BIOLOGIA P L A N T A R U M (PRAHA) 18 (3): 195-199, 1976 Activity of Humus Acids from Peat as Studied by Means of Some Growth Regulator Bioassays HOANG KIM PHUO~O and V TICH~ Department of Plant Biology, Faculty of Natural Sciences, J E Purkyn6 University, Brno* Dedicated to Academician S Prdt on the occasion of his 80th birthday Abstract Biological activities of humm acid (Na salt), hymatomelanie acid (Na salt), lignofulvie acid and of fulvie acids isolated from peat were studied by means of the auxin-, gibboreUin-, and cytokinin- bioassays All the four tested fractions showed higher or lower stimulating activity in these btoassays However, the stimulating effect is considerably lower and cannot be interpreted as phytohormone activity Some fractions in some concentrations also showed inhibitory effects The manner of biological action of the studied fractions might be the result of interaction of their respective components The way in which humus acids influence plant metabolism and physiological processes in plants is so far still unclear, despite the efforts of many authors Moreover, some of the recently published papers draw attention to further biological processes which are influenced by these substances They also show that humus acids can influence not only plant organisms (see e.g PR~T 1964, GUivII~SKI 1967 and other), but also animals including man (VISSER 1973) The result of one of the earlier attempts to explain the effects of humus substances on plants is the idea that they can act as growth stimulators (BoTTOMLEY 1914) PRAT (1964), when reviewing his own results as well as those of his co-workers, showed that humic acids cannot be classified as auxin-like growth regulators, though their gibberellin-likc activity cannot be excluded From the results published by PRXT (1964) it also follows that humus substances not enhance the growth of meristems either, and that they therefore have no cytokinin activity On the other hand other papers indicate (e.g SLADKY and TICH~ 1959) that some humus fractions can, in certain cases, exert some effects comparable to the action of plant growth stimulators of the auxin type Received M a r c h 3, 1975 * Address: Kotlh~skh 2, 611 37 Brno, Czechoslovakia 195 196 H K PHUONG, V TiCH~? These discrepancies led us to a profound investigation of the biological activities of humie, hymatomelanic, lignofulvic and fulvie acids by means of the auxin-, gibberellin-, and cytokinin-bioassays Material and Methods Humus acids were prepared from peat (P~YI'2~6KOV)~ and TICH~ 1971, TICH~ and CECHOVX 1974) Humie acid and hymatomclanic acid were used in the form of sodium salts, lignofulvie acid and fulvic acids were tested as free acids The concentrations of the solutions of the acids were in all cases: (control); 1; 5; 10; 50; 100 and 500 mg 1-1 The solutions of indole-3acetic acid (IAA), gibberellic acid (GA3) and kinetin (KIN) were used as standards for comparison at 0.1 and mg 1-I concentrations The auxin bioassay was carried out according to the method described by BENTLEY and I~IOUSLEY (1954) Each experimental variant included 30 coleoptile segments (n = 30) and the activity of each solution was expressed in segment length in m m after a 24 h incubation in the solution tested Lettuce seedlings were used in the gibberellin bioassay performed according to FRANKLAND and WAREING (1961) There were 30 plants in each variant (n = 30) and the activity of each of the solutions was expressed in segment length in mm at the end of the assay, i n the cytokinin bioassay barley leaf segments were used according to the method described by OSBORNE and MCCALLA (1961) 30 leaf segments were used in each variant from which samples consisting of 10 segments were taken for the determination of chlorophyll (n = 3) The activity of each solution was expressed in chlorophyll concentration in mg 1-~ in extracts from the leaf