Effects of a short-term p-hydroxybenzoic acid application on grain yield and yield components in different tiller categories of spring barley

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Effects of a short-term p-hydroxybenzoic acid application on grain yield and yield components in different tiller categories of spring barley

O. C H R I S T E N and J.V. LOVETT

Institute of Crop Science and Plant Breeding, Christian-Albrechts-University Kiel, W24118 Kiel 1, Germany, and Department of Agronomy and Soil Science, University of New England, Armidale, N.S.W. 2351, Australia

Received 16 September 1992. Accepted in revised form 3 April 1993

Key words: allelopathy, grain yield, phytotoxicity, phenolic compounds, p-hydroxybenzoic acid, spring barley, yield components, yield losses

Abstract

The aim of these experiments was to evaluate how thresholds for phytotoxic substances obtained in seedling bioassays relate to yield losses or changes in yield components of mature barley crops after a short-term exposure to p-hydroxybenzoic acid. Under laboratory conditions a treatment with 1.81 mM p-hydroxybenzoic acid significantly reduced the radicle length of barley, whereas coleoptile elongation was less sensitive. The inhibition of the radicle length and coleoptile elongation was greater if the pH of the test solution was not buffered at pH 5.5. In a glasshouse trial the effect of p-hydroxybenzoic acid on the radicle and coleoptile elongation of spring barley was compared with the yield response after a three day exposure either during germination or at the double ridge stage of apex development. Applications of 0.72mM, 1.44mM and 3.62mM p-hydroxybenzoic acid averaged over the treatments during germination or at the double ridge stage of development caused a yield reduction in the single ear weight of 5%, 13% and 19% in comparison with the control, respectively. The higher tiller categories in general showed a greater sensitivity towards an application of p-hydroxybenzoic acid and, therefore, could not compensate for the yield decrease of the main stem tiller. A single application of p- hydroxybenzoic acid either at germination or at the double ridge stage may cause yield losses, as reported from no-till systems or cereal monocultures. The data have implications for the interpretation of seedlings bioassays in allelopathic research and their applicability in estimating yield losses caused by phytotoxic substances.

Introduction

Substantial evidence has been accumulated that yield reduction of cereals in no-tillage systems short cereal rotations or monocultures is at least partly caused by phytotoxic substances released during the decomposition of plant residues (McCalla and Norstadt, 1974; Mfiller-Wilmes et al., 1977; Wolf and H6flich, 1983). However, the exact contribution of allelochemicals to yield losses is unknown, since the most common

methods of identifying phytotoxicity have been germination or seedling growth bioassays using extracts from cereal straw and other residues (Guenzi et al., 1967; Mason-Sedun and Jessop, 1989). A response in a bioassay is normally considered as an indication for aUelopathy (Kimber, 1973; Leather and Einhellig, 1986;

Stowe, 1979).

Extensive research has identified the chemical nature of allelochemicals associated with decomposing cereal residues as being mainly phenolic

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280 Christen and Lovett

and short chain aliphatic acids (Chou and Pat- rick, 1976; Guenzi and McCalla, 1966; Tang and Waiss, 1978; Wojcik-Wojtkowiak et al., 1990). In addition, Norstadt and McCalla (1963) isolated the antibiotic patulin produced by a number of different soil fungi as another cause for toxicity problems in mulch farming.

According to a recent review by Siqueira et al.

(1991), most phenolic acids present in crop residues have also been isolated from soils, however, their concentration varies considerably depending on soil type, amount of organic matter, previous cropping, type and amount of retained residues and extraction method. Based on the reviewed literature Siqueira et al. (1991) concluded that the amount of p-hydroxybenzoic acid might peak at 1400 nM in the soil solution.

But other phenolic acids will contribute to phytotoxic effects, since in a soil environment normally a mixture of phenolic acids can be isolated.

In a series of experiments McCalla and as- sociates (Ellis and McCalla, 1973; McCalla and Norstadt, 1974; Norstadt and McCalla, 1971) quantified the effect of a single application of the microbial product patulin on wheat grown to maturity.

