Influence of humic acids on laurel growth, associated ... - Springer Link

Biology and Fertility

Biol Fertil Softs (1993) 16:1-4

~ Soi]s 9 Springer-Verlag1993

Influence of humic acids on laurel growth, associated rhizospheric microorganisms, and mycorrhizal fungi G. Vallini 1, A. Pera 1, L. Avio 1, M. Valdrighi 1, and M. Giovannetti 2 1Soii Microbiology Center, National Research Council (CNR), and 2Institute of Agricultural Microbiology, University of Pisa, Via del Borghetto 80, 1-56124 Pisa, Italy Received: July 17, 1992

Summary. Increasing concentrations of humic acids were tested in order to determine their effects on the microbial rhizosphere and the growth of laurel ( L a u r u s n o b i l i s L.). Plants that were treated with 300 mg kg-1 of humic acids had the heaviest weights of both fresh and dry shoots; however, doses of 3000rag kg -1 were highly phytotoxic and inhibited the growth o f laurel. Total aerobic bacteria and actinomycetes were stimulated by doses of 1500 and 3000 mg k g - 1 of humic acids at the first harvest. Nevertheless, at the end of the experiment no significant differences were found a m o n g the different doses. The number of fungi living in the laurel rhizosphere was not affected by any concentration of humic acids. Vesicular-arbuscular mycorrhizal (VAM) colonization was only slightly affected by the addition of increasing concentrations of humic substances to the soil, while the hyphal growth of G l o m u s m o s s e a e was reduced. Key words:

G l o m u s m o s s e a e - H u m i c acids - L a u r u s Rhizospheric microorganisms - Vesicular-arbuscular mycorrhizae nobilis -

Interest in humic substances, as soil amendments for crop production, has increased considerably in the last few years due to progress in understanding the importance of humified organic matter to soil fertility (Vaughan and Ord 1985) and to plant biochemical processes (Vaughan et al. 1985). Many authors have reported that humic substances positively affect plant growth by increasing soil aggregation, aeration, and permeability (Tan and N o p a m o r n b o d i 1979a; Mylonas and McCants 1980; Rauthan and Schnitzer 1981; Mt~ller-Wegener 1988). Humic substances were found to stimulate plant growth since they increased the absorption of soil nutrients (Vaughan and McDonald

Correspondence to: G. Vallini

1971), allowed a greater distribution of metal ions (i. e., heavy metals) as chelates within the plant (Kononova et al. 1966; Weber 1988), and affected metabolic reactions (Flaig 1970; Cacco and Dell'Agnola 1984). Humified organic matter is also known to increase microbial growth and activity. Visser (1985 a) pointed out that humic acids, if added to selective media, could increase the growth of a wide range of taxonomic and functional groups of soil bacteria and he hypothesized that a modification of cellular activity and growth might be promoted by humic substances through their influence on cell membrane permeability or on nutrient absorption (Visser 1985 b). Very few results on the effect of humic acid amendments on mycorrhizal infection have been reported (Tan and N o p a m o r n b o d i 1979b; Schisler and Linderman 1989), though estimates of the relationships between humic acids and mycorrhizal infection might be o f great interest. In the present study, an experiment performed in vivo, we examined the interactions a m o n g humic acids, rhizospheric microorganisms, and plant growth in laurel. In the same experiment, we evaluated the effects of increasing concentrations of hnmic acids in soils on VAM infection in laurel. We also tested the in vitro spore germination and hyphal growth of G. m o s s e a e in the presence of increasing concentrations of humic acids.

Materials and methods Experiment i

The soil used was an alluvial sandy loam from the banks of the Arno river near Pisa. Physical and chemicalproperties of the soil are reported in Table 1. This pot trial was conducted to evaluate the effects of increasing concentrations of humic acids on rhizospheric microorganisms and the growth of laurel (Laurus nobilis L.). Humic acids at 0, 300, 700, 1500, and 3000mgkg -1 (humic acid, sodium salt, Aldrich-Chemic, Steinheim, Germany) were added to the experimental soil. The pH values of the soil, after addition of the humic acids, ranged between 8.2 (at the lowesthumic acid dose) and 8.7 (at the highest humic acid dose). The soil was then transferred to plastic pots, each with a capacity of 0.8 litre. Each dose was tested with and without the addition of a selected

