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Barley developmental mutants tweaky spike (tw) with disturbed auxin pathways possess a unique feature of an increased level of mouldy germinating grains (MGG), which serves as a convenient model to investigate the effects of plant immunity-related substances. The effects of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D), auxin inhibitors, salicylic acid (SA), and trans-cinnamic acid (TCA) were studied using the tw-WT system in surface-sterilized and unsterilized germinating grains under high rates of natural infection. Significant differences among the allelic tw mutants were revealed at the natural MGG level and in response to 2,4-D, SA, and TCA. The most effective means against MGG were sterilization and TCA. 2,4-D inhibited root growth in tw and tw2 mutants, occurring only in unsterilized and not sterilized germinating grains, while the opposite was observed for TCA and SA. The tw mutations influenced variations in the seed-borne fungal spectra, decreasing the frequency of Bipolaris sorokiniana and increasing Fusarium spp. Hypochlorite-based surface sterilization methods should be used with caution in studies where the action of exogenous 2,4-D will be analysed in germinating grains. Auxin pathway disturbances specific for pleiotropic tw mutants are generally restricted to organogenesis but not to germination events.
- barley
- MGG assay
- salicylic acid
- trans-cinnamic acid
- tweaky mutants
- 2
- 4-D
1. Introduction
Grain contamination with fungi and their produced mycotoxins is not only a problem for organic producers but also for conventional agriculture [1]. For barley, special attention is required for malt and beer contamination with mycotoxins and their proper control and avoidance [2].
2. Analysis on Results
2.1. The Action of 2,4-D, SA and TCA on MGG and Root Growth of Allelic Tweaky Spike Mutants
Naturally, grain sterilization before germination is an effective treatment against MGG. In most cases studied, the MGG level was significantly lower in surface-sterilized grains than in unsterilized grains under the same experimental conditions, and independent of the plant genotype and the studied 2,4-D concentrations (Figure 1a and Figure 2a, Tables S1a and S2a).
Figure 1. Effects of 2,4-D over a 10–50 mg L−1 range on barley tweaky-type mutants. (a) The frequency of mouldy grains (%) and (b) the root lengths (cm) of unsterilized and sterilized germinating grains measured after 5 days. For mouldy grains, n = 10 Petri dishes (100 grains); for root length n = 30; for tw2, n = 15 Petri dishes (150 grains) and n = 60. WT2016 and tw22016, grain reproduction in 2016; other material, grain reproduction in 2013; the MGG assay was performed in 2017. The asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) between the control and the 2,4-D treatment. The numbers denote significant differences (1 p < 0.05; 2 p < 0.01) between the controls of the tw-type mutant and the WT.
Figure 2. Effects of 2,4-D over a range of 50–800 mg L−1 and the auxin inhibitors HFCA and PCIB on (a) the frequency of mouldy grains (%) and (b,c) the root length (cm) of unsterilized germinating grains of tw mutants and the WT, as measured after 5 days. For mouldy grains, n = 10 Petri dishes (100 grains); for root length, n = 30. Grain was produced in 2013, and the MGG assay was performed in 2017. The asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) between the control and the 2,4-D treatment. The numbers denote significant differences (1 p < 0.05; 2 p < 0.01) between the controls of the tw-type mutant and the WT.
In the range of 10–50 mg L−1, an appreciable inhibitory effect of 2,4-D on the root growth of allelic tw-type mutants depended on (1) the plant genotype and (2) the sterilization status of grains (Figure 1b and Table S1b). In most cases, 2,4-D significantly inhibited root growth only in unsterilized germinating grains of all tested genotypes, including the nonallelic twN18 and twmk mutants. The twmk mutant was the most sensitive to 2,4-D among all tested tweaky-type mutants. In contrast, 2,4-D-induced root growth inhibition in sterilized germinating grains mostly in a nonsignificant manner (Figure 1b and Table S1b).
The tested lower 2,4-D concentrations (10–50 mg L−1) revealed an interesting dependence of 2,4-D-induced root growth inhibition on grain sterilization status; consequently, the effect of elevated 2,4-D concentrations was investigated in further experiments. In the range of 50–800 mg L−1 2,4-D, a significant effect of 2,4-D on MGG was observed only in unsterilized grains of WT (p = 0.0026), in which 2,4-D increased the level of MGG (Figure 2a and Table S2a), while in the range of 10–50 mg L−1 2,4-D, a significant increase in MGG was observed only in sterilized grains of the tw2 genotype (p = 0.024; Figure 1a and Table S1a).
