Effects of growing medium, temperature and light intensity on the HAs in the leaf tissue

In the field experiment lower levels of HAs were recorded than previously measured in the plants growing in the growth cabinet. An experiment was set up to test how different temperature / light intensity and growing medium can affect the HA levels in the hexaploids Tybalt and Solstice, in the tetraploid Alifen and the diploid Ae. speltoides. In a controlled cabinet we tried to simulate the outside conditions (a cloudy day in the autumn) for this reason we reduced the temperature and the light intensity (Story 2003). Our hypothesis is the low light intensity and temperature will reduce the HAs level in the plants.

Plants were allowed to develop in the different environment straight after sowing. Because in the cold environment development is slowed down we sampled the plants twice. In the optimal growing condition when the plants were 7 days old the height was measured and leaf samples were taken. In the cold environment we sampled the plants at the first time (2 weeks after sowing) when they reached the same plant height as we previously measured in the warm environment. The second sampling (20 days after sowing) was carried out when the growth stage matched the stage recorded in the warm environment at the sampling time.

Overall, the statistical analysis showed the temperature and the light intensity had a significant effect on DIMBOA-glucoside and DIMBOA (+HDMBOA-glucoside) levels (P<0.008) and the plant height (P<0.001) (Figure 85-86.). The DIMBOA-glucoside level was significantly higher in the warmer environment with high light intensity compared to the level found in the plants in the cold under low light intensity (P<0.001). A decrease in the glucoside level was noticed in the cold environment by the second sampling time compared to the first time point. The DIMBOA (+HDMBOA-glucoside) level increased while the DIMBOA-glucoside decreased in the cold environment, which could be a stress reaction to the low light intensity and temperature. This stress reaction shows in the plant height as well, since in the cold environment seedlings were taller because they lacked light (etiolation) and the development of the plants was slower than in the optimal growing conditions.

Figure 85. HA level differences in the leaf tissue in different growing environments (20°C, 9500 Lux, 16:8 D:L and 10°C, 1150 Lux, 12:12 D:L). All varieties were sampled twice in the cold environment (10CI. and

10CII.).

Figure 86. Plant height differences in different growing environments (20°C, 9500 Lux, 16:8 D:L and 10°C, 1150 Lux, 12:12 D:L). All four varieties were measured twice in the cold environment

(10CI. and 10CII.).

The growing medium had no effect (P=0.9) on the DIMBOA (+HDMBOA-glucoside) level, but a significantly lower level of DIMBOA-glucoside (P<0.001) was recorded in plants grown in compost (Figure 87.). This may have been due to a fungal infection, which was noticeable a couple of days after germination on some of the seeds.

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Mean of combined DIMBOA and HDIMBOA-glu (mmol/kg FW)

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Figure 87. The mean of DIMBOA (+HDMBOA-glucoside) and DIMBOA-glucoside levels in the leaf tissue of plants (Tybalt, Solstice, Alifen and Ae. speltoides) growing in compost and vermiculite. The DIMBOA-glucoside level was significantly lower in the compost but no difference was found in the DIMBOA level.

A significant difference was recorded between the varieties (P<0.001), but they reacted similarly to the different test environments in the different media.

Tybalt

In the optimal growing conditions in compost Tybalt leaf tissue (GS11) contained 5.2 mmol/kg FW DIMBOA-glucoside. This was significantly greater (P<0.006) compared to the two samples taken in the cold environment with low light intensity where the DIMBOA-glucoside level was 0.7 mmol/kg FW by the time the plant reached the 1+¹/2 leaf stage (Figure 88.). The DIMBOA (+HDMBOA-glucoside) level was similar in all growing conditions ≈12mmol/kg FW. Plants growing in vermiculite showed similar changes in the compounds; at 20°C plants contained 5.1 mmol/kg FW of DIMBOA-glucoside, which was significantly higher (P=0.003) compared to the plants at the same growth stage grown in the cold environment (2.4 mmol/kg FW). The DIMBOA (+HDMBOA-glucoside) level remained very similar in the plants growing in both media ≈12 mmol/kg FW (P>0.2)

0 2 4 6 8 10 12

DIMBOA-glu DIMBOA (+HDMBOA -glu)

mmol/kg FW

compost vermiculite

a b

a a

Figure 88. Hydroxamic acid level in the leaf tissue of Tybalt in the different growing environment. Warm env. 20°C, 9500 Lux, cold env. I.= first sampling time in the cold environment, 10°C, 1150 Lux, cold env. II.

second sampling time in the cold environment.

Solstice

In the warm environment in compost, 2.7 mmol/kg FW of DIMBOA-glucoside was recorded, which was similar to the level found in plants growing in the cold environment.