2 Table 1. Physicochemical Characteristics of the soil used in experiment 1 (values on a dry matter basis)

Results and discussion

Plant growth and mycorrhizal infection Texture

Sandy loam

Clay Silt Sand Limestone Moisture pH (H20) Total N Total P Soluble P Soluble K

7.0% 16.0% 76.0% 2.0% 1.6% 8.1 0.103% 0.139% 0.07% 1.2%

mycorrhizal inoculum, represented by a soil/sand mixture harbouring spores and external mycelium of G. mosseae (Nicol. and Gerd.) Gerd. and Trappe, which was grown in lucerne stockplants. Thirty grams of this inoculum was added to each G. mosseae-treated pot, while 30 g of the same mixture, after sterilization, was added to the pots containing the natural soil with only the autochthonous mycorrhizal endophytes. Three laurel seeds were sown in each pot. After germination, which occurred in April 1989, the seedlings were reduced to one per pot. Each treatment consisted of 40 replicates. The pots were arranged in a random block design and kept in an open nursery for 14 months.

Experiment 2 The objective of this experiment was to evaluate the effects of increasing concentrations of sodium humate on the germination of G. mosseae spores and on its hyphal growth. Sporocarps, containing 8 - 1 0 spores each, were obtained by wet-sieving infested soil from lucerne stockplants. Afterwards, these were placed between two millipore membranes and put into Petri dishes, which were then covered by sterile sand containing 0, 25, 50, 100, 200, 400, 800, and 3200 mg kg- i of humic acids. The sand was moistened with an equal quantity of distilled water per dish. To check the effect of Na + when introduced with humic acid sodium salt, a similar test was performed using sodium sulphate in Na concentrations corresponding to those that were applied with 50, 200, 800, and 3200 mg kg -1 of sodium humate. Controls were prepared by moistening the sand only with distilled water. Spore germination and hyphal growth were estimated after 3 weeks.

Results obtained from experiment 1, after periods of 7 and I4 months, are shown in Tables 2 - 4 . As expressed by plant shoot fresh and dry weights (Tables 2, 3), maximum growth responses were observed only in those plants that received 300 mg kg -1 sodium humate. These data are in accord with previous results showing progressive and significant increases in biomass production in different plant species amended with several humic substances in doses from 0 to 500mgkg -t (Mylonas and McCants 1980; Rauthan and Schnitzer 1981). A concentration of 3000 mg kg -~ of humate was highly phytotoxic and inhibited the growth of laurel plants, which were all dead by the second harvest. This strong inhibition confirms previous findings that doses higher than 2000 mg kg -a strongly inhibited plant growth (Mylonas and McCants 1980; Rauthan and Schnitzer 1981). Recently, doses higher than 2000 mg litre-t were found to stimulate the pri-

Table 2. Effect of increasing concentrations of humic acids on the growth of laurel plants in the natural soil

Humic acids Harvest (months Shoot fresh Shoot dry Root fresh added after planting) weight (g) weight (g) weight (g) (nag kg - l) 0 300 700 1500 3000

7 14 7 14 7 14 7 14 7 14

1.22b 1.45B 2.08a 2.88A 1.34b 1.47B 1.56b 1.62B 0.42c -

0.48b 0.58B 0.96a 1.22A 0.48b 0.57B 0.60b 0.63B 0.11c -

2.46b 2.69B 3.50a 4.20A 2.68b 2.78B 2.63b 2.59B 0.88c -

At each harvest, values in the columns followed by different letters are significantly different at P = 0.05

Harvests and measurements The pH, total organic C, N, P, and K contents of the soil were determined following the procedures suggested by the Italian Society for Soil Science (1985). The determination of humic acid content was carried out as reported by Riffaldi et at. (1986). The humic acid content of the soil was 0.6% of the entire measured organic C, this value corresponding to approximately 57 mg kg- 1 dry matter. In experiment 1 the plants were harvested after 7 and 14 months. Ten plants were used in each trial, both for the evaluation of plant growth and for microbiological analyses. Rhizosphere microorganisms were determined as follows: Ten root apices, 1 cm long, were cut from each plant and put into vials containing 10 ml sterile distilled water. The vials were shaken for 2 min in a vortex and known aliquots of the suspensions were taken in order to count the total number of aerobic bacteria, actynomycetes, and fungi, according to the methods outlined by Pochon and Tardieux (1962). Ligninolytic fungi were determined following the method described by Eggins (1964). Mycorrhizal infection was assessed on five plants in each trial, after clearing and staining the root system (Phillips and Hayman 1970), by the grid-line intersect method (Giovannetti and Mosse 1980). In experiment 2, spore germination and hyphal growth were assessed under a dissecting microscope, after staining the samples with a few drops of 0.05% Trypan blue in lactic acid, using a microscope eyepiece grid.

