Scientia Horticulturae, 20 (1983) 361--375 361 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

ALLELOPATHY IN CITRUS ORCHARDS

W.P. BURGER and J.G.C. SMALL I

Citrus and Subtropical Fruit Research Institute, Private Bag Xl1208, Nelspruit 1200 (Repu blic of Sou th A frica)

I Department of Botany, University ofPretoria, Pretoria 0002 (Republic of South Africa)

(Accepted for publication 15 December 1982)

ABSTRACT

Burger, W.P. and Small, J.G.C., 1983. Allelopathy in citrus orchards. Scientia Hortic., 20: 361--375.

The involvement of allelopathy in citrus orchards is indicated by the discovery of a phenolic phytotoxin in soils and partly decomposed citrus root residues. The phytotoxin, which was tentatively identified as homovanillic acid, is produced by the anaerobic decomposit ion of citrus roots in the deeper soil horizons. It caused an arrest in rough lemon (Citrus jambhiri) radicle elongation and a severe swelling of the root tip.

Keywords: allelopathy; Citrus jam bhiri; homovanillic acid; replanting.

INTRODUCTION

The expansion of citrus production in many parts of South Africa leaves citrus growers with no alternative but to establish new orchards on sites where citrus had been grown for 30 years or more. In the Western Transvaal, Central Transvaal and the Eastern Cape, where the replant problem is prevalent, the economic lifetime of such orchards is usually short and production low.

Although the poor citrus tree performance associated with the replant problem is caused by a complex interaction between root parasites and unfavourable soil conditions (J.P. Martin, Unpublished report, 1959; Burger, 1971), there is abundant evidence that allelopathy is involved in "soil sick- ness", or the replant problem of various crops (Rice, 1974).

The involvement of allelopathy in the replant problem of fruit trees has only been investigated in detail on peaches and apples. In a summary of the results of the peach replant problem, Patric et al. (1964) stated that regard- less of the causal organism involved, the production of toxic substances through the hydrolysis of amagdalin appears to be the main mechanism involved in peach root degeneration. With apples, BSrner (1959) came to

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the conclusion that microbial decomposit ion products of the flavanoid glycoside, phlorizin, are involved in the apple replant problem.

In the case of citrus, Waiters (1917) isolated the phytotoxin , p-hydroxy benzoic acid from soil in citrus orchards suffering from "die-back" disease. Prevatt (1959) reported the presence of toxins in extracts of waterlogged soil in which citrus roots were incubated. Although no conclusive evidence is available involving allelopathy in the citrus replant problem, Martin and Batchelor (1952), Burger (1971) and Rice (1974) considered it very likely that phytotoxins are produced by microbial action from citrus plant residues.

In reviewing the literature on the physiology and biochemistry of allelopathy in relation to disease resistance, it became abundantly clear that phenolic substances play an important role in both subjects (BSrner, 1960; Farkas and Kir~ly, 1962; Kosuge, 1969; Rice, 1974, 1979). There is also evidence that the uptake of exogenous phenolic compounds can alter the resistance of plants to diseases and nematodes (Patric et al., 1964; Linder- man, 1970; Feldman and Hanks, 1971).

Research concerning the citrus replant problem has been focussed mainly on the inorganic chemistry, soil physics and biological aspects of old citrus soil. No research of significance has been done to investigate the possible involvement of allelopathy in the growth reduction of citrus trees under replant conditions. A study of this nature could therefore contr ibute a great deal towards a bet ter understanding of the basic causes of the problem. In the present investigation, at tention was focussed on the role of phenolic compounds as allelopathic agents in the replant problem of citrus. This involved the detect ion of phyto toxic compounds in citrus orchard soils and in citrus root residues in the soil. The effect of these toxins on the root morphology and histology of citrus seedlings was studied and, finally, an effort was made to identify the toxins present in decomposed citrus root residues.

MATERIALS AND METHODS

Sample preparation. -- Soil samples as well as fresh and partly decomposed rough lemon (Citrus ]ambhiri) roots were collected on several farms in the Sundays River Valley (SRV) in the Eastern Cape, and at Zebediela Estates in the Northern Transvaal, where citrus trees have been grown on sites for at least 20 years. With the soil samples, all recognizable root debris were removed, while in root samples, soil particles were removed before analysis.