In contrast to the effect of the lower concentrations, the higher concentrations (50–800 mg L−1) of 2,4-D induced a significant decrease in root length independent of the plant genotype and the grain sterilization conditions, and the inhibitory effect of 2,4-D on root growth was dose-dependent (Figure 2b,c and Table S2b). The root growth of the tw and tw2 allelic mutants, but not the tw1 allelic mutant, was weaker than that of WT germinating grains. An unsterilized grain background better revealed the inhibitory effect of 2,4-D on the germination rate, which was uniform independent of the plant genotype. In turn, grain sterilization revealed better differences among allelic mutants in the response to 2,4-D according to the germination rate (Table S2c). In general, 2,4-D in the range of 50–800 mg L−1 inhibited root length independent of the plant genotype (Figure 2c), while in the range of 10–50 mg L−1 2,4-D, differences between WT and tw-type mutants and among allelic tw mutants themselves were observed (Figure 1).
Despite the proposed opposite effects to the action of 2,4-D, the auxin inhibitors HFCA and PCIB did not show a significant effect on MGG, except PCIB in sterilized grains of the tw2 mutant, in which the MGG level decreased (Figure 2a and Table S2a). However, both auxin inhibitors suppressed root growth similarly to 2,4-D (Figure 2c and Table S2b).
Theoretically, the effects of TCA and SA are supposed to be opposite to those of 2,4-D, and TCA decreased the frequency of MGG in unsterilized germinating grains of the WT and all allelic tw-type mutants (Figure 3 and Table S3).
Figure 3. Effects of salicylic (SA) and trans-cinnamic (TCA) acids on (a) the frequency of mouldy grains (%) and (b) the root lengths (cm) of unsterilized and sterilized germinating grains of barley tw mutants and the WT, as measured after 5 days. For mouldy grains, n = 10 (100 grains); for root length, n = 30. Grain was produced in 2016, and the MGG test was performed in 2020. The asterisks represent significant differences (* p < 0.05; ** p < 0.01) between the control and the SA or TCA treatment. The numbers denote significant differences (2 p < 0.01; 3 p < 0.001) between the controls of the tw-type mutant and the WT.
However, in sterilized germinating grains, TCA decreased MGG at a significant level only in the allelic tw mutant, while SA decreased MGG at a significant level only in unsterilized grains of the tw2 mutant (Figure 3 and Table S3a). Similar to 2,4-D, SA and TCA also inhibited root growth, but only in the allelic mutant tw2 (Figure 3b and Table S3b). Neither compound showed any effect on the grain germination rate (Table S3c).
2.2. Fungi Spectrum in the Internal Grain Tissues of Barley tw-Type Mutants and Revertants
To reveal the possible differences in the fungal diversity in surface-sterilized mouldy germinating grains of tested barley genotypes, the spectrum of fungi species was investigated. Among the fungi that frequently reside in the internal tissues of barley grains, B. sorokiniana prevailed in all the tested plant genotypes (Figure 4 and Table S4). In addition to the WT, tw1 and tw2 mutants, the fungi spectra were also studied in several revertants that arose during the phase of stabilization of tw1 and tw2 mutants. The revertant studies allowed for a broader understanding of the differences between tw-type mutants and the WT.
Figure 4. Spectra of fungi (%) in the internal grain tissues of barley tw mutants and the WT. The middle row—revertants from tw1; the lower row—revertants from tw2. N—revertants with normal spike and floral structure, C—revertants with normal floral structure but compactoid spikes. Grain was produced in 2013, and the analysis was performed in 2014. The asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) between the tw-type mutant and the WT (in the upper row) or between the revertant and the respective initial tw mutant (N1, N13 and C1 derived from tw1, N46, C6 and C7—from tw2).
