Plant and Soil 131: 11-19, 1991. 1991 Kluwer Academic Publishers. Printed in the Netherlands.

PLSO 8507

Growth and nutrient status of citrus plants as influenced by mycorrhiza and phosphorus application
V. ANTUNES and E.J.B.N. C A R D O S O

Department of Soil Science, Escola Superior de Agricultura Luiz de Queiroz, University of Sao Paulo, 13400 Piracicaba, S.P., Brazil
Received 28 October 1989. Revised June 1990

Key words:

Glomus etunicatum, parasitism, Rangpur lime (Citrus limonia, Osbeck), rock phosphate,

vesicular-arbuscular mycorrhiza
Abstract

To test the hypothesis that high levels of soluble phosphate applied in combination with VAM fungi, to citrus plants, can cause growth depression even in the absence of other limiting factors, and also to test if rock phosphate, under these conditions, may be a satisfactory P source, a greenhouse experiment was conducted using sterilized soil with four levels of phosphate (0, 50, 100 and 200 ppm P) supplied either as soluble P or as rock phosphate. Citrus seedlings were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus etunicatum or left uninoculated. Six months after the start of the experiment, the plants were harvested and shoot dry weight, P and K uptake, root colonization and the number of spores in 50 cm 3 of soil were determined. Significant increases were found in dry matter yields and in P and K contents, due to VAM fungus inoculation, at the zero and 50 ppm soluble P levels and at all rock phosphate levels. At 100 ppm soluble P, the development of VAM plants was equilvalent to that of non-VAM plants, and at 200 ppm, growth was significantly less than that of non-VAM plants. Root colonization and sporulation were reduced at higher P levels. The absolute growth depression of VAM plants at the higher P level was likely due to P toxicity. In addition, high leaf P and K concentrations may have interfered with carbohydrate distribution and utilization in these symbioses. Rock phosphate may be used with VAM citrus to substitute for medium amounts of soluble phosphate.

Introduction

According to Reed and Fremont (1935), the occurrence of vesicular-arbuscular mycorrhiza in citrus was reported for the first time in 1933. With the introduction of soil fumigation for citrus production in the USA, both stunting and nutrient-deficiency symptoms were observed, and these were attributed to the occurrence of toxic substances in the soil induced by fumigation. However, Kleinschmidt and Gerdemann (1972) found that such symptoms were due to the elimination of vesicular-arbuscular mycorrhizal (VAM) fungi. After that, much more at-

tention was given to the importance of these fungi in citrus culture (Menge et al., 1978; Timmer and Leyden, 1980). The feasibility of VAM inoculation is much greater for nursery-grown than for field-grown citrus seedlings, as the quantity of inoculum required in the nursery is much smaller. The combination of VAM fungi and rock phosphate can lead to remarkable yield increases, as reported by several authors (Cardoso, 1985; Ezeta and Santos, 1980; Pairunan et al., 1980; Sparling and Tinker, 1978). Soluble fertilizer phosphates are usually too expensive for agriculture in developing countries. Rock phosphates, although

12

Antunes and Cardoso

relatively insoluble, are abundantly found and easily mined. The use of P fertilizers is especially important in the highly acidic and usually infertile soils of the tropics. The combination of low-cost rock phosphates and VAM fungi presents an attractive, ecologically sound alternative to the intensive use of manufactured P fertilizers for subsistence farmers in the developing world. Graham and Timmer (1984) have commented on the economical advantages of the use of rock phosphate in citriculture but, only very few experimental data are available about the benefits of rock phosphate in combination with VAM fungi for citrus or even for other perennial crops. Not only the phosphate source, but also the P level, should be taken into account in an inoculation program. Various combinations of P sources and levels should be tested in combination with an effective VAM fungal strain, as previously demonstrated by Cardoso et al. (1986). The present study was undertaken to assess the degree of effectiveness of VAM fungi in citriculture when high- and low-solubility P sources are used.