In order to obtain sufficient quantities of decomposed root matter for bio-assays and the identification of phytotoxins, the method of Toussoun et al. (1968) was used to decompose fresh roots under anaerobic conditions in the laboratory. Roots with a diameter of 3 mm or less were cut into small pieces, mixed with virgin soil in a ratio of 1:3 and placed in plastic con- tainers. This root--soil mixture was saturated to field capacity, sealed with

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black plastic sheeting and incubated in the dark at 25°C. After 8 -10 weeks, when maximum soil toxicity occurred (Burger, 1981}, the samples were pro- cessed for analysis. Control treatments were prepared in the same way, but without the roots.

E x t r a c t i o n procedures . - - After evaluating the various methods suggested in the literature, it was decided to use the following procedure for the extraction of roots, soil and decomposed root--soil mixtures (Burger, 1981). Soil and soil--root samples were thoroughly mixed with distilled water until saturated. After 1 h, the mixture was adjusted to pH 7.5 with HC1 or NaOH. A volume oI 95% ethanol was added to give an estimated final concentration of approximately 60% ethanol water extraction solvent. In the case of root material, the sample was directly homogenised at 20 000 r.p.m, for 1 min in 60% ethanol and adjusted to pH 7.5. After 15 h, the samples were centrifuged and the clear supernatant was suction-filtered through filter aid to remove any remaining insoluble matter. The ethanol was evaporated in vacuo at 45°C. The crude aqueous extract was partit ioned 3 times with equal volumes of petroleum ether (40- 60°C BP) and subsequently with di-ethyl ether. The aqueous phase was then adjusted to pH 2 and left overnight. The humic acid precipitate was removed by filtration and discarded. The aqueous phase was finally partit ioned 3 times with di-ethyl ether.

The 3 organic phases, petroleum ether (I), di-ethyl ether at pH 7.5 (II) and di-ethyl ether at pH 2 (III) were reduced to dryness in vacuo at 45°C and dissolved in a small volume of 95% ethanol. These fractions were used for chromatographic studies and bio-assays.

Th in layer c h r o m a t o g r a p h y . - - Plant or soil extracts were applied in narrow bands, 10--15 mm in length, at the origin of the TLC plate (Merck Si G60 pre-coated plates with fluorescent indicator). The solvent, cyclohexane-- tertiary amyl alcohol--acetic acid (30:1 .2:0 .05) , was used to develop the plate 6 times to 80 mm from the origin. Between each development, the plate was dried under a current of dry nitrogen.

Using standard techniques suggested by Harborne (1973), phenolic sub- stances were detected on TLC plates by means of UV fluorescence (365 nm), UV absorbtion (254 nm) and colour reactions with Gibb's spray reagent. The procedure with the Gibb's reagent was modified by substituting the ammonia vapour fumigation with a 10% sodium carbonate spray.

High pressure l iquid c h r o m a t o g r a p h y . - - The HPLC system used was of a modular design, comprising a reciprocating pump (LDC Constametric II), a fixed wavelength UV absorbance monitor (ISCO UA-5) and an integrating computer (Spectra Physics SP 4100). Analytical grade solvents were used for the mobile phase, i.e. acetonitrile (MeCN), te t rahydrofuran (THF), methanol (MeOH) and acetic acid (HOAc). For analytical analysis, a 4 × 250-mm RP8 column was isocratically eluted at a flow-rate of 1.5 ml/min with the solvent

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H20--MeCN--THF--HOAc (80 :12 :3 :1 ) . A 9 X 500-mm RP18 column was used for semi-preparative separations at a flow-rate of 1.5 ml/min with the solvent H20--MeCN--THF--HOAc (30 :12 :3 :1 ) . Peaks were monitored at 280 nm UV absorbance.

Identification o f toxic compounds. -- Commercially available phenolic standards were used as markers to make tentative identification of unknown compounds by comparing fluorescence colours, Rf values (TLC), retention times (HPLC) and colour reactions with Gibb's spray reagent. Absorption spectra were determined in the 200--600 nm range with a PYE Unicam SP8- 200 spectrophotometer.

