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Revista de Microbiologia

Print version ISSN 0001-3714

Rev. Microbiol. vol. 29 n. 4 São Paulo Oct./Dec. 1998 



Marco A. Martins* , Andre F. Cruz
Universidade Estadual do Norte Fluminense, Centro de Ciências e Tecnologias Agropecuárias, Setor de Microbiologia do Solo, Campos, RJ, Brasil

Submitted: July 07, 1997; Returned to authors for corrections: June 04, 1998;
Approved: July 23, 1998




An experiment under greenhouse conditions was carried out to evaluate the relative contribuition of arbuscular mycorrhizal fungi (AMF) in the process of nitrogen transfer from cowpea to maize plants, using the isotope 15N. Special pots divided in three sections (A, B and C), were constructed and a nylon mesh screen of two diameters: 40µm (which allowed the AMF hyphae to pass but not the plant roots) or 1µm (which acted as a barrier to AM hyphae and plant roots) was inserted between the sections B and C. Section A had 25.5 mg of N/kg using (15NH4)2SO4 as N source. Two cowpea seedlings inoculated with Rhizobium sp. were transplanted with their root systems divided between the sections A and B. Ten days later, 2 seeds of maize were sown into the section C which was inoculated with Glomus etunicatum. Thirty-five days after transplanting, the maize plants were harvested. AMF inoculation increased dry weight and 15N and P content of maize plant shoots. Direct transfer of 15N via AMF hyphae was 21.2%; indirect transfer of 15N mediated by AMF mycelium network, was 9.6%, and indirect transfer not mediated by AM mycelium network , was 69.2%.

Key words: mycorrhizae, cowpea, maize, nitrogen transfer




Associations between arbuscular mycorrhizal fungi (AMF) and plant roots are usually non-specific in terms of the fungus-plant pairings that are compatible (24). This apparently low host specificity shown by many of the fungi that form AMF, may allow mycelial network of a particular fungus in the soil to be connected directly to the fungal structures within the roots of two or more different plants forming hyphal links between their mycorrhizal roots (25, 26, 28). These links provide a pathway that mediates the inter-plant transfer of nutrients directly through the fungal mycelium. Transfer of nutrients from one plant to another via AM hyphal connections was shown with carbon (8, 20, 21, 22), phosphorus (23, 31) and nitrogen (1, 15, 34).

In intercropping systems with grass and legume plants it is possible that the nitrogen derived from the biological fixation of nitrogen (BFN) by the legume can be transferred to the grass. Brophy et al. (3) detected a transfer of 40 kg of N from a legume to a grass, and Rao and Giller (27) observed a high content of N in grass intercropped with a legume. The flow of N between plants may happen through several pathways. The legume may release N to the soil through exsudates from the roots, leaves, nodule debris and roots tissues (5, 7, 14). Some works with 15N dilution (4) and subdivided roots (15) have shown increases of N transfer between plants associated with AMF. The AMF may promote a high uptake of N in grass intercropped with a legume, even though there may be no significant differences in the shoot dry weight (10). The N transferred may be enough to improve the growth of the receiver plants (9). The nitrogen transfer between plants mediated by the AMF may occur by the following pathways: 1) direct transfer (DT) of nitrogen through mycelium which interconnects the roots of two plants; 2) indirect transfer (ITM), involving leakage of nitrogen from one root, its absorption by AMF hyphae scavenging in the rhizosphere, and transfer to neighbouring plants. In addition to these two pathways, there is a third mechanism which is not mediated by AMF, which involves the leakage of nitrogen from roots of one plant and its subsequent absorption by the roots of neighbouring plants. To evaluate the relative contribution of each pathway to the total N transferred from one plant to another is very important because the direct transfer of N by AMF mycelium reduces the loss of N in the soil (leaching and immobilization) and also could improve the N cycling. Our purpose was to examine the contribution of the three possible pathways to the N transfer process between cowpea (donor plant) and maize (receiver plant), using the isotope 15N.



Rectangular pots were constructed and each pot was divided longitudinally into three identical sections (2 dm3 of capacity) A, B and C (Fig. 1). Between sections B and C two procedures were adopted: a) a nylon mesh screen of 1µm was inserted to act as a barrier to AMF hyphae and plant roots; b) a nylon mesh screen of 40µm was inserted to allow the AMF to pass but not the plant roots. A solid barrier was also inserted between sections A and B to prevent any physical contact between the two sections.


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Figure 1. Design of the split pots


Each pot section was filled with a soil: sand substrate (1:2, v/v) previously sterilized with CH3Br. The substrate properties were: pH 5.3; organic C, 0.41%; total N, 0.041%; P and K content 8 and 19 mg/kg, respectively. The substrate in each pot section was amended with: 50 mg P/kg, 50mg K/kg and 0.6 ton/ha of Ca and Mg in the proportions of 4:1. At planting, 25 mg/kg of N was added into the section A, using as source (15NH4)2SO4 with 10% of 15N. Fifteen days later, 0.5 mg/kg of N was once again added into the section A, using the same source, but with 99% of 15N.