Materials and methods

The experiment was conducted according to a completely randomized factorial 2 4 x 2 design with 4 replicates, consisting of a total of 64 plants. The factors were: a) 2 phosphate sources; Patos de Minas rock phosphate (RP) of Brazilian origin, a fluor-apatite with a P content of 35 percent and a particle size of 200 mesh, and triple superphosphate (SP); b) 4 levels of each P source, (0, 50, 100 and 200 ppm P, equivalent to 0, 230, 460 and 920kg P205 ha-X); and c) inoculated (VAM) and not inoculated (nonVAM) with Glomus etunicatum (Becker and Gerdemann). A sandy soil (Quartzipsament) was sterilized by steaming (100C) for 2 hours a day on 3 consecutive days in order to eliminate the indigenous endophytes. Native P content of the soil was 5 ppm by the resin method. Lime (with a dolomitic carbonate) was applied to obtain a soil pH of 6.2, and 20 days afterwards nutrients were mixed with the soil in the following amounts based on soil weight; 50 ppm K as KCI, 40 ppm

N and 48ppm S as ( N H 4 ) 2 S O 4. Micronutrients were supplied as H3BO 3 (0.25 ppm B), CuSO 4 (0.75ppm Cu), FeSO 4 (2.Sppm Fe), ZnSO 4 (2.Sppm Zn) and H 2 M o O 4 (0.05ppm Mo). Then, phosphate, either as rock phosphate (RP), or as soluble phosphate (SP), was mixed with the soil at the rate of 0, 50, 100 or 200 ppm P, or/zg p g-i soil, according to the treatment. Two and four months after transplanting an additional N fertilization was given, as 40 ppm N in the form of NH4NO 3. The fertilized soil was apportioned into 3 kg holding pots and uniform Rangpur lime seedlings were transplanted, one into each pot. The seedlings had been raised in a sterilized sandbed, sown two months before transplanting. For the VAM treatments a 5 0 c m 3 soil portion from a stock culture (corn as host plant) was added below each seedling, containing about 500 spores, hyphae and infected root fragments. The uninoculated pots received an identical amount of the same soil but without the fungal structures. The pots were kept in the greenhouse from December 1984 to June 1985, at a temperature fluctuating between 25 and 45C, and at high light intensity, high relative humidity and with a mean photoperiod of 12 hours. The plants were watered every day to field capacity. Plant height was measured monthly and the diameter of the trunk, taken 10cm above soil level, was measured at the age of 6 months. At this date the plants were harvested and the shoot dry weight, P and K uptake (Sarruge and Haag, 1974), root colonization (Ambler and Young, 1977; Phillips and Hayman, 1970) and number of spores (Gerdemann and Nicolson, 1963) were determined. All the data were subjected to analysis of variance by the SAS general linear models procedure. Student's t test was used to compare means pair-wise, for significant differences between RP and SP treatments. Graphic analysis of the data for additional information was performed on regression curves.

Results Inoculation with G. etunicatum increased shoot dry matter significantly at 0 and 50 ppm of SP (Fig. 1). At 100ppm ~the difference was not

Mycorrhiza in citrus
25

13

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. . . . .

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I " I I 10() 114135150 P level ( p p m )

I 200

Fig. I. Regression curves for shoot dry weight of VAM and non-VAM plants, fertilized with soluble phosphate ( - - ) or rock phosphate ( - [ ] - ) at four P levels. SP-VAM: 0.00085 x 2 + 0.21 x + 6.8; r 2 = 0.99*. SP-non-VAM: 0.57 + 1.6 v'g; r 2 = 0.94*. RP-VAM: 6.88 + 0.41 V-g; r 2 = 0.79. RP-nonVAM: 1.47 + 0.0026 x; r 2 = 0.39. Arrows point at maximum values.

significant, and at 200ppm, there was an inversion (control plants had significantly higher shoot dry matter than the inoculated ones). For RP fertilized plants, there were significant differences between inoculated and control plants at all P levels. Only VAM-inoculated plants responded to RP fertilization. Non-mycorrhizal plants did not benefit from any RP level (Fig. 1). Similar observations were made for plant height at the end of the experiment (Fig. 2).