Significantly higher amount of DIMBOA (+HDMBOA-glucoside) was measured (P<0.0006) in the plants growing in the cold environment compared to the optimal conditions where the level reached 5.4 mmol/kg FW (Figure 89.). At 20°C in plants growing in vermiculite the DIMBOA-glucoside level was 3.9 mmol/kg FW, which was significantly higher (P=0.001) compared to the plants growing in the cold environment at the second sampling stage. The DIMBOA (+HDMBOA-glucoside) level was similar in all stages and growing conditions ≈4mmol/kg FW. We recorded a significant difference between the growing media in the DIMBOA (+HDMBOA-glucoside) level; in the warm environment we found less (P=0.01) aglucone in the plants growing in compost compared to vermiculite, however in the second sampling time at 10ᵒC the aglucone (P=0.048) and the glucoside (P=0.001) level was lower in the plants growing in vermiculite than in the

Figure 89. Hydroxamic acid level in the leaf tissue of Solstice in the different growing environment. Warm env. 20°C, 9500 Lux, cold env. I.= first sampling time in the cold environment, 10°C, 1150 Lux, cold env. II.

second sampling time in the cold environment.

Alifen

Plants growing in vermiculite and compost did not show a significant difference in the DIMBOA-glucoside levels, which was between 3.1 and 4.8 mmol/kg FW under the different growth regimes (Figure 90.). Significantly higher level of DIMBOA (+HDMBOA-glucoside) was recorded (P=0.03) in the cold environment at the first sampling stage (4.6 mmol/kg FW) in vermiculite compared to the optimal growing environment (1.7 mmol/kg FW) however no statistical difffernces was recorded in compost between the different environment. In this case the medium did not have an effect on the HA levels.

Figure 90. Hydroxamic acid level in the leaf tissue of Alifen in the different growing environment. Warm env. 20°C, 9500 Lux, cold env. I.= first sampling time in the cold environment, 10°C, 1150 Lux, cold env. II.

second sampling time in the cold environment.

Aegilops speltoides

The greatest differences were recorded in this high HA producing diploid variety. In the optimal warm growing conditions in compost there was 19.4 mmol/kg FW of DIMBOA-glucoside in the leaf tissue, which was significantly higher compared to the cold environment where the glucoside reached only 10.2 mmol/kg FW (P<0.01). The DIMBOA (+HDMBOA-glucoside) level remained between 10.8 and 15.9 mmol/kg FW (Figure 91.).

In vermiculite, levels of both compounds were significantly different (P<0.0001) between the cold and the warm environment. The DIMBOA-glucoside level was 22.7 mmol/kg FW in the leaf tissue of the plants growing in the favourable condition while in the cold environment it was only 11 mmol/kg FW. The DIMBOA (+HDMBOA-glucoside) level was higher in the cold environment (14.8-16.2 mmol/kg FW) but remained low (7.9 mmol/kg FW) in the warm environment. A significant difference was recorded between the growing media in the DIMBOA-glucoside level at 20ᵒC, we measured lower (P=0.02) amounts of glucoside in the plants growing in the compost than in vermiculite.

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Figure 91. Hydroxamic acid level in the leaf tissue of Ae. speltoides in the different growing environment.

Warm env. 20°C, 9500 Lux, cold env. I.= first sampling time in the cold environment, 10°C, 1150 Lux, cold env. II. second sampling time in the cold environment.

Summary

This experiment was intended to provide more evidence of how temperature, light intensity and growth medium can affect the level of the HAs in the plants. The lower temperature and light intensity slowed down the development of the plants which caused etiolation and possibly a stress reaction that resulted in a decrease in the DIMBOA-glucoside level and an increase in the DIMBOA level compared to the optimal growing condition. These observations are consistent with our hypothesis. However, more sampling times are required in the different growing conditions from the germination stage all the way through the experiment to make a standard curve which would present the HA level in the different growth stages related to the age of the plant. This was a good provisional experiment to show the kind of problems the different growing conditions may produce.