Other experiments with short term exposure of crops to a phytotoxic chemical were either conducted with other plant species, different chemicals or the plants were not allowed to grow to maturity. Cochran et al. (1983) compared the influence of various concentrations of acetic acid on the average number of first stem tillers of wheat and described an almost 30% reduction after a 4 day exposure with 10 mM compared with the control, but lower concentrations of a short term exposure with acetic acid did not cause long-term effects in their experiments. In contrast, Waters and Blum (1987) report that phaseolus bean plants recovered from 1.0 mM or 2.0 mM two day treatment with ferulic acid at seedling or flowering. Only an exposure at pod- fill caused a significantly lower leaf area and plant dry weight at maturity due to water stress and a loss of turgidity. Using cucumber in a series of experiments Blum and Rebbeck (1989) observed a rapid recovery of roots after a short- term exposure with ferulic acid, but did not relate this result to later growth, development or yield parameters.

In contrast, research on cereal development and organogenesis with respect to reaction towards fertilizer (Langer and Liew, 1973; Thorne et al., 1988) or herbicide (Tottman, 1977) applications has focused on stage specific responses and identified differences in susceptibility depending on the developmental stage of the shoot apex. In particular the transition from the vegetative to the generative stage, the so called 'double ridges stage', seems to be a period with a particularly high sensitivity towards external factors, but so far no experiments in the area of allelopathy have addressed this.

The aim of this study was to compare the thresholds of a single application of p-hydroxybenzoic acid as an example for a phenolic compound which has been frequently isolated from soil or soil water on grain yield and yield components of spring barley with the degrees of inhibition in a coleoptile and radicle elongation bioassay with special emphasis on the yield of the different tiller categories.

Methods

Coleoptile and radicle elongation bioassay In order to obtain some estimates about the magnitude of effects of a phenolic compound in a bioassay using a spring barley (cv. Lara), a series of preliminary experiments to optimize cultural conditions, i.e. temperature and solution volume, was conducted. Based on these results a two factorial experiment was designed comparing the interaction between different concentrations of p-hydroxybenzoic acid (Sigma Chemi- cal Company, St Louis, Missouri) and the pH of the test solution. P-hydroxybenzoic acid was dissolved in hot deionized water in concentrations of 0.72 mM and 3.62 mM to 7.24 mM. In one treatment the pH was allowed to drop according to the concentration of p-hydroxybenzoic acid from 5.5 in the control to 4.1, 3.7 and 3.5 in the different p-hydroxybenzoic acid treatments, respectively. In the other treatment the pH was buffered with a 0.9 mM N H 4 H 2 P O 4 and 0.1 mM ( N H a ) 2 P O ~ buffer system at a pH of 5.5. The coleoptile and radicle elongation bioassay was conducted with 10 seeds of barley in 9 cm diameter petri dishes placed on Whatman

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filter paper No. 1 using a volume of 5 mL test solution for each petri dish. Treatments were replicated 4 times and randomly arranged in a growth chamber with 25°C constant temperature and darkness. The elongation of the radicle and the coleoptile was measured after 72 h. In this experiment all seeds germinated.

Glasshouse experiment

Based upon the results of the petri dish experiment, a glasshouse trial with the same spring barley variety comparing three different factors was designed. According to the methods described previously the factors consisted of 4 different concentrations of p-hydroxybenzoic acid (control, 0.72 mM, 1.4 mM and 3.62 mM), the optional control of the pH was included and therefore the pH was either allowed to drop according to the concentration of p-hydroxybenzoic acid or was buffered with a 0.9mM N H 4 H 2 P O 4 and 0.1mM (NH4)2PO 4 buffer system at a pH of 5.5.

Additionally two different developmental stages for the application of p-hydroxybenzoic acid were compared. In the first treatment p- hydroxybenzoic acid was applied with the nutrient solution for three days during germination, in the second treatment p-hydroxybenzoic acid was applied with the nutrient solution for three days at the sensitive double ridge stage of the main stem tiller, which marks the transition of the apex from the vegetative to the generative development (Kirby and Appleyard, 1984; Land- es and Porter, 1988). At the second application date (double ridges) the nutrient solution includ- ing p-hydroxybenzoic acid was applied to the sand, avoiding direct contact with the above- ground parts of the barley. This application method should simulate the leaching of a phytotoxic substance in a single exposure following a rain event.