81

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VAM plants were significantly taller than the uninoculated controls up to 100 ppm SP, while growth inversion occurred at 200 ppm (as for dry weight). Increasing RP levels did not improve height significantly, but VAM plants were 5 to 6 times taller than the controls at all P levels. After 6 months growth, stem diameters were large enough for grafting, (minimum diameter of about 5.0 mm) with VAM-plants (Table 1) at all P levels and with both P sources, with non-VAM plants at 50, 100 and 200ppm SP, while the non-VAM plants at 0 ppm SP and at all RP levels were stunted and less than 10 cm tall. It is very important to get plants ready for budding as quickly as possible because this results in a significant economic advantage for the nursery. There were also pronounced differences between the two plant groups, with VAM-inoculated citrus having deeper green, larger, and more numerous leaves, indicating improved plant vigor. Height of VAM and control plants taken at monthly intervals in relation to the SP level, is shown in Fig. 3. At 0 ppm SP, growth of nonVAM plants was severely limited during the whole period, while growth of the VAM plants increased up to 216% between 75 and 105 days after transplanting, the period corresponding to the maximum growth-burst in VAM- and nonVAM plants. VAM-plants showed a tendency towards growth depression at 200 ppm P from the first measurement onward, and this became more pronounced with age. The largest increases in plant height occurred at 100 ppm P in VAM plants. Non-VAM plants showed maximum growth increases at 200 ppm P (Fig. 3). Phosphate source and level, and the presence of the endophyte also affected P concentration of
Table 1. Stem diameter (mm) of citrus seedlings 10 cm above soil level, 165 days after transplanting, with (VAM) and without (non-VAM) inoculation of G. etunicatum, and with increasing soil P levels (means of four replicates)

P level Ippml
Fig. 2. Regression curves for plant height of VAM and
non-VAM plants, fertilized with soluble phosphate ( - O - ) or rock phosphate ( - D - ) at four P levels. SP-VAM: -0.0025 x z + 0.57 x + 45.29; r 2 = 0.98. SP-non-VAM: 8.82 + 4.56 v ~ ; r 2 = 0.99**. RP-VAM: 32.45 + 0.061 x; r 2 = 0.21. RP-nonVAM: 5.93 + 0.014 x; r 2 = 0.97*. Arrows point at maximum values.

P level (ppm) 0 50 100 200

Soluble phosphate VAM 5.0 7.4 7.9 6.6 non-VAM NM a 6.1 6.7 7.5

Rock phosphate VAM 5.1 6.4 4.5 6.5 non-VAM NM NM NM NM

a NM (not measured). These plants were less than 10 cm tall.

14

Antunes and Cardoso

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AM
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100 150 SP level (ppm) b) Non- VAM plants ~ A ~ . ~ - ' ~ - ~ ~ . . . . . . . . . . ~

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45 days I 200 I I 100 150 SP level(ppm)

Fig. 4. Regression curves for total leaf P content of VAM and non-VAM plants, fertilized with soluble phosphate ( - O - ) or rock phosphate (-Fq-) at four P levels. SP-VAM: -0.00062 x 2 + 0.17 x + 1.54; r 2 = 0.99*. SP-non-VAM: 0.035 + 0.93 x/-~; r 2 = 0.98*. RP-VAM: 1.78 + 0.30 v ~ ; r 2 = 0.87. RP-non-VAM: 0.016 +0.0021 v'~; r 2= 0.93*. Arrows point at maximum values.
P in non-VAM plants (Table 2). In most cases, VAM plants had higher P concentrations than the control plants. With RP, there was only a significant difference in leaf P concentration of VAM plants between 0 and 200 ppm P. RP level had no effect on leaf P concentrations in nonVAM plants which were much lower at all P levels than in VAM plants. In SP treatments, total P content of leaves increased up to 100 ppm in VAM plants and up to 200ppm in control plants (Fig. 4). In RP treatments, P-fertilized VAM plants had much higher total leaf P contents than non-VAM plants, due to higher tissue P concentrations and 4- to 8-fold greater dry weight. Leaf K concentration varied little among different treatments (Table 2). On average, VAM plants had slightly higher leaf K concentrations than non-VAM plants; this difference was significant at 0 ppm P with SP and RP, and at 200 ppm P with SP. With non-VAM plants, there was a tendency towards higher leaf K concentrations in the RP treatments than in the SP treatments, probably as a consequence of the poorer growth of the non-VAM/RP plants. This was clearly expressed in the total K content of the RP plants which showed extreme differences between VAM and non-VAM plants (Fig. 5). With the SP treatments the differences in K content between