Table 3. Effect of increasing concentrations of humic acids on the growth of laurel plants in the natural soil inoculated with Glomus mosseae

Humic acids Harvest (months Shoot fresh Shoot dry Root fresh added after planting) weight (g) weight (g) weight (g) (mg kg- l) 0 300 700 1500 3000

7 [4 7 14 7 14 7 14 7 14

1.58b 1.71B 2.52a 3.35A 1.57b 1.74B 1.64b 1.80B 0.9tc -

0.62b 0.71B 1.21a 1.64A 0.60b 0.69B 0.61b 0.66B 0.20c -

2.56b 2.81B 3.96a 4.95A 2.40b 2.73B 2.52b 2.79B 0.94c -


Table 4. Effect of increasing concentrations of humic acids on mycorrhizal colonization of laurel roots in pots with only natural mycorrhizal endophytes or inoculated with G l o m u s m o s s e a e % Infection in % Infection in Humic acids Harvest pots inoculated added (months after pots with only (mg k g - 1) planting) natural endophytes with G. m o s s e a e 0 300 700 1500 3000

7 14 7 14 7 14 7 14 7 14

56+6.0 64+5.1 64 + 4.0 52_+3.7 50 _+5.5 46+-5.1 48+- 11.6 46+-6.8 38 +_10.6 -

58+_3.7 70_+3.2 70 +_3.2 44+5.1 52 _+6.6 58+-5.8 46+ 11.6 50+_6.3 24 _+4.0 -

Means + SE

mary root growth of onion, lettuce, and cantaloupe, although these doses did not represent the active fraction of the oxicoal product used (Van De Venter et al. 1991). Indeed, it is possible that different plant species react differently to the presence of humic substances, as suggested by Vaughan and Malcolm (1985). The addition of humic acids at concentrations from 300 to 1500 mg kg -t did not affect mycorrhizal infection due to either G. mosseae or the naturally occurring endophytes, whereas concentration of 3000 mg kg -t of humic acids had a slightly depressing effect on the percentage of root length infected by VAM fungi (Table 4). The addition of an infective, selected strain of G. mosseae to the naturally occurring endophytes did not produce any change in the spread of mycorrhizae in laurel roots. This confirms the findings of a previous study on the ectomycorrhizae of Douglas fir, which showed that they were not affected by humic amendments (Schisler and Linderman 1989). The variation in soil pH obtained after the addition of humic acids did not influence the growth and the root infection of G. mosseae nor of the naturally occurring endophytes. This is not surprising, because many VAM fungi can grow at pH levels higher than 8.0 (Giovannetti 1983; Giovannetti 1985). Since the addition of humic acids to the soil at doses higher than 1500mgkg -t proved dangerous to plant health, it is possible that the inhibition of mycorrhizal development is mediated by the plant. In conclusion, our data show that the addition of humic acids to soil at concentrations of 300 mg kg -1 can considerably improve plant biomass, while mycorrhizal activity is retained.

concentration of humic acids. The highest number of ligninolytic fungi was recorded at the first harvest. Schisler and Linderman (1989) observed that humic substances did not affect soil microbial populations consistently. It is not possible to establish a Cause-effect correlation between a given concentration of humic acid and the response of microorganisms; in fact, differential responses by specific microbes to the same dose of a humic fraction depend on several factors, soil characteristics representing the main factor. Growth of mycorrhizal fungi

The germination of G. mosseae spores was not inhibited by increasing doses of humic acids (Table 7), whereas hyphal growth was reduced. Regression analysis showed a negative linear relationship between the log of humic acid concentration and hyphal growth (y=615.2-70.6x; r = -0.65). Indeed, germination and hyphal growth were

Table 5. Total numbers of rhizosphere microorganisms of laurel plants growing in the natural soil added with increasing doses of humic acids (cells g - 1 soil, dry weight) Humic acids added (mg kg - l)

0 300 700 1500 3000

Total aerobic bacteria ( x 106)