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5. DISCUSSION

Hydroxamic acids are secondary metabolites, which can be found in the cultivated monocotyledonous plants maize, wheat and rye. They can play an important role in the development of resistance against bacteria, nematodes, fungi and insects (Thackray et al., 1990, Nicol et al., 1992 & 1993; Gianoli et al., 2000; Niemeyer, 2009). In the hexaploid and tetraploid wheat varieties tested, mainly DIMBOA-glucoside and smaller amount of combined DIMBOA and HDMBOA-glucoside were detected in the undamaged leaf tissue samples. In the leaf tissue of the hexaploid varieties, levels of DIMBOA-glucoside ranged from 1.2 to 4.7 mmol/kg FW in 6 day old plants, and these levels decreased by the time the plants were 13 days old. Combined levels of DIMBOA (+ HDMBOA-glucoside) ranged from 1.4 to 7.8 mmol/kg FW. The highest level of HAs was recorded in the RWA resistant variety Fiorello, but low level was detected in the other RWA resistant variety Turksikum which suggests that HAs are not directly related to RWA resistance as expressed in such varieties. In the tetraploid varieties tested, the DIMBOA-glucoside level was twice as high (from 11.3 to 14.3 mmol/kg FW) as the maximum level found in the hexaploid varieties indicating that the tetraploids may have greater potential for natural defence. This reduction in the level of HAs from the 4x to 6x species was also reported by Niemeyer and Jerez (1997). The maximum level of DIMBOA (+ HDMBOA-glucoside) in the tetraploids was 4.6 mmol/kg FW, which was comparatively lower than that recorded in the hexaploid varieties. The study demonstrated that levels of the HAs vary within and between hexaploid and tetraploid wheat varieties and that they decrease as the plant ages and were therefore insufficient in these plants to affect longer term aphid development and fecundity.

Most of the B genome species (Aegilops searsii, Ae. longissima, Ae. bicornis and Ae.

sharonensis) possess only very low or non-detectable levels of the known HAs in the leaves of seedlings. However, the accession of the B genome species Ae. speltoides, which is the closest ancestral species of the B genome donor of the hexaploid wheat, contained over 20 mmol/kg FW in the seedling leaves, which is a considerably higher level of HAs than reported previously in either the 6x or 4x species. It is possible that there is variation

in the HA level within the Ae. speltoides species, and the ancestral B genome donor was a lower HA producer variety than the accession we tested. Gianoli et al. (1997) suggest further reduction was caused by the incorporation of the D genome which may inhibit the accumulation of HAs in the hexaploid varieties.

In the A genome species (Triticum monococcum and T. boeoticum) no known HAs were detected in the leaf tissue. Similar studies by Nomura et al. (2007), found no HAs in T. boeoticum. This was also confirmed by the genomic DNA amplifications of the BX gene sequences which showed the absence of the Bx3 gene, indicating that the HA pathway is incomplete and neither the glucoside nor the aglucone can be produced in T. boeoticum.

Thus, in the A genome species there were no corresponding links between aphid feeding and increased levels of the known HAs. Two unknown compounds were detected by HPLC, which may have an effect on aphid behaviour and development. The production of both compounds was followed up in two T. monococcum varieties (MDR 037 and MDR 049). Of the two novel compounds, the expression pattern of compound II is similar between the two lines tested, in that it builds continuously in the leaf, and would suggest that this is not responsible for any differences in aphid growth between the lines MDR 037 and MDR 049. Compound I on the other hand is expressed differently between the lines, with the more aphid resistant line MDR 049 showing higher concentrations in progressively older leaves. .

Niemeyer’s publications indicate that the higher level of HA in the leaf tissue cause reduction in nymph production. It is possible that the range of levels of HAs presents in the healthy leaf tissue of the tetraploid and hexaploid varieties tested was insufficient to have a negative effect on R. padi behaviour or development. For example, 14% more alatae settled on Humber, which contained 3.3 mmol/kg FW DIMBOA-glucoside and 4.9 mmol/kg FW of DIMBOA (+ HDMBOA-glucoside) in the leaf tissue of 6 day old seedlings, compared to Solstice, which contained only 1.9 mmol/kg FW DIMBOA-glucoside and 1.7 mmol/kg FW of DIMBOA (+ HDMBOA-DIMBOA-glucoside). In addition, the average nymph production on Humber in the fecundity test was 69.6, which is 33% higher than the average for the other hexaploid varieties tested. The varieties carrying resistance genes to other insect pests did not have negative effects on aphid settling behaviour. The greatest number of alatae was noted on the RWA resistant hexaploid variety PI 137739 which has the Dn1 resistance gene.

The least preferred varieties belong to the A and B genome diploid species. Significantly lower numbers of aphids settled on these plants than on the control. In particular, significantly fewer aphids settled on line 8404 (T. boeoticum) and MDR043 (T.

monococcum) than on the control. MDR040 and MDR037 were highly attractive to R. padi and 2.5-3 times more aphids settled on those varieties than on Solstice. All tested B genome varieties, whether HA producer or non-producer, were less attractive to R. padi alates, since significantly fewer aphids settled on the test plants than on the control, but when the diploid varieties were tested against each other there was no preference between them. The negative effect of the diploid varieties could be caused by the morphological, chemical and nutritional attributes of the species, which makes the adaptation and acceptance of those plants as a host more difficult to R. padi aphids or it could be due to the aphids being reared on a hexaploid variety and they prefer what they are used to. The development/fecundity tests are a better measure of acceptance in this instance as they reflect the antibiotic effects, although the unidentified compounds which were detected on the HPLC could play a role in the reduction in the settling.