I 50

Fig. 3. Plant height taken at monthly intervals of VAM and

non-VAM plants, fertilized with soluble phosphate at four P levels.

citrus leaves (Table 2) and total leaf P content (Fig. 4). With SP, leaf P concentration increased corresponding to the amount of applied P up to 100 ppm P in VAM plants and only up to 50 ppm
Table 2. P and K concentration (%) of citrus leaves with (VAM) and without (non-VAM) inoculation of G. etunicatum, and with increasing soil P levels (means of four replicates)
P level (ppm) 0 50 100 200 Soluble phosphate VAM non-VAM 0.04 aB 0.14 bA 0.17 bB 0.18 bB Rock phosphate VAM 0.09 aA 0.14 abA 0.14 abB 0.16 bB non-VAM 0.04 aB 0.03 aB 0.05 aC 0.04 aC

P(%)
0.09 aA a 0.16 bA 0.21 cA 0.24 cA

K(%)
0 50 100 200 2.8 aA 2.1bA 1.6 bA 2.3 aA 1.6 aB 1.7aA 1.6 aA 1.4 aB 2.8 aA 2.0bA 2.8 aB 2.3 bA 1.8 aB 1.9aA 2.2 aB 2.5 aA

a Values followed by the same letters do not differ by the t test (P < 0.05). Small letters compare values in columns and capital letters compare values in rows.

M y c o r r h i z a in citrus

15

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P level ( p p m )

100 150 P level(ppm)

200

250

Fig. 5. Regression curves for total leaf K content of VAM

and non-VAM plants, fertilized with soluble phosphate ( - O - ) or rock phosphate (-I-l-) at four P levels. SP-VAM: 65.9 + 3.0 w'-~; r 2 = 0 . 6 5 . SP-non-VAM: 9 . 0 + 8 . 1 ~/~; r 2 = 0 . 8 2 . RP-VAM: 39.7 + 3.7 V-~; r 2 = 0.82. RP-non-VAM: 1.0 + 0.068 x/2; r 2 = 0.59. Arrows point at m a x i m u m values.

Fig. 7. Sporulation (spore number/50cm3 soil) of Glomus etunicatum on VAM citrus plants fertilizedwith solublephos-

phate (-O-) or rock phosphate (-El-) at four P levels. Arrows point at maximumvalues.

VAM and non-VAM plants followed the differences in shoot dry weight (Fig. 1). VAM colonization varied between 74 and 23% (Fig. 6) and spore numbers varied between 1118 and 156 per 50 cm 3 of rhizosphere soil (Fig. 7). High levels of SP were correlated with significantly lower infection percentages and lower spore numbers, whereas the high RP level had no pronounced effect on VAM growth, and depressed spore number considerably less than SP. Figures 1, 2, 4, and 5 permit several corn-

80 -------~'~'--'--'--'----- . . . . . . . . . . . i "~
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40200 0

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100

150

200

250

P level ( p p m )

Fig. 6. Regression curves for root infection percentage of VAM plants, fertilized with soluble p h o s p h a t e ( - O - ) or rock phosphate ( - E l - ) at four P levels. SP: 7 1 . 6 - 0 . 2 5 x; r 2= 0.99**. RP: 71.0 - 0.022 x; r: = 0.93*. Arrows point at maxi m u m values.

parisons between the treatments. For example, in the SP treatments one may establish the P level at which VAM and non-VAM plants have an equivalent development. In the SP treatments non-VAM plants reached the same P content as VAM plants at 183 ppm P (Fig. 4), the same height at 170 ppm (Fig. 2), and the same shoot dry weight at 135 ppm P (Fig. 1), while the same K content was reached at l l 8 p p m P (Fig. 5). Non-VAM plants reached the shoot dry weight of VAM plants without P fertilization, at 27 ppm P (Fig. 1) and the same height as VAM plants at 44 ppm P (Fig. 2) in SP treatments, and the same P and K contents at 10 and 23 ppm P respectively (Figs. 4 and 5). In RP treatments non-VAM plants never reached an equivalent development to VAM plants. When combining VAM fungal inoculation and soluble P, the maximum plant response for plant height occurred at 100 ppm (Fig. 2), while the maximum response for shoot dry weight (114 ppm), total P content (150 ppm) and total K content (200 ppm) all occurred at higher levels (Figs. 1, 4 and 5). Maximum VAM response in combination with RP occurred at 200 ppm P for all variables. VAM plants with RP fertilization reached the maximum shoot dry weight at about 120 ppm P, the same weight showed SP-fertilized VAM plants at 18 ppm P, and SP-fertilized non-VAM plants at 62 ppm P (Fig. 1). VAM plants with RP fertilization reached the maximum height some-