segments These bioassays were described in more detail by SLADK~" (1971) The significance of the differences of the individual variants from the control was evaluated statistically Results anti Discussion The numerical data arranged in Table are the results of 12 independent experiments carried out in most cases at different consecutive time intervals For this reason each of the experiments included its own control and standards Each of the four investigated fractions shows to a greater or lesser extent the stimulating activities of all three types However, their activity is in most cases statistically significant only at higher concentrations The highest activity in the auxin bioassay could be detected with the mg 1-1 solution of fulvic acids The fact t h a t the stimulating activity of fulvic acids changes with increasing concentration in the auxin bioassay into inhibition (at 500 mg 1-1) without achieving a higher stimulating effect could be explained by the fact t h a t this fraction contains substances with weak auxin-like effects, different fl'om the heteroauxin Besides that, the presence of compounds with antiauxin effccts m a y be supposed and, of course, the two cases m a y occur simultaneously Hymatomelanic and lignofulvic acids showed an even lower activity in the auxin bioassay These fractions provoke a strange phenomenon, namely inhibition (significant in the gibberellin bioassay), at lower concentrations of the tested substances We had observed this phenomenon, called in other cases a "paradoxical effect" by SCnATZ et al (1964) even earlier, when 197 BIOASSAYS OF HUMUS ACIDS TABLE A c t i v i t y (x • s.p) of the s o d i u m salt of h u m i c acid ( N a H U ) , of the s o d i u m salt of h y m a t o m e l a n i e acid ( N a H Y ) , of lignofulvic acid (LF) a n d of fulvic acids (FU) isolated f r o m p e a t in auxin, gibberellin a n d cytokinin bioassays ST = s t a n d a r d s Difference f r o m the control : -b = P < 0.05; 5=-k = P < 0.01 Concentration [rag 1-1] ST Control NaHU ST Control NaHY ST Control LF ST Control FU Auxin bioassay [mm] Gibberclhn bioassay [mm] Cytokinin bioassay [rag 1-1] 0.1 (0) 10 50 100 500 5.8 6.8 4.9 4.6 4.5 4.4 4.7 5.2 6.3 • 0.12 ++ 5= 0.19 ++ 5= 0.09 5= 0.07 5= 0.07 ++ 5= 0.05 ++ 5= 0.08 5= 0.11 + 5= 0.13 ++ 10.6 16.2 6.0 5.5 6.3 6.3 7.9 8.0 11.3 =k =k 5= • 5= • 5= =k 5= 0.37 ++ 0.44 ++ 0.30 0.26 0.28 0.24 0.26 ++ 0.31 ++ 0.30 ++ 13.8 16.4 8.7 8.0 7.9 10.2 ll.2 11.3 13.6 5= • • 5= 5= -9 5= 5= 5= 0.82 ++ 1.14 ++ 0.24 0.31 0.95 0.43 + 0.79 + 0.91 + 0.62 ++ 0.1 (0) l0 50 100 500 5.3 6.1 4.8 4.6 5.0 4.8 5.5 5.3 5.3 5= 5= 5= • • • 5= 5= 5= 0.11 ++ 0.11 ++ 0.09 0.07 0.13 0.08 0.10 ++ 0.08 ++ 0.09 ++ 13.1 18.5 9.1 8.8 7.6 8.2 8.4 8.6 12.6 5=4-0.53 ++ 5= 0.32 ++ 5= 0.44 5= 0.45 5= 0.30 ++ 5= 0.41 5= 0.31 5= 0.36 =k 0.44 ++ 7.9 12.2 5.7 5.2 6.0 7.9 10.6 9.4 8.2 5= • 5= • • 5= • • 5= 0.56 + 0.66 ++ 0.26 0.65 0.89 0.35 ++ 1.61 + 1.37 + 0.42 ++ 0.1 (0) 10 50 100 500 5.4 6.1 4.7 4.6 4.9 4.9 5.1 5.2 5.1 4- 0.08 ++ -4- 0.11 ++ 5= 0.06 5= 0.09 5= 0.07 + 5= 0.09 5= 0.12 ++ • 0.09 ++ 5= 0.09 ++ 13.1 20.0 10.1 8.3 9.2 10.2 10.8 10.7 15.8 5= 0.30 ++ • 0.50 ++ -4- 0.44 5= 0.35 ++ =J= 0.40 -4- 0.59 4- 0.35 =k 0.49 • 0.67 ++ 12.8 14.8 10.5 11.1 9.5 11.3 11.6 12.7 11.8 • • -4• 5= • 5= 5= 5= 0.59 + 0.85 ++ 0.33 0.32 0.59 0.67 0.50 0.55 + 0.34 + 0.1 (0) 10 50 100 500 5.4 6.1 4.7 4.9 5.1 4.9 4.9 4.8 4.3 5= 0.08 ++ • 0.11 ++ • 0.06 5= 0.09 i 0.10++ ~= 0.09 + 5= 0.06 + • 0.07 • 0.05 ++ 12.6 19.0 8.5 10.2 9.7 11.5 12.5 11.8 12.9 5= 0.26 ++ • 0.34 ++ 5= 0.38 • 0.42 ++ -4- 0.44 + 5= 0.37 ++ 5= 0.43 ++ 4- 0.45 ++ 5= 0.67 ++ 13.5 17.0 11.3 10.9 11.1 14.0 15.6 16.3 15.8 5= 0.58 • 1.22 + 5= 0.46 5= 0.32 -4- 0.30 5= 0.55 + =k 1.02 + 5= 0.55 ++ 5= 0.55 ++ 198 ]~ K PHUONG, V TICHY studying the effects of lichens on wood-destroying fungi (TICHff 1955) We attempted to explain it as a mutual interaction of several components of the