Bio-assays. --- A bio-assay, using rough lemon seedlings grown in ~ nutrient solution in 5-ml test tubes, was developed to determine the effect of phyto- toxins on radicle growth. For this purpose, a specially designed black perspex container with a clear perspex cover was used to house 20 X 5-ml test tubes. The seedlings with a radicle length of 10--15 mm were grown at 28°C in heat-sterilised vermiculite from manually shelled fresh seed sterilised for 5 min in 0.15% sodium hypochlorite solution.

A saline nutrient solution was used to represent the saline soil conditions found in the regions where the replant problem occurs. The elemental composition of the solution in mg dm -3 was: Ca, 100; Mg, 24; K, 40; P, 8; N, 116; S, 105; Na, 150; Fe, 5; Mn, 0.2; B, 0.1; Zn, 0.1; Mo, 0.1. Other chemical characteristics were SAR = 3.5; conductivity = 157 mS m-l ; pH = 7.2; osmotic potential = 55 kPa.

The test compounds, which were generally poorly soluble in water, were introduced into the nutrient solution by applying the desired volume of ethanolic extract of known concentration to a strip of Whatmann No. 3MM chromatographic paper (5 X 50 mm). The paper strips were placed in the nutrient solution after drying under a current of air.

The seedlings were left in the test solution for 4 days at 28°C before the total radicle growth was measured.

RESULTS AND DISCUSSION

Phyto tox ic i ty o f soil and root residues from citrus orchards. -- It was estab- lished by Patric (1971), that extracts obtained from soil and plant residues, in the relative proportions encountered in the field, had little effect on tobacco and lettuce growth. A marked root injury and growth reduction was, however, found with extracts of decomposing residue fractions. To test this possibility in the case of citrus, the phytotoxic i ty of fresh roots, partly decomposed roots and soil (from the 200--400 mm horizon) from a 30-year citrus orchard and adjacent virgin soil was compared (Table I). The samples were extracted as described, and assayed for toxicity to rough lemon roots. Only the extract of the virgin soil did not show any significant reduction in

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radicle growth as compared to the control. The greatest growth reduction was found in Fraction III, followed by Fraction II of the root residues and citrus soil extracts.

It is significant to note that Fraction III of the fresh roots had little effect on radicle growth, whereas no growth occurred with the same fraction from partly decomposed root residues. This observation supports the supposition that phytotoxins are formed during the decomposit ion of citrus roots. There is abundant evidence in the literature that the complex phenolic compounds, including lignin in plant material, are decomposed to simpler structures such as phenolic acids (Patric, 1971; Rice, 1974, 1979). Most of these compounds can be extracted from aqueous solutions at pH 2, i.e. Fraction III.

In order to determine the relationship between soil depth and the accumu- lation of phytotoxic compounds, additional soil samples were collected at 4 depths ( 0 - 2 5 0 , 250--500, 500--750 and 750 ~1000 mm) in a citrus orchard at the Sundays River Research Station, near Addo.

The results (Fig. 1) show that the extract from the 0 -250- ram zone caused the greatest growth reduction. At the 250--500-mm zone, growth reduction was the least, but toxici ty then increased gradually with increasing depth.

The fact that samples collected at 500--1000-mm depth showed consider- able phyto toxic i ty supports the theory that allelopathic toxins in citrus orchards might accumulate in the fine-textured sub-soils, where their degra- dation could be retarded by environmental factors such as poor aeration and the absorbent properties of clay particles. This could also explain why deep ploughing of old citrus soils alleviated the effects of the replant problem (Burger and Bruwer, 1979).

T A B L E I

G r o w t h r e s p o n s e o f r ad i c l e s t o e x t r a c t s o f so i l , f r e s h r o o t s a n d r o o t residues

S a m p l e N o . a n d c o n e . in m m 3 / 5 c m 3 n u t r i e n t

Radicle g r o w t h ( m m ) in e x t r a c t f r a c t i o n s L S D P = 0 . 0 5

0 I II III

%CV

F r e s h r o o t s 39 18 14 34 4 12 20 m m 3 =- 1 0 0 m g

R o o t r e s i d u e s 31 22 9 1 4 .3 23 50 m m 3 ~ 2 .5 g

C i t r u s so i l 34 26 19 8 5 15 1 0 0 m m 3 -= 3 0 0 g

V i rg in so i l 31 29 31 29 NS 1 0 0 m m 3 -= 3 0 0 g

14

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40

3o g to

20

"d

10

0

0 250 500 750 1000

Soil depth (mm)

Fig. 1. Ef fec t o f soil d e p t h on t he t o x i c i t y o f ac id - - e the r soil ex t r ac t s ( ex t rac t cone. 130 g soi l /5 crn 3 n u t r i en t ) .