16

Antunes and Cardoso

20
15

~' I0

0,10

0,15
Leof P %

0,20

0,25

Fig. 8. Shoot dry weight of SP-fertilized VAM plants at

different leaf P concentrations.

where close to 150 ppm P, but with SP fertilization the same height was reached by VAM plants at 10 ppm P, and by non-VAM plants at 60 ppm P (Fig. 2). Maximum P content of RP-fertilized VAM plants occurred at 200 ppm P, corresponding to the same P content of SP-fertilized VAM plants at 33 ppm P and SP-fertilized non-VAM plants at 72 ppm P (Fig. 4). Maximum K content of RPfertilized VAM plants occurred at 200ppm P, corresponding to the same K content of SPfertilized VAM plants at 72 ppm P and of SPfertilized non-VAM plants at 100 ppm P (Fig. 5). When compared with VAM plants that received SP, 200 ppm P in the form of RP can substitute for 10 or 18 ppm P of SP, for height (Fig. 2) and shoot dry weight (Fig. 1) respectively. To study the nature of the relationship between shoot weight and P concentration in shoots, these values were plotted one against the other (Fig. 8). Figure 8 shows that shoot dry weight increases with leaf P concentration up to a value close to 0.20% P (about the average leaf concentration in healthy citrus plants, according to Delfs-Fritz, 1970). At this point there is a sharp inversion in the response curve and a higher leaf P concentration induces lower dry matter production.

Discussion

Weaker growth of VAM plants than of non-VAM controls, usually under high P-fertilization and reduced photosynthetic conditions, has been re-

ported by several authors (Bethlenfalvay and Pacovsky, 1983; Buwalda and Goh, 1982; Diederichs, 1982; Koide, 1985; Siqueira and Colozzi-Filho, 1986; Smith et al., 1986; Snellgrove et al., 1982; Son et al., 1988; Tester et al., 1985). Although most of these papers discuss a growth reduction by VAM with various host plants, all of them differ from the results of this experiment in one aspect; we observed a growth depression of the plants not only in relation to the non-VAM control, but also relative to VAM plants at lower P levels. This phenomenon has been also observed earlier (Cardoso et al., 1986). Abbott and Robson (1986) presented a discussion on several hypotheses that could explain this parasitism, such as, introduction of plant pathogens together with the mycorrhizal inoculum, competition for P, P toxicity, and competition between endophyte and the host plant for carbohydrates. However, they stated that the C-drain hypothesis seemed to be very improbable in cases in which there was also a pronounced reduction in root colonization by the VAM fungus. When analysing the hypotheses above, we can dismiss the first one because there was no disease incidence in the plants, and at lower P levels VAM fungi promoted significant growth increases. P toxicity seems to be the most plausible explanation, because VAM citrus at 100 and 200 ppm soluble P presented a significantly higher leaf P concentration than at lower soil P (Table 2). An average of various analyses of citrus leaves quotes a leaf P concentration of 0.18% (Dells-Fritz, 1970). In the present experiment, VAM plants at 50ppm soluble P and non-VAM plants in the range of 50 to 200 ppm P, had a similar concentration, whereas VAM plants at 200 ppm soluble P reached a leaf P concentration of 0.24%, that is 30% above the highest control plant level. When P is not limiting for plant growth, (and perhaps when it is in excess), more P will be available for carbohydrate phosphorylation, which is fundamental for active carbon transport from the host to the endophyte (Callow et al., 1978). Therefore, at higher soil P levels, active carbon transport can possibly proceed at higher rates, resulting in a greater carbon drain from the host.