Nine seeds of barley were planted 2 cm deep in each of the 30 cm diameter pots filled with sand. This sand was unwashed river sand, that is, essentially a coarse siliceous, inert material which may have a small amount of organic matter as a contamination but no significant content of nutrients. It did not have any significant sorptive capacity either.

The pots were randomly arranged in a glass-

house with a day/night temperature setting of approximately 24/14°C. Apart from the treatment periods the pots were watered every day and once a week received watering with a standard nutrient solution. After the first leaf was visible the three innermost plants were marked; only these plants were used for further analysis to avoid border effects. With these plants single ear analyses were conducted. The total growing period from planting to maturity was 113 days. The double ridge treatment was applied 25 days after planting. At maturity all shoots having fertile ears were harvested indi- vidually and, after drying at 80°C for 48 h, the grain weight and ear structure (kernels per ear) were determined. The thousand grain weight (TGW) was calculated. All tillers of higher categories than T2 are included in the tiller category T2.

All data were subjected to a statistical analysis using PROC GLM (Generalized Linear Models), option LSMEANS of the SAS package (SAS 1985; Searle 1987). The statistical proce- dure GLM was applied to allow for the unequal numbers of observations in the cells.

Results

Radicle elongation of barley seeds was more sensitive in the petri dish bioassay to p-hydroxybenzoic acid treatment than was coleoptile length, however, the results of the experiment indicate a strong interaction between the phenol concentration and the pH of the test solution (Table 1).

A treatment of 1.81mM p-hydroxybenzoic acid caused a significant reduction in the radicle length (20% compared with control) if the pH was adjusted at 5.5. The highest concentration of p-hydroxybenzoic acid resulted in a more than 40% reduction of the radicle length. If the pH was allowed to drop according to the concentration of the p-hydroxybenzoic acid, radicle elongation showed an even greater sensitivity in the treatments with 3.62mM and 7.24 mM. At the highest concentration the radicle length measured an average of 8.5cm, only 14% of the elongation of the untreated control. Though less sensitive compared with radicle elongation the coleoptile responded in a similar way. In the

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Table 1. Effects of p-hydroxybenzoic acid ( H B A ) and p H adjustment on coleoptile and radicle elongation (mm) of barley seeds

T r e a t m e n t Concentration (mM)

Con trol 1.81 3.62 7.24

Coleoptile

H B A ( p H adj. to 5.5) 33.7" 33.6 a 30.9 "°c 29.0 bc

H B A ( p H according to H B A 32.3 a 31.3 ab 28.0 bc 14.8 d

concentration) Radicle

H B A ( p H adj. to 5.5) 57.3 a 46.0 b 39.0 c 32.9 d

H B A ( p H according to H B A 57.9 a 48.6 ° 30.9 d 8.5 e

concentration)

Values followed by the same letter within the same feature are not significantly different at p < 0.05 level.

treatment with the buffered test solution only the highest concentration of p-hydroxybenzoic acid caused a significant reduction (14%). Without pH adjustment p-hydroxybenzoic acid affected the coleoptile elongation more severely, causing 14% reduction compared with the control at 3.62 mM and 55% at 7.24 mM, respectively.

Based on the results of the petri dish bioassay some lower concentrations of p-hydroxybenzoic acid were included in the development experiment.

Averaged over the two different application stages the single ear yield showed a typical response after the treatment of p-hydroxybenzoic acid (Table 2). Compared with the control an application of p-hydroxybenzoic acid caused a yield decrease between 5 to 19%. It is noteworthy that, regardless of whether p-hydroxybenzoic acid was applied during germination or later, at the double ridge stage yield

losses showed a similar trend, thus demonstrat- ing a similar sensitivity at the two compared developmental stages. The reaction of the yield components 'number of kernels per ear' and 'thousand grain weight (TGW)' showed a similar pattern compared with the single ear weight, however, it could be argued that a slight nonsig- nificant compensating effect occurred at the concentration of 1.44mM p-hydroxybenzoic acid. The thousand grain weight (TGW) com- pensated partly for the lower number of kernels per ear. Such a compensation was not observed at the highest concentration of p-hydroxybenzoic acid and the lower single ear weights were caused by a decrease in both thousand grain weight (TGW) and kernels per ear. Neither during germination nor after an application at the double ridge stage was the effect of p-hydroxybenzoic acid modified by the pH of the test solution, a fact which stands in contrast with the