In the fecundity test, aphids had no choice but to feed on the test plants and substantial differences in the rate of reproduction of R. padi between varieties was seen.

The intrinsic rate of population increase values for R. padi on the A genome diploid varieties was 4-15% lower than on the hexaploid control and nymph development and maturation was slower. In fecundity test III, the average daily nymph production on the non HA producing diploid varieties (B genome: Ae. longissima, Ae. sharonensis and the A genome: T. monococcum) was 3.9 compared to 5.2 on the hexaploid variety Solstice.

Although seemingly small differences, this reduced fitness of the aphids if translated to field conditions would substantially alter the dynamics of aphid population growth. Aphids on Ae. speltoides (B genome), which had higher levels of HAs, had a significantly lower intrinsic rate of population increase compared to the those on the hexaploid control (Solstice). The average daily nymph production on Ae. speltoides during the test period was 2.05 compared to 5.2 on Solstice. Certainly the development and reproduction of the aphids on this diploid species was substantially reduced to a level which if replicated in the field would greatly facilitate protection of the crop from aphid damage. However aphids have a great potential to adapt to different conditions because of the high number of

generations per year and they may be able to adapt to the higher level of HAs which have been found in Ae. speltoides.

As with the settling test, the reproduction of R. padi on the wheat accessions selected because of resistance to other insect pests was not reduced significantly compared to the control.

Studying the effect of R. padi feeding, the HPLC peak representing DIMBOA (+HDMBOA-glucoside) levels in the hexaploid varieties showed a higher level after 24 hours of aphid feeding and was maintained after 48h, while the DIMBOA-glucoside level was lower compared to plants with no feeding damage. This effect was seen most strongly in the variety Tybalt. These effects within the host plant were localised near the aphid feeding site and were not found at the base of the leaf or in the rest of the plant. Although the actual levels of each compound (DIMBOA and HDMBOA-glucoside) could not be determined, the reduction in DIMBOA-glucoside is consistent with the hypothesis that aphid feeding caused sufficient cell damage to release DIMBOA-glucoside from the cell vacuoles enabling contact with β-glucosidase enzymes, which transformed it into DIMBOA to protect the plants against further insect damage (Niemeyer, 2009 and papers therein). Previously Oikawa et al. (2002) showed a decrease in DIMBOA-glucoside and an increase in HDMBOA-glucoside in wheat plants after induction with jasmonic acid, indicating that the induction of the methyltransferase enzyme activity was responsible for the accumulation of HDMBOA-glucoside. This current study has not resolved the relative importance of these two alternative fates for DIMBOA-glucoside owing to the co-incidence of DIMBOA and HDMBOA-glucoside in the HPLC analysis. Therefore, the possibility that HDMBOA-glucoside increases in wheat on aphid feeding as occurs in maize (Ahmad et al., 2011) should be followed up in future work in order to be clear as to which toxic HA aphid pests are most exposed to.

The response of the diploid Ae. speltoides to aphid feeding was not as great as that seen in the hexaploids, possibly because the HA levels in Ae. speltoides are already comparatively high and the plant is not able to increase the level of the compounds further. Follow up work is recommended to test if the defence reaction is systemic. Aegilops sharonenesis is a null HAs expressor in the leaf, but it can produce both compounds in the root and coleoptile therefore the metabolic pathways are intact and there is a difference in the

regulatory control of expression leading to the substantial difference in expression of HAs in leaf tissue compared to Ae. speltoides.

Niemeyer and Givovich (2000) suggested that the level of HAs in the host plant directly affects the feeding behaviour and performance of R. padi. However, we observed no clear relationship between the levels of HAs in leaf tissue and aphid settlement and reproduction. Many of the accessions tested have high HA levels (such as Fiorello, Humber, Turcikum 57) and these levels should be toxic (if compared to the artificial diet results), but showed no significant effect on aphid settling and fecundity.

In our artificial diet tests the levels of DIMBOA used were lower than those found in wheat leaf tissue in these current studies and by Argandona et al. (1981 and 1983) or Nicol and co-workers (1992). On the diet containing 2 mM DIMBOA (below the level found in most of the tested varieties) only 10.7% of the aphids were alive after 72 hours. A possible explanation of this apparent discrepancy is that the aphids do not come directly into contact with sufficient levels of aglucone in the leaf tissue before reaching the phloem,

In our artificial diet tests the levels of DIMBOA used were lower than those found in wheat leaf tissue in these current studies and by Argandona et al. (1981 and 1983) or Nicol and co-workers (1992). On the diet containing 2 mM DIMBOA (below the level found in most of the tested varieties) only 10.7% of the aphids were alive after 72 hours. A possible explanation of this apparent discrepancy is that the aphids do not come directly into contact with sufficient levels of aglucone in the leaf tissue before reaching the phloem,