Mycorrhiza in citrus

17

P toxicity may also be related to assimilation or immobilization of other plant nutrients, such as Zn and Cu. There may have occurred some chemical reactions between P and other plant nutrients, especially heavy metals, which became inhibitory in the presence of the VAM fungus. This point should be further investigated. K nutrition is also directly tied into carboyhydrate production, transport and utilization. Total K content increased continuously with soil P increases (Fig. 5). VAM plants at 200ppm SP also contained a higher leaf K concentration than at 100 ppm P (Table 2). Perhaps the combination of a high P and K concentration can interfere with the carbohydrate metabolism in a way that reduces carbohydrate utilization by the host, while allowing for more transport to the endophyte. Therefore, extra P given to VAM citrus, possibly increases P and K concentrations to a point at which there is an inhibition of the growth promoting ability of the VAM fungus, resulting in growth depression. The relationship observed between leaf P concentration and dry weight of the shoots (Fig. 8) reinforces the idea that up to a point greater P concentrations lead to higher dry matter accumulations. However, at concentrations above approximately 0.18% P there is a growth inversion, i.e., very high P concentrations seem to be toxic for plant growth and these exceedingly high P concentrations in the plant are a consequence of the combination of an effective VAM fungus with high soluble P levels in the soil. Even at 200 ppm SP in the soil, non-VAM plants did not surpass the expected value of 0.18% P in leaves, while VAM plants reached 0.21 and 0.24%, respectively at 100 and 200 ppm SP in the soil. Different P equivalents for different parameters show that VAM plants are relatively taller and contain more P than P-fertilized citrus with equivalent dry weight, implying that there are morphological and physiological differences between P-fertilized and VAM plants (Pacovsky et al., 1986). Increased uptake of several macroand micro-nutrients (Rhodes and Gerdemann, 1980) and alterations in chemical composition (Nagy et al., 1980, Nordby et al., 1981), may also play an important role in the growth response of VAM plants. In this experiment the 100ppm soluble P level promoted maximum height and

dry matter of VAM citrus, while higher P levels further increased P and K accumulations. With respect to root colonisation rates, Johnson et al. (1982), Cardoso et al., (1986) and many other authors previously observed a reduction due to increases of soluble P in the soil with citrus plants. However, the development of intraradical structures of the VAM fungus may differ from the development of extraradical mycelium (Abbot et al., 1984; Graham et al., 1982a). Also, growth of external hyphae on troyer citrange was extremely variable according to the species or isolate of the endophyte (Graham et al., 1982b). Since there are no data available for the present experiment, it would be impossible to speculate on a possible C-drain for these structures. Root respiration rates for VAM- and non-VAM plants would also be required. Reduction of percentage VAM colonization, due to P fertilization, is very variable according to the host plant or fungal isolate. There may be a special ionic balance for every VAM fungushost plant combination that allows for maximal expression of the root colonization character or of the growth promoting ability (Lambais and Cardoso, 1988). Also, as reported in Cardoso et al. (1986), different combinations of VAM fungi and citrus species led to a reduction in percentage root infection due to soil P increases from 0 to 100 ppm P with Rangpur Lime and with Sweet Orange. Growth depression occurred in Sweet Orange but not in Rangpur Lime when inoculated with Glomus etunicatum. Conversely, the VAM fungus Scuttelospora gilmorei caused no growth depression of either host plant, and reduced percentage root infection only with Rangpur Lime (Cardoso et al., 1986). From all these evidences, one may infer that, different citrus species or varieties, at higher, but differentiated levels of P fertilization and in combination with the VAM fungus Glomus etunicatum, absorb excess P which is toxic and results in growth depression. With exception of the low P supply (50 ppm P), the SP-fertilized plants were always superior to the RP-fertilized and VAM-infected plants. VAM inoculation was beneficial also for SPfertilized plants at 100 ppm P in respect of each of the characters investigated, but not at

18

Antunes and Cardoso long-term sustainable agricultural practice, creating an economic, advantageous and ecologically sound, procedure for citrus cultivation in the future.