Experience has shown that the effect of the citrus replant problem can of ten still be observed when an old orchard site is replanted 5--10 years after the removal of the previous orchard. Few known organic compounds impli- cated in allelopathy would be stable enough to resist degradation over such a long period when exposed to the normal environmental conditions existing in the upper soil horizons. By implication, this would mean that allelopathic compounds involved in the citrus replant problem could only exist in a sub- soil environment for an extended period of time. It must be added that factors such as salinity, pH and the nature of the microflora populations present in the soil, could also be involved in preventing the degradation of phytotoxins.

The toxici ty of the 0--200-mm sample shown in Fig. 1 can possibly be explained by the chance inclusion of anaerobic pockets or clumps of decom- posed roots in the samples. The fact that these samples were collected in an existing orchard lends some support to this explanation. These explana- tions are supported by the reports of Patric et al. {1964}. Studies on soil aeration furthermore suggest that localised pockets of anaerobioses are wide- spread in soil {Greenwood, 1961}. Anaerobic conditions might therefore prevail for a considerable period of time after irrigation in fine-textured soils, such as are found in the Sundays River Valley.

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The phyto tox ic i ty o f citrus root residues decomposed under anaerobic soil conditions. -- The extraction of soil samples requires large quantities of expensive organic solvents, and the quanti ty of phytotoxins recovered were often too small for bio-assays or identification purposes. Suitable methods for the product ion of phyto toxic root residues under laboratory conditions were therefore investigated. Various investigators have indicated that soil toxici ty due to organic compounds is most frequently associated with heavy, poorly aerated or waterlogged soil (Toussoun et al., 1968; Shindo and Kuwatsuka, 1975; Chou and Patric, 1976). Patric et al. (1963) also reported that the toxici ty of compounds from plant residues is enhanced by increasing salinity. The replant problem of citrus is associated with poorly- aerated fine-textured soils and salinity (J.P. Martin, unpublished report, 1959; Martin et al., 1961). Under these conditions, it seems possible that phytotoxins might be produced from decomposing citrus plant residues. It was established that the phyto toxic i ty of extracts of citrus residues reached a peak at 8--10 weeks of incubation under anaerobic conditions (Burger, 1981).

The results of extractions made during 1978--1980 are presented in Table II. The rough lemon roots used for the production of phytotoxins during July 1978 to March 1980 were collected in various old citrus orchards in the Sundays River Valley. Only the roots for extract No. 1980/05 were collected from an orchard at the Citrus and Subtropical Fruit Research Institute, Nelspruit. Note that the concentrat ions-in Table II are expressed as the equivalent mass of fresh roots originally incorporated in the root--soil mix- ture. This procedure facilitated the comparisons between the extracts. As compared to the first extract, No. 1978/07, Extract 1979/01 was relatively non-toxic, bu t bet ter results were obtained with a subsequent repetition of the experiment (extract No. 1979/05). Extracts 1980/02 and 1980/03 were as phyto toxic as the first extract. Extracts made from roots collected from orchards at Nelspruit (1980/05), where the replant problem is of no impor- tance, were relatively non-toxic. A concentration nearly 8 times higher than

TABLE II

Effect o f extracts of root residues, decomposed under anaerobic conditions, on rough lemon radicle growth

Extraction Decomposition Conc. in date time equivalent mass (month/year) (weeks) fresh roots (rag)

Radicle growth (ram) in fractions

0 I II III

7/1978 9 600 23 15 20 0 1/1979 9 1200 33 19 25 7 5/1979 9 800 30 18 20 0 2/1980 8 600 31 18 11 0 3/1980 10 400 33 21 20 0 5/1980 10 3200 32 18 2 9

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that which was used for the extract made of roots from the SRV (1980/03) was used before some reduction in root growth was observed. The acid-- ether fraction (III) caused the greatest reduction in growth.