In the grains of the tw1 and tw2 mutants, the B. sorokiniana frequency was lower than that in the WT strain, but the difference was only statistically significant in tw1. Interestingly, the level of B. sorokiniana was only significantly lower in grains of revertant N1 compared to WT (p < 0.05) and remained the same as in grains of its parental mutant tw1. Such a tendency was not observed. A similar tendency was also observed in other revertants, except for the compactoid (C)-type revertants from tw2, but only in an insignificant manner. However, a decrease in the Bipolaris proportion occurred at the expense of the increasing Fusarium portion in the fungi spectra, and the observed effect was statistically significant (Figure 4). Comparable results were also obtained after analysing the fungi spectra in the internal grain tissues in our previous studies [22]. This finding provided a pretext for studying the effects of grain meals made from the different tw-type allelic mutants and the WT on B. sorokiniana growth.
2.3. The Impact of Meals from Grains of tw-Type Mutants on the Colony Growth of Bipolaris sorokiniana
The growth of B. sorokiniana colonies on MEA media supplemented with meals prepared from the grounded dry grains of allelic mutants tw, tw1, and tw2 was compared with B. sorokiniana growth on MEA medium containing meals from the grains of WT. Additionally, the effects of the SA and TCA concentrations were investigated on such media (Figure 5). The meals from the allelic tw mutants significantly decreased the growth of B. sorokiniana colonies. In most cases, a further statistically significant decrease in Bipolaris colony growth occurred only after TCA but not SA addition. SA decreased the growth of B. sorokiniana in a concentration-dependent manner only in tw2. TCA also showed a strong inhibitory effect on B. sorokiniana growth on MEA media with meals from the WT. Even the lowest concentration of TCA, 0.05 mM, significantly decreased the growth of Bipolaris colonies, and a further increase in TCA concentration did not enhance its inhibitory effect on B. sorokiniana growth in WT media (Figure 5).
Figure 5. Effects of salicylic acid (SA) and trans-cinnamic acid (TCA) on the growth of Bipolaris sorokiniana after 7 days of growth on medium containing meal from tw. (a) The morphology of B. sorokiniana colonies grown on MEA media with different supplements. (b) Size of the colonies 7 days after inoculation. MEA, malt extract medium. Grain was produced in 2013, and the experiment was performed in 2014. The asterisks represent significant differences (* p < 0.05; ** p < 0.01; *** p < 0.001) between the control and the SA or TCA treatment. The numbers denote significant differences (2 p < 0.01) between the controls of the tw-type mutant and the WT.
3. Current Insights
The present study showed that more appreciable conclusions on the action of the modifying factors could be made when the MGG assay is performed in parallel with the root growth test. The inhibition of root elongation of germinating grains is one of the earliest and most distinct symptoms exhibited in response to auxins, especially 2,4-D [18], but auxin promotes lateral root formation [13], and this auxin feature is related to pathogen invasion [17]. In the present study, significant differences in 2,4-D-induced root growth inhibition among the different allelic tw-type mutants were revealed only in the lower range (10–50 mg L−1) of 2,4-D concentrations (Figure 1b). Moreover, significant root length inhibition with 2,4-D in the allelic mutants tw and tw2 and the nonallelic mutant twN18 was only observed in unsterilized germinating grains, whereas in sterilized grains, root growth inhibition in these mutants was absent (Figure 1). While differences in MGG frequency between sterilized and unsterilized grains are naturally expected, the dependence of root growth after 2,4-D treatment on grain sterilization status was quite an unexpected phenomenon. Hypochlorite-based agents, including commercial bleach, are routinely used for surface sterilization of various plant materials [32,52]. Hypochlorite was proposed to react with the seed surface, forming a chlorine cover that is not completely removed by rinsing, and subsequently can be converted into highly toxic chloramines that easily penetrate plant tissues [32]. Furthermore, various salts are known to antagonize the phytotoxicity of several herbicides, including 2,4-D [53,54]. After surface sterilization with commercial bleach, nonremovable chlorine compounds can antagonise exogenously applied 2,4-D and subsequently diminish the effect of root growth inhibition in comparison to that of unsterilized 2,4-D-treated grains. This observation highlights the importance of comparing sterilized and unsterilized grain conditions in studies of plant-auxin–pathogen relationships where the action of exogenous auxin will be analysed in germinating grains since hypochlorite-based sterilization itself can lead to underestimation of the 2,4-D
This entry is adapted from the peer-reviewed paper 10.3390/agronomy11112180
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