200 ppm P. RP at higher levels, together with VAM inoculation, can substitute for 60 to 72 ppm of SP applied to non-VAM citrus or for 10 to 72ppm of SP, applied to VAM plants, according to the variable under investigation. Patos de Minas RP by itself did not improve plant growth or phosphate nutrition, probably due to low P availability for nonoVAM citrus. According to different authors (Gianinazzi-Pearson et al., 1981; Hayman and Mosse, 1972; Sanders and Tinker, 1971) VA fungi cannot increase the solubility of rock phosphates but do increase the uptake of slowly released P ions from the soil solution by increasing the soil volume that is exploited by the host. This point has become more controversial. Short-term experiments done with soluble 32p sources, perhaps, cannot be used to demonstrate uptake from the same P pool in the soil. New research has cast doubt on these earlier studies (Bolan et al., 1984). The addition of 100 ppm P as RP, equivalent to approximately 500 kg RP per hectar, together with effective VAM inoculum can apparently satisfy a reasonable plant-P requirement for the first year's growth.

Acknowledgements
The award of a post graduate grant to the First author by the Science Foundation of the State of Sao Paulo, FAPESP, is gratefully acknowledged. Thanks are also due to Marcio R Lambais for help with the statistical analysis and to Dr Raymond S Pacovsky who revised the first draft and made valuable comments. The authors also wish to thank Professor Emeritus Dr S Rehm for reviewing the manuscript.

References
Abbott L K and Robson A D 1986 The effect of mycorrhiza on plant growth. In VA Mycorrhiza 2nd ed. pp 113-130. Eds. C L Powell and D J Bagyaraj. CRC Press. Boca Raton, FL. Abbott L K, Robson A D and De Boer G 1984 The effect of phosphorus on the formation of hyphae in soil by the vesicular-arbuscular mycorrhizal fungus, Glomus fasciculatum. New Phytol. 97, 437-446. Ambler J R and Young J L 1977 Techniques for determining root length infected by vesicular-arbuscular mycorrhizae. Soil Sci. Soc. Am. J. 41, 551-556. Bethlenfalvay G B and Pacovsky R S 1983 Light effects in mycorrhizal soybeans. Plant Physiol. 73, 969-972. Bolan N S, Robson A D, Barrow N J and Aylmore L A G 1984 Specific activity of phosphorus in mycorrhizai and non-mycorrhizal plants in relation to the availability of phosphorus to plants. Soil Biol. Biochem. 16, 299-304. Buwalda J G and Goh K M 1982 Host-fungus competition for carbon as a cause of growth depressions in vesiculararbuscular mycorrhizal ryegrass. Soil Biol. Biochem. 14, 103-106. Callow J A, Capaccio L C M, Parish G and Tinker P B 1978 Detection and estimation of polyphosphate in vesiculararbuscular mycorrhizas. New Phytol. 80, 125-134. Cardoso E J B N 1985 Effect of vesicular-arbuscular mycorrhiza and rock phosphate on the soybean-Rhizobium symbiosis. R. bras. Ci. Solo 9, 125-130. Cardoso E J B N, Antunes V, Silveira A P D and Oliveira M H A 1986 Efici~ncia de fungos micorrizicos vesiculo-arbusculares em porta-enxertos de citros. R. bras. Ci. Solo 10, 25-30. Dells-Fritz W 1970 Citrus. Ruhr-Stickstoff A.G., Bochum, FRG 230 p.

Conclusions
Growth improvement of VAM citrus at low soil P levels is primarily correlated with a better plant P nutrition, although there are other factors involved. Growth depression of VAM plants at higher levels of available soil P appears to be a result of exceedingly high P and K concentrations in the leaf tissue which in combination may impair the carbohydrate metabolism of the plants. For commerical VAM inoculation programs, it is fundamental to test various endophytes over a broad range of P levels to avoid 'fertilizerinduced pathogenicity of mycorrhizal fungi' (Kiernan et al., 1983). Rock phosphate in combination with VAM inoculation can give results comparable to the application of medium amounts of a soluble P source, even when the apatite reacts very slowly and induces no growth responses in the absence of the VAM fungus. Thus, fertilisation with rock phosphate together with VAM fungi, can be used as a viable,

M y c o r r h i z a in citrus
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