The influence o f phytotoxic compounds on root morphology and histology. - Extract No. 80/03 III (Table II) was added to the nutrient solution in

concentrations of 5, 10 and 15 mm 3 per 5 cm a nutrient solution. These concentrations represented an equivalent of 100, 200 and 300 mg fresh roots decomposed under anaerobic conditions, as described under Methods. The effect of the extract on the growth of rough lemon seedlings as recorded over a period of 4 days is shown in Fig. 2A. The growth of both the hypo- cotyl and the radicle was inhibited progressively more with increasing extract concentration. At a concentrat ion of 15 mm3/5 cm a, growth was almost completely arrested. On exposure to the toxic extract, a swelling of the root tip occurred at all 3 concentrations.

The swollen roots, as indicated in Fig. 3B, show that the swelling was initiated abrupt ly on exposure to the toxic extract. At very high concentra- tions (30 mm3/5 cm3), the apical meristem became necrotic and all growth ceased.

Various other morphological changes were observed on affected roots. A brown discolouration, the formation of brown scales on the root and sometimes shallow longitudinal cracks were observed on the swollen tissue (Fig. 3B).

The morphological changes in rough lemon radicles exposed to toxic extracts are very similar to those found by Patric et al. (1963, 1964} and Svensson (1971) on exposure to phenolic phytotoxins.

In Fig. 4, the longitudinal section through the transitional zone, from the normal to the swollen region of the root, is shown. The region above the swollen part represents the normal growth before exposure to the toxin. The section indicates that the response to the toxin was practically imme- diate. Cell elongation was arrested and radial expansion was initiated. These observations, which were reported in detail by Burger (1981), are in agree- ment with the observations of Svensson (1971), who found a similar change in the polarity of cell growth on exposure of maize roots to coumarin.

The isolation and identification o f toxic compounds in citrus root residues. -- A series of 24 phenolic compounds, which are known to occur in citrus or in decomposing organic matter, were evaluated by means of the rough lemon bio-assay (Burger, 1981). Only 4 compounds, i.e. homovanillic acid, phloretic acid, o-coumaric acid and coumarin, caused a severe swelling and reduced growth comparable to that by the toxic extract. An effort was sub- sequently made to determine which of these compounds, if any, were present in the toxic extract. The initial step taken in the isolation of the toxic compounds in the extract was to separate the extract by means of TLC. Six bands were isolated from the chromatogram and evaluated for

B!i:i

HOMOVANILLIC

370

Fig. 3. Growth response of rough lemon root tips exposed to a nutrient solution treated with phytotoxic extracts. A, normal root tip from untreated nutrient solution. B, swollen root tip from toxin-treated nutrient solution.

371

Fig. 4. Longitudinal section through the transitional zone from the normal to the abnormal region of the root (A, x 130; B, x 50).

TABLE III

Effect of the isolated TLC fractions of root residue extracts on radicle growth

TLC band No. Radicle growth (mm)

Extract 78/07 III Extract 80/03 III

Con~ol 33 25 B1 30 14 a B2 19 a 4 b B3 24 13 a B4 26 25 B5 19 a 15 a B6 25 18

a Slightly swollen root tip. bSeverely swollen root tip.

t o x i c i t y to rough l e m o n radicles. T h e resul ts indica te 2 m a j o r tox ic zones on the c h r o m a t o g r a m , i.e. B2 and B5 (Table I I I ) . Band B2 s h o w e d the g rea tes t inh ib i t ion o f g row t h and caused the charac ter i s t ic swelling response of the r o o t t ip p rev ious ly observed.

A H P L C eva lua t ion o f Band B2 s h o w e d t h a t it c o n t a i n e d a t least 15 c o m - p o n e n t s (Fig. 5A}. The b a n d was subsequen t ly subd iv ided in to 4 f rac t ions by m e a n s o f p repa ra t ive H P L C and t e s t ed fo r t ox i c i t y (Table IV). T h e frac- t ions B2-2 and , t o a lesser e x t e n t , B2-4 inh ib i ted radicle g rowth .

