was effective in quantifying inA gene copy number from grain samples exposed to moderate to severe levels of frost events during grain filling. Assuming one inA copy per bacterial genome, the result of the qPCR analyses is in good agreement with INB count from the replica freeze technique for 2018 samples However, detection capability of the qPCR assay in quantifying samples with inA copy numbers below ca 3000 per ml (late sown samples from 2018 and some of the 2019 early sown samples) requires further refinement such as, concentrating bacterial cells, increasing sample size and modifying the protocol. Because the technique is unable to discriminate between live bacteria and dead bacteria, care must be taken interpreting the result.
Frosted grains harbour more INB population than unfrosted grain
In both 2018 and 2019, the proportion of grains affected by frost was higher when the wheat was sown early (Figs. 1 and 2). Additionally, both the replica plate freezing assay and qPCR analysis revealed that INB populations are higher in seed samples from earlier TOS (April sown) compared to late sown (around mid-May) wheat in both years (Figs. 1 and 2). Early sowing usually increases the likelihood of overlapping frost events with the susceptible stage of wheat plants. For all genotypes, the 2018 samples had a higher INB population compared to the 2019 samples, which corresponds to the greater proportion of frosted grains for these genotypes.
At present, the correlation between INB population and percentage of frosted grain is not understood. For instance, it is not clear if the presence of INB on the seed caused the frost or if frost affected seeds provide favourable conditions for opportunistic INB to flourish. Grains that are frosted during early grain fill have a typical pinched appearance that forms creases along the axis, and this crease may provide habitat for the bacteria. Frosted grains also have a high sugar content, potentially serving as a nutrient source for INB and other microbes. The cause and effect of the presence of INB on frosted grains needs to be investigated further.
Seed-borne INB population varies among wheat genotypes
We accounted for the effect of variations in wheat phenology in INB populations in this study by collecting seed samples from genotypes that flowered at approximately the same period. Therefore, the variations in INB populations are likely to be due to genotype effect rather than the environment. Unlike the early sown 2018 samples where severe grain frost (80-100%) probably masked genotype variation for INB count, there was a moderate level of grain frost (5-20%) in early sown 2019 samples which allowed for discrimination among the genotypes. In these samples, wheat genotypes show differences in the INB populations present on their seeds. For instance, in the early sown 2019 samples, Wyalkatchem and Young had the highest and lowest lNB populations on their seeds, respectively (Figure 2). The qPCR analysis also detected higher copy numbers of the inA gene (log10 copy number 4.4) on Wyalkatchem seeds in TOS 3 and none for Young TOS 4 (data not shown). The analyses did not detect inA genes in the remaining genotypes. Previous studies on frost induced floret sterility as well as metabolite and lipidomic analysis suggest there is genetic variation for frost tolerance among wheat genotypes (Cheong et al 2019). For instance, Wyalkatchem responds to cold exposure by accumulating osmolytes such as fructose and glucose while in Young, changing the ratio of unsaturated to saturated fatty acids helps to maintain membrane fluidity and avoids the membrane damage and water stress observed for Wyalkatchem (Cheong et al 2019). Hence in Wyalkatchem, increasing sugar levels followed by membrane damage could make nutrients available for microbial growth including for INB. The fundamental difference in acclimatization mechanisms by these genotypes might explain the differences in INB population present on their seeds which may also determine the success of colonization by this group of bacteria (Figure 3). Further measurements on the amount and type of sugar accumulation in response to frost may shed light on the role of sugars in modulating INB populations on the plant and subsequent frost damage.
Conclusion
Grain frost appears to be corelated with (or associated with higher) INB colonization on the seed surface but the number of INB populations varies depending on wheat genotypes and the severity of frost damage. For instance, Wyalkatchem showed high levels of INB present on the grain when exposed to a moderate level of frost damage and there were significantly less INB present on Young under the same conditions. An increased level of sugars in frosted grains and plant tissue in response to cold exposure might hold the key to understand the link between INB colonisation and genetic tolerance. The role of seed-borne INB in repopulating the subsequent crop and sustaining the frost cycle for the next crop remains to be investigated. In the meantime, it would be safer to avoid keeping frosted grain for seeding purposes to reduce frost risk. The use of both molecular and microbiological techniques will be a powerful tool in detection and quantifying seed borne INB population with prospects for the technique to be used in other environmental samples.
Acknowledgments
This work was supported by the Council of Grain Grower Organisations, DPIRD, WA and many growers including Bill, Anne Cleland and families, Dale WA.
Paper reviewed by
George Mwenda, DPIRD and Steve Curtin, ConsultAg
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