Table 2. Effects of p-hydroxybenzoic acid and the developmental stage of application on grain weight, kernels per ear and t h o u s a n d grain weight ( T G W ) of barley

Application Concentration ( m M )

stage Control 0.72 1.44 3.62

Grain weight (g)

Kernels per ear

T G W (g)

Germination 0.67 a 0.62"b 0.55b 0.54b

Double ridge 0.65" 0.65" 0.61 "° 0.55 °

Average 0.66" 0.63a~ 0.58bc 0.54 c

Germination 14.7" 14.2 ab 12.5 b 13.6 "h

Double ridge 14.5" 14.8 a 13.1 "b 12.5 ab

Average 14.6 a 14.5 a 12.8 b 13.08

Germination 45.0 abe 42.4 bc 43.3 abe 40.7 c

Double ridge 48.0 a 44.7 abe 47. l"b 43.7abc

Average 46.5 a 43.5 ab 45.2 "b 42.2 b

Values followed by the same letter within the same feature are not significantly different at p < 0.05 level.

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results o b t a i n e d in the coleoptile and radicle elongation bioassay. T h e total n u m b e r of tillers p e r plant (data not shown) were slightly reduced in the highest concentrations, h o w e v e r , the differences were not statistically significant.

T h e previously described response of the single ear weight after an application with p- h y d r o x y b e n z o i c acid is confirmed for all tiller categories, but the magnitude of the reaction differed b e t w e e n the categories (Table 3).

T h e highest concentration of p-hydroxy- b e n z o i c acid only caused a yield decrease of 8%

in the main s t e m tillers ( M ) c o m p a r e d with the u n t r e a t e d control, whereas tillers of the second c a t e g o r y (T2) suffered a 20% reduction in weight. T h e yield of the first tillers (T1) aver- a g e d 13% less at the highest concentration of p - h y d r o x y b e n z o i c acid. T h e slight yield increase at a c o n c e n t r a t i o n of 0.72 m M p-hydroxybenzoic acid was only based on a higher grain weight of m a i n s t e m tillers (M). T h e first (T1) and second (T2) tiller categories showed a yield decrease c o m p a r e d with the control. In general higher tiller categories suffered m o r e severely after an application of p - h y d r o x y b e n z o i c acid and therefore were not able to c o m p e n s a t e for the detri- m e n t a l effects of the phenolic acid. In the u n t r e a t e d control the second category tillers (T2) yielded 53% of the main stem tiller (M). This p r o p o r t i o n d r o p p e d with increasing concentration of p - h y d r o x y b e n z o i c acid f r o m 0 . 7 2 m M , 1.44 m M and 3.62 m M to 4 7 % , 45% and 41%, respectively. T h e yield c o m p o n e n t s b e h a v e d the s a m e way as previously described for the aver-

aged tiller categories. T h e different c o m p o n e n t s of yield showed the s a m e b e h a v i o u r previously described for the single ear yield of the different tiller categories, that is, no c o m p e n s a t i o n a l effect occurred (data not shown).

Discussion

A comparison of the quantitative r e s p o n s e of barley seedlings in a petri dish bioassay t o w a r d s p-hydroxybenzoic acid with the effect on yield and yield c o m p o n e n t s after a s h o r t - t e r m exposure either during germination or at the double ridge stage of apex d e v e l o p m e n t reveals specific thresholds for the sensitivity depending on the technique and the o b s e r v e d p a r a m e t e r s . Radicle elongation, which was significantly decreased at a concentration of 1.81 m M , was m o r e sensitive than coleoptile length, w h e r e a s a significant yield reaction could already be d e t e r m i n e d at 1.44 raM. This result confirms reports f r o m various authors describing the effect of phenolic acids on germination and early seedling develop- m e n t ( C h o u and Patrick, 1976; Einhellig et al., 1982;/Wang et al., 1967). T h e difference in the susceptibility of the coleoptile c o m p a r e d with the radicle elongation might be due to a different Sensitivity of cell elongation c o m p a r e d with cell division, which occurs sequentially in the growing tissues.