A c o m p a r i s o n b e t w e e n the c o m p o n e n t peaks o f B2 (Fig. 5A) and the 5 pheno l i c s t andards {Fig. 5B) did n o t s h o w any s ignif icant peaks

372

TABLE IV

The effect of the 4 fractions of Band 2 on radicle growth

HPLC retention time (rain)

Preparative Analytical

HPLC fraction Radicle growth (ram)

Control 0--24 0.- 4 B2-1

24--26 4-- 8 B2-2 26 -28 8--12 B2-3 28--40 12--30 B2-4

33 28

7 b 25 19 a

a Slight swelling of root tip. b Severe swelling of root tip.

B2-2 - - g l g l l

v g

I" g l

B2-4 B

t~

A Fig. 5. High-pressure liquid chromatograrns showing the HPLC purification of compounds occurring in TLC Band 2. A, Band B2. B, Phenolic standards: homovaniUic acid; p-hydroxybenzoic acid; phloretic acid; coumarin; o-coumaric acid.

c o r r e s p o n d i n g to o - coumar i c acid or ph lo re t i c acid. T h e peaks , a c c e n t u a t e d wi th b l ack spots , had r e t e n t i o n t imes c o r r e s p o n d i n g to those o f homovan i l l i c acid, p - h y d r o x y b e n z o i c acid and poss ib ly coumar in .

In o rde r to f ind add i t iona l s u p p o r t fo r these t en t a t ive ident i f ica t ions , the original e x t r a c t 80 /03 I I I and a series of au then t i c pheno l i c c o m p o u n d s were c o - c h r o m a t o g r a p h e d on T L C and s p r a y e d wi th G i b b ' s reagent (Fig. 6). Acco rd ing to the R f values and the co lour reac t ions of the original T L C plate ,

373

Fig. 6. Th in layer c h r o m a t o g r a m o f r o o t res idue ex t rac t and phenol ic s tandards . B2 and B5 = Toxic zones (4 , ) . HV = homovani l l ic acid; OC -- o-coumar ic acid; PC = p-cournaric acid; CO = cournarin; PH = ph lore t ic acid; PHB = p - h y d r o x y b e n z o i c acid.

p-hydroxybenzoic acid and coumarin were not present in the original extract. It seems certain that homovanillic acid was the major toxin present in the

extract. The evidence can be summarised as follows:

(1) homovanillic acid caused growth inhibition and a swelling response simi- lar to that by the acid--ether extract of root residues (Figs. 2 and 3);

(2) the suspect inhibitory compound isolated by means of HPLC had the same retention t ime as that of homovanillic acid (Fig. 5};

(3) the UV absorption spectra of the unknown compound corresponded exactly with that of homovanillic acid, i.e. it had a maximum at 277 nm and a small shoulder at 280 nm;

(4) homovanillic acid and the inhibitory Band B2 occupied the same Rf positions on the TLC plate (Fig. 6);

(5) homovanillic acid, a relatively rare phenolic acid in higher plants, was identified by Feldman and Hanks (1968) in the ether fraction of acid- hydrolysed rough lemon roots.

CONCLUSIONS

This a t tempt to explore the dimensions of allelopathy in citrus orchards has shown that potent phytotoxins can be produced in the fine-textured

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alkaline soils where the replant problem is prevalent. Homovanillic acid has been tentatively identified as the primary toxin in the acid--ether fraction of partly decomposed rough lemon roots. Laboratory studies have shown that anaerobic soil conditions, such as are found in the subsoil after heavy irriga- tions, are a pre-requisite for the accumulation of these phytotoxic sub- stances.

Although the results of this investigation cannot be directly extrapolated to field conditions, a hypothesis involving two possible mechanisms of allelopathy in citrus orchards can be suggested. When citrus soils in the Sundays River Valley are cropped to citrus, a marked decline in the numbers of beneficial fungi such as Trichoderrna viride occurs (Martin, 1960). As these fungi are capable of breaking down phenolic compounds in the soil (Leon, 1976), their absence could lead to the accumulation of toxins. In sufficient quantities, these toxins could affect the citrus roots directly by causing a reduction in growth, as was shown in this investigation. Indirectly, these compounds could cause the decline of citrus trees by rendering the root system more susceptible to a variety of soil organisms (Patric et al., 1964). These could include relatively low-grade pathogens such as Fusarium fungi, which in fact becomes the dominant fungus species in soils cropped for some time to citrus (Martin, 1960).

It is therefore suggested that the interaction between allelopathic agents and organisms occurring in citrus soil might be the primary cause of the replant problem of citrus.

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