substance studied differing in solubility and we assume that this explanation could also be used when explaining the influence of the fractions of hymatomelanic and lignofulvic acids observed in the auxin bioassay and in the gibberellin bioassay The paradoxial effect becomes clearly apparent in the auxin bioassay in the presence of humic acid The result of this assay can be considered a support of PRXT'S theory (e.g 1960) that humic acids can function as auxin antagonists especially at lower concentrations On the other hand, it is necessary to state (at least in the case of lignofulvic and hymatomelanic acids) the positive effect in the auxin bioassay, however much less pronounced, and only apparent at higher concentrations Humus substances also show activity in the gibberellin bioassay, fulvic acids being most conspicuous among them Hymatomelanic and lignofulvic acids showed positive activity in the gibberellin bioassay only at the 500 mg 1-1 concentration, whereas at lower concentrations they exerted an inhibitory influence The retention chlorophyll bioassay was also influenced by each of the four fractions Fulvic acids were again the most active substances in this respect and hymatomelanie acid as well The fractions of humic acid and of lignofulvic acid showed a somewhat lower activity Statistically significant inhibitions were not established in the cytokinin bioassay A considerable vacillation in the significance in this bioassay results on the one hand from the small number of repetitions (n = 3), and on the other hand from the nature of the bioassay itself Thus all the four tested fractions have a significant statistical influence on the bioassay system in the oat coleoptile bioassay, in the lettuce bioassay and in the retention chlorophyll bioassay From the dependence of the extent of the stimulation effect on the concentration of the respective fraction it follows that indole-3-acetic acid, gibberellic acid, kinetin or other substances related to these growth regulators, which could survice in peat or which could be formed in it during its ripening, were not the source of this activity There are obviously other substances there, the activity of which is on the one hand lower than that of the common growth regulators, but which on the other hand has a much more complex character So far we not understand exactly the chemical nature of the biologically active substances contained in peat which could cause the biological effects observed The chemical structure of the proper humus acids which could be the carrier of the activity of these acids is not understood either The biologically active components of peat mentioned in some papers (e.g L~.CHLEXT~ER and LACHMANN 1960) are probably of importance rather in medicine than in plant physiology Nevertheless it can be considered indubitable that peat like humus is a very heterogeneous system from the point of view of its biological action, which contains compounds with both stimulatory and inhibitory effects (TRoJAI~OWSKI1954, TOLPA and CZYZEWSKI 1960) We can therefore explain the results of our experiments not only as the effect of specific substances different from the commonly known growth stimulators but as a consequence of the interaction of different components of the tested fractions as well BIOASSAYS OF HUMUS ACIDS ] 99 Aeknowled9ement The authors wish to thank Mrs Helena Salajkovg~ for careful technical assistance References B e 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BIOASSAYS OF HUMUS ACIDS TABLE A c t i v i t y (x • s.p) of the s o d i u m salt of h u m i c acid ( N a H U ) , of the s o d i u m salt of h y m a t o m e l a n i e acid ( N a H Y ) , of lignofulvic... results of our experiments not only as the effect of specific substances different from the commonly known growth stimulators but as a consequence of the interaction of different components of the