T h e evidence p r e s e n t e d d e m o n s t r a t e s the potential of p-hydroxybenzoic acid to substan- tially reduce grain yields of spring barley u n d e r Table 3. Effects of p-hydroxybenzoic acid application on grain weight, kernels per ear and thousand grain weight (TGW) of different tiller categories of harley

Tiller Concentration (raM)

category Control 0.72 1.44 3.62

Grain weight (g)

Kernels per ear

TGW (g)

Main stem tiller 0.96 ab 1.04" 0 . 9 5 ahc 0.89 b~

First category 0.86 t'~d 0.82 d l).79 d 0.75 ~

Second category 0.51~ l).49 ~ 0.43 ~ 0.41 f

Main stem tiller 20.2 ~'b 21.5 ~' 19.4 "~'~ 19.6 "be

First category 1 8 . 6 bc 17.5 ~ 17.4 ~ 17.6 ~c

Second category 11.8 '~ 1 1.9 ~ 10.0 e 10.3 ~

Main stem tiller 47.7 "h 50.6 ~ 49.2" 4 5 . 8 abe

First category 46.6 ~b 46.5 ~,b 45.6 "~'~ 43.1 ,he Second category 46. l"b 41.0 ~ 44. I a~,c 41. i bc Values followed by the same letter within the same feature are not significantly different at p < 0.05 level.

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controlled conditions. We believe this to be the first report of deleterious effect of a phenolic acid in a single application on grain yield and yield components of spring barley. These results, however, have to be interpreted very carefully in relation to yield effects observed in field experiments, since sand with only negligible organic content, as we have used here, does not com- pletely reflect the situation in the field. Addition- ally the concentration of p-hydroxybenzoic acid chosen in this experiment is considerably higher than concentrations found in the soil. However, this concentration of phenolic acids in the bulk soil and the soil solution is affected by various parameters and estimates of the active part are still subject of considerable dispute and specula- tion in the literature (for review see Siqueira et al., 1991). On the other hand, in the field plants are exposed to a great number of different phenolic acids at the same developmental stage, which, in combination, according to Einhellig et al. (1982), affect germination and early growth well below their separate thresholds.

Though not statistically significant, a negative response in grain weight of 5% in our experiment after an application of p-hydroxybenzoic acid with a concentration of 0.72 mM averaged over the two treatment would relate to a considerable yield depression in a field situation. Con- sidering other possible causes of lower yields of cereals in a no-till situation or grown in monoculture (e.g. pathogens, nitrogen deficiencies, changes in soil structural properties etc.), a yield depression of that magnitude solely caused by allelochemicals would be remarkable.

The finding that both yield components, i.e.

kernels per ear and thousand grain weight, were affected by a single exposure to p-hydroxybenzoic acid in the development experiment confirms reports from Ellis and McCalla (1973) applying patulin to wheat in a similar experiment. However, no research so far has considered the reaction of the different tiller categories after a short term application of phytotoxic substances in respect to yield components in different tiller categories.

Although the application date at the double ridge stage of the apex was based on the development of the main stem tiller, the higher tiller categories proved to be more sensitive and

showed a larger yield decrease. It could be argued that the development of the main stem tiller was enhanced after the application of p- hydroxybenzoic acid at double ridge and, sub- sequently, the higher tiller categories suffered a yield reduction due to interplant competition.

Another possibility would be a larger sensitivity of the higher tiller categories at early stages of the apex development which consequently increased the single ear weight of the main stem tiller. Both effects might also appear in combination. The so-called "sensitive stages" and, espe- cially, the transition from the vegetative to the generative development of the apex, are solely based on experiments comparing herbicide or fertiliser treatments and the cereal crop will not necessarily show sensitivity at similar stages towards allelochemicals.