Two seedlings of cowpea inoculated with Rhizobium sp. strain BR-2001, from the National Centre of Agrobiology Research - EMBRAPA - RJ - Brazil, were carefully planted with their roots divided so that half of their root systems were growing in each of sections A and B. The pots were then placed in a green house.

Ten days later, two seeds of maize (receiver plant) were sown into the section C. The inoculation with AMF was made in half of the pots by adding into the substrate of section C, approximately 20 g of soil containing spores and hyphae of Glomus etunicatum (Becker and Gerdemann). In the case of non-mycorrhizal plant pots, approximately 25 ml of filtrate solution (filter paper Whatman n° 1) derived from infected soil, was added into section C to ensure that control plants received a bacterial population comparable with that present in the mycorrhizal medium, but lacking AM propagules.

Thirty-five days after maize planting, the plants were harvested. A sub-sample of cowpea and maize roots was taken to determine the root colonization by AMF (11). The shoot of maize plants was oven-dried (48h at 80°C), weighed and ground. The 15N content was determined by mass spectrometer and P content colorimetrically by spectrophotometer.

The relative contribution of each of the three processes of N transfer to the total amount 15N transferred to maize was obtained using the procedures of Martins (22) for 14C, as follows: 

a) Direct transfer pathway via AMF hyphae (DT):

0011fo4.gif (716 bytes)


NM40 = 15N content in maize shoots grown in pots with a nylon mesh screen of 40µm and inoculated with AMF;

NM1 = 15N content in maize shoots grown in pots with a nylon mesh screen of 1µm and inoculated with AMF.

b) Indirect transfer pathway mediated by AMF mycelium network (ITM)

0011fo5.gif (811 bytes)


NM1 = 15N content in maize shoots grown in pots with a nylon mesh screen of 1 µm and inoculated with AMF;

NNM40= 15N content in maize shoots grown in pots with a nylon mesh screen of 40 µm and not inoculated with AMF;

NM40 = 15N content in maize shoots grown in pots with a nylon mesh screen of 40 µm and inoculated with AMF.

c) Indirect transfer pathway not mediated by AMF (ITNM)

0011fo6.gif (657 bytes)


NNM40= 15N content in maize shoots grown in pots with a nylon mesh screen of 40 µm and not inoculated with AMF;

NM40 = 15N content in maize shoots grown in pots with a nylon mesh screen of 40 µm and inoculated with AMF.

Data were subjected to an analysis of variance (ANOVA test); least significant differences were calculated by an F test.



The levels of arbuscular mycorrhizal infection in either maize or cowpea plants were satisfactory (Fig. 2). The detection of infection in the roots of cowpea confirmed that transfer of infection had occurred from maize plants. The observation of no colonization in cowpea roots grown in pots with a nylon mesh screen of 1µ, confirmed that this screen was efficient to prevent invasion of external hyphae from section C into B, so that, maize and cowpea plants were not interconnected by a common mycelium. No colonization was observed in non-mycorrhizal treatments.


0011i02.gif (17541 bytes)

Figure 2. Percentage of cowpea and maize root length infected by Glomus etunicatum. Each bar represents the mean of 4 replicates.


The mycorrhizal infection led to a significant increase of both dry weight (Fig. 3) and P content (Fig. 4) of maize shoots. There was no effect due to the barrier between the sections B and C on dry weight of maize shoots. However, P content in the pots inoculated with AMF (+AMF) was significantly lower in the pots with a nylon mesh screen of 1 µm.


0011i03.gif (25868 bytes)

Figure 3. Dry weight of maize shoots. Each bar represent the mean of 4 replicates.



0011i04.gif (59612 bytes)

Figure 4. Phosphorus content of maize shoots. Each bar represent the mean of 4 replicates.


The 15N content in maize shoots was not significantly affected by the presence of the imposed barrier (Fig. 5). Mycorrhizal infection led to a significant increase of 15N content in maize shoots.


0011i05.gif (17164 bytes)

Figure 5. 15N content of maize shoots. Each bar represent the mean of 4 replicates.


The relative contribution of each pathway to the total amount of 15N transferred to maize shoots mediated by AMF, including the direct (DT) and indirect process (ITM), was lower (21.2 + 9.6 = 30.8%) than the contribution not mediated by the fungi (ITNM = 69.2%) (Table 1). Thus the main route by which 15N was transferred from cowpea to maize was via the indirect transfer not involving the AMF, where the 15N may have been exudated from the cowpea root into soil solution being subsequently uptaken by the roots of maize plants.


0011i06.gif (12468 bytes)


0011fo1.gif (576 bytes)

0011fo2.gif (600 bytes)

0011fo3.gif (550 bytes)



The presence of infection in the roots of cowpea plants in section B of pots confirmed that the AMF had grown from maize roots (section C) to cowpea (section B) (Fig 2). Bethlenfalvay et al. (2) and Camel et al. (6) have shown that the hyphae can spread from their associated roots into the soil over distances of 6 to 9 cm. Since the onset of sporulation of AMF requires 4-8 weeks and new spores have an endogenous dormancy cycle of 6 weeks to 6 months (33), it seems that the colonization observed in cowpea roots is attributable to growth of mycelium from roots of maize plants.