Actinomycetes ( • 103)

Fungi ( x 103)

Ligninolytic fungi ( x 103)

7 14 7 14 7 14 7 14 7 14

35a 182A 70a 198A 165a 197A 346b 374A 309b 318A

154a 281A 191ab 341A 106a 324A 254b 301A 308b 360A

118a 49A 147a 71A 145a 76A 148a 50A 232a 47A

75a 22A 84a 36A 57a 55A 63a 40A 126a 46A


Table 6. Total numbers of rhizosphere microorganisms of laurel plants growing in the natural soil added with increasing doses o f humic acids and inoculated with G l o m u s m o s s e a e (cells g - 1 soil, dry weight)

Humic acids added (mg k g - 1)

0 300

Rhizosphere microorganisms

The number of rhizosphere microorganisms at 7 and 14 months is reported in Tables 5 and 6. Humic acid amendments of 1500 and 3000 mg kg -~ led to a stimulation of bacterial and actinomycete growth that was evident at the first harvest, whereas at the end of the experiment no significant differences were found among the different trials. The number of fungi was not affected by any tested

Harvest (months after planting)

700 1500 3000

Harvest (months after planting)

Total aerobic bacteria ( x 106)

Actinomycetes ( x 103)

Fungi ( x 103)

Ligninolytic fungi ( x 103)

7 14 7 14 7 14 7 14 7 14

77a 151A 65a 295A 82a 224A 277b 290A 258b 283A

103a 335A 234ab 302A 18lab 363A 295b 297A 290b 320A

105a 70A 145a 49A 149a 51A 136a I00A 265a 105A

36a 33A 120a 40A 49a 33A 88a 54A 118a 54A


Table 7. Germination of spores and hyphal length of Glomus mosseae growing in the presence of increasing concentrations of humic acids Dose of humic acids (rag kg- 1)

Spore germination (%)

Hyphal length (mm)

0 25 50 100 200 400 800 3200

93.3_+ 6.6 80.0_+ 0.0 66.6 _+18.5 93.3_+ 6.6 80.0_+ 0.0 56.6_+ 12.0 60.0_+20.0 60.0_+ 5.7

855_+211 495_+122 515 _+175 553 _ 126 350_+113 137+ 22 317+115 258_+ 27

Means + SE

u n a f f e c t e d by N a 2 S O 4, d e m o n s t r a t i n g t h a t t h e l o w q u a n tities o f N a + i n t r o d u c e d w i t h t h e h u m a t e d i d n o t depress p r o p a g u l e g r o w t h , a n d h e n c e t h e d e p r e s s i n g e f f e c t was p r o b a b l y d u e to t h e o r g a n i c i o n s a l o n e ( H i r r e l 1981).

Conclusions I n this p a p e r we h a v e s h o w n t h a t (1) h u m i c a c i d s a d d e d to t h e soil at 300 m g kg -1 i n c r e a s e t h e g r o w t h o f l a u r e l p l a n t s ; (2) i n c r e a s i n g d o s e s o f h u m i c a c i d s s t i m u l a t e t h e growth of rhizospheric bacteria and actinomycetes, while they do not affect the number of rhizosphere and l i g n i n o l y t i c f u n g i ; (3) h i g h d o s e s ( 3 0 0 0 m g k g - t ) o f humic acids depress laurel growth, slightly depress m y c o r r h i z a l d e v e l o p m e n t in l a u r e l r o o t s , a n d d o n o t affect s p o r e g e r m i n a t i o n o f t h e m y c o r r h i z a l e n d o p h y t e G. m o s s e a e , w h e r e a s t h e y i n h i b i t its h y p h a l g r o w t h ; a n d (4) t h e i n h i b i t i o n o f h y p h a l g r o w t h is n o t d u e to N a + i n t r o duced with the humates. Acknowledgments. The Regional Government of Tuscany, Environment Protection Department, partially supported this research by granting Dr. Manuela Valdrighi a 2-year fellowship (Grant BURT no. 41/Jul. 11, 1990). We thank Dr. Francesca Romana Pittaluga who coordinated, on behalf of the Regional Government, all relations with CNR. We also thank Mr. Sauro Ciampalini of the Vival Ciampalini Romano, Marciana di Cascina (Pisa), who provided laurel seeds and allowed the pot trial to be carried out at his nursery farm.

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