The specific response of different categories in respect to grain yield following an exposure to p-hydroxybenzoic acid clearly demonstrates the need for considering more agronomic and husbandry factors in. the design of experiments investigating allelopathy. Since higher tiller categories showed a larger yield decrease after short-term stress caused by p-hydroxybenzoic acid, the yield depression in a field situation interact with seed density, thus modifying the ability of different tiller categories to compensate for the detrimental effects of allelochemicals.

The great sensitivity of cereal plants to stress at the transition of the shoot apex from the vegetative to the generative development ('double ridges stage'), which has been confirmed in our experiment with p-hydroxybenzoic acid, is not fully understood. The observed effects of p-hydroxybenzoic acid on barley yield and yield components are the integrated results of specific biochemical and cytological reactions of the plant. It has been demonstrated that phenolic acids might affect processes like cell division, cell elongation, membrane permeability and mineral uptake. Tottman (1977) investigating the effect of herbicides on wheat, argues that the yield decrease caused by a stress at 'double ridges' is due to interference with the extreme sensitive process of primordium formation on the shoot apex. It therefore seems also possible, that phenolics like p-hydroxybenzoic acid which might interact with the hormone synthesis will

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Effect of p-hydroxybenzoic acid on yield of spring barley

affect a cereal plant at this developmental stage by changing its hormonal balance.

The only other experiment we know of investigating the response of a cereal crop to the application of a phytotoxic substance in the context of yield decline caused by crop residues is reported by McCalla and colleagues. They compared germination bioassays with the response of wheat grown to maturity after a single application with the microbial product patulin in order to quantify yield losses caused by phytotoxic substances. The response in tillering revealed an interaction with the medium, thus patulin reduced the number of tillers in sand, but increased tillering of wheat in soil. An effect which the authors attribute to the ability of soil microorganisms to inactivate patulin in soils. In both treatments, however, the patulin application caused a reduction in the kernel weight, due to a lower number of kernels per head. Based on further experiments, McCalla and co-workers (Ellis and McCalla 1973; McCalla and Norstadt 1974) concluded that spring wheat was most sensitive to an application of patulin at germination or at heading. Other application dates based on the external development of the wheat plant at the second node stage (EC 32) and the flat leaf sheath opening (EC 49) were less suscep- tible. Due to the different chemicals used in the bioassays, a direct comparison is restricted to the general approach, however, it is confirmed that a short time exposure with a phytotoxic substance during the early development of the cereal crop might cause substantial losses in grain yield.

In order to relate these findings to yield depressions described in field situations, quantitative analyses of the phenolic content in soils a n d / o r soil water based on sequential sampling dates are required. But so far most analyses have been restricted to soil samples taken only once or twice during the growing season (Whitehead et al., 1981; Whitehead et al., 1983). Few reports have attempted to relate changes in concentrations of phytotoxic substances in the soil to the crop development in field experiments.

M/iller-Wilmes et al. (1977) found highest concentrations of phenolics in an experiment with winter barley grown in monoculture or following potatoes from early spring until ear emergence. They explain the yield depression of

10% to 23%, depending on the level of nitrogen application, to differences in the chemical composition of the phenolic compounds rather than to the actual concentration in the soil. In contrast, Wolf and H6flich (1983) comparing the inhibition of soil collected in a wheat monoculture with wheat grown in rotation using a radish germination bioassay reported the largest degree of toxicity in October and April. How- ever, they did not isolate or identify the chemical nature of the effects. These results indicate a shift in phytotoxicity during the growing season, although the flow rates between the different pools of phenolics in the soil and conditions affecting these processes are little understood (Blum et al., 1991).

A quantification of the yield decrease caused by phytotoxic substances released from crop residues under field conditions is only possible if sequential soil sampling is accompanied by a detailed observation of the crop development, since differences in the apex development are most important for an understanding of yield differences in respect to environmental conditions and husbandry factors (Kirby and Ap- pleyard, 1984; Landes and Porter, 1989).

On the evidence presented this is an approach which has to be considered in allelopathic research.

Acknowledgements

The senior author of this project was financially supported by a grant of the German Research Council (Deutsche Forschungsgemeinschaft).

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