The external mycelial network of AMF growing from the section C into the section B can promote links between the two plant species. Read et al. (30) observed that most plants in seminatural grassland become heavily infected very soon after seed germination. They suggested that infection of the developing root system must arise from contact with mycelium spreading from plants with established infection, and that as a consequence of this pattern of infection, many plants within the community must be inter-linked by mycorrhizal hyphae. This has since been confirmed by studies of the development of infection of seedlings in the field (29).

This work showed that the maize shoot dry weights were higher in mycorrhizal plants than those of their non-mycorrhizal counterparts (Fig. 3). However there is no evidence that this fact is attributable to the N transferred from legume to grass. It is more probable that this plant growth is due to a higher P content observed in mycorrhizal plants (Fig. 4). Hamel and Smith (12) concluded that the higher growth of maize plants intercropped with soybean under mycorrhizal condition was due mainly to a better P uptake by mycorrhizal plants and the N-transfer to maize plants had low significance to that growth. In practice the N content transferred between plants may not be enough to improve the growth or mineral nutrition of the receiver plant, because perhaps the maize plants need a higher quantity of N than that available from the cowpea plants.

The results revealed that AMF was enable to promote direct transfer of N from donor to receiver plants, because there was an increase of 15N transferred to receiver when they were separated from the donor plants by a nylon mesh screen of 40µm, which allowed only the AMF mycelium to pass. The involvement of AMF in the process of N transfer between plants remains controversial. Some researches have shown a positive effect (17, 19). On the other hand, others have not observed any influence of mycorrhizae in this process (16). Hamel and Smith (13) showed that even though the AMF had established links between plants, they did not increase N transfer. According to Johansen et al. (18), the transfer of N from one plant to another mediated by arbuscular mycelium depends on the demand of plants. Tomm et al. (32) have also pointed out that this process can be bi-directional depending of the demand of receiver plants.

The transfer of substances between plants mediated by AMF have been also shown for other elements, such as carbon and phosphorus, by using radioisotope techniques. Francis and Read (8) and Martins (22) have demonstrated that the 14C transfer between plants occurred directly via AMF mycelium. Whittingham and Read (35) and Martins and Read (23) showed that direct transfer of 32P between living source and sink plants occurs mainly by hyphae connecting the two plants.

The present study revealed that the AMF can provide channels for transfer of N between plants cultivated under intercropped systems. However, the most significant route by which 15N was transferred from cowpea to maize was by the indirect process not involving the fungus. Further studies are now required to determine if the transferred N is enough to stimulate the growth of the receiver and it is also necessary to evaluate the behaviour of this fungus under other environmental circunstances.



We are extremely grateful to CNPq (Brazil) for financial support.




Significância do micélio externo dos fungos micorrízicos arbusculares. III. Estudo da transferência de nitrogênio entre plantas inter-conectadas por um mesmo micélio.

Conduziu-se um experimento em casa de vegetação para avaliar a importância dos fungos micorrízicos arbusculares (FMA) sobre o processo de transferência de nitrogênio de plantas de caupi para o milho, utilizando-se o isótopo 15N. Foram construídos vasos compartimentalizados, compostos de 3 seções (A, B e C) de 2 dm3 de capacidade. Entre as seções B e C inseriu-se uma tela de nylon com 2 diâmetros de abertura de malha: 1 µm (que atuou como barreira para o micélio fúngico e para as raízes das plantas), ou 40 µm (que atuou como barreira somente para as raízes das plantas). Adicionou-se 25,5 mg/kg de N na forma de (15 NH4)2SO4 na seção A dos vasos. A seguir, 2 plântulas de caupi previamente inoculadas com Rhizobium sp. foram plantadas com seus sistemas radiculares divididos entre as seções A e B. Após 10 dias, duas sementes de milho foram semeadas na seção C, onde se efetuou a inoculação com o fungo Glomus etunicatum no orifício de plantio. O experimento foi coletado 35 dias após, e os resultados demonstraram que a presença do FMA aumentou a matéria seca e o conteúdo de 15 N e P da parte aérea das plantas de milho. A transferência direta de 15 N via hifa fúngica, foi de 21,2%; a transferência indireta mediada pelos FMA, foi de 9,6%; e a indireta não mediada pelos FMA, foi de 69,2%.

Palavras-chave: micorrizas, caupi, milho, transferência de nitrogênio.




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* Corresponding author. Mailing address: Universidade Estadual do Norte Fluminense, Centro de Ciências e Tecnologias Agropecuárias Setor de Microbiologia do Solo, Av. Alberto Lamego, 2000, CEP 28015-620, Campos, RJ, Brasil. Fax (+5524) 726-3746. E-mail: 

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