1

 

 

 

Fixação de nitrogênio e micorrização em 

leguminosas de mata ciliar 

 

 

 
CAMILA MAISTRO PATREZE 

 
 
 
 
 
 
 

Dissertação apresentada ao Instituto 
de Biociências da Universidade 
Estadual Paulista “Júlio de Mesquita 
Filho”, Campus de Rio Claro, para a 
obtenção do título de Mestre em 
Ciências Biológicas (Área de 
Concentração: Biologia Vegetal). 
 
 
 
 
 
 
 

 
Rio Claro 

Estado de São Paulo – Brasil 
Janeiro de 2003 

 



    

 

2

 

 

 

Fixação de nitrogênio e micorrização em 

leguminosas de mata ciliar 

 

 
CAMILA MAISTRO PATREZE 

 
 
 

Orientadora: Profa. Dra. LÁZARA CORDEIRO 
 
 
 
 
 
 

Dissertação apresentada ao Instituto 
de Biociências da Universidade 
Estadual Paulista “Júlio de Mesquita 
Filho”, Campus de Rio Claro, para a 
obtenção do título de Mestre em 
Ciências Biológicas (Área de 
Concentração: Biologia Vegetal). 
 
 
 
 
 
 
 

Rio Claro 
Estado de São Paulo – Brasil 

Janeiro de 2003 
 

 



    

 

3

 
 
581.13        Patreze, Camila Maistro 

P314f                 Fixação de nitrogênio e micorrização em leguminosas de 
mata ciliar / Camila Maistro Patreze. – Rio Claro : [s.n.], 2003 

100 f. : il., gráfs, tabs. + fots. 
 

Dissertação (Mestrado) – Universidade Estadual Paulista, 
Instituto de Biociências 

                          Orientador: Lázara Cordeiro 
 
1. Plantas – Nutrição. 2. Ecofisiologia vegetal. 3. Ribózio. 4. Micorriza. 5. 
Nodulação 6. Dupla inoculação. 7. Simbiose.  I. Título. 
 
                 Ficha catalográfica elaborada pela STATI – Biblioteca da UNESP 
                                                 Campus de Rio Claro/SP 

 



    

 

4

 
 

ÍNDICE 
 
 

Página 
 

AGRADECIMENTOS...............................................................................................iii 

RESUMO.................................................................................................................v 

ABSTRACT.............................................................................................................vii 

1-INTRODUÇÃO....................................................................................................01 

 1.1. Considerações sobre as matas ciliares e as leguminosas...................01 

 1.2. A Simbiose rizóbio-leguminosas..........................................................02 

 1.3. Micorrizas vesículo-arbusculares (MVA)..............................................04 

 1.4. As relações rizóbio-micorriza-leguminosas..........................................06 

2- OBJETIVOS.......................................................................................................09 

 2.1. Objetivos gerais....................................................................................09 

 2.2. Objetivos específicos............................................................................10 

3- LITERATURA CITADA......................................................................................11 

CAPÍTULO 1. Nitrogen-fixing and vesicular-arbuscular mycorrhizal symbiosis in 

tropical tree of tribe Mimoseae (Leguminosae-Mimosoideae)………………..........17 

CAPÍTULO 2. Nodulation, mycorrhizal colonization and growth of Enterolobium 

contortisiliquum and Inga laurina seedlings in the nursery…...……………… …...45 

CAPÍTULO 3. Inoculation of Lonchocarpus muehlbergianus and Platypodium 

elegans with rhizobia and/or mycorrhizal fungi……………....….………………..…67 

4. CONSIDERAÇÕES FINAIS...............................................................................84 

5. APÊNDICE.........................................................................................................86

 



    

 

i 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Aos meus pais João e Ângela  
e minhas irmãs Flávia e Larissa, 
- minha família querida - 
dedico este trabalho. 

 
 
 
 

 

 



    

 

ii 

 

 

Metade (Oswaldo Montenegro) 
 
Que a força do medo que tenho não me impeça de ver o que anseio. 
Que a morte de tudo que acredito não me tape os ouvidos e a boca. 
Porque metade de mim é o que eu grito, 
mas a outra metade é silêncio. 
Que a música que eu ouço ao longe seja linda, ainda que triste. 
Que a mulher que eu amo seja sempre amada, mesmo que distante. 
Porque metade de mim é partida e a outra metade é saudade. 
Que as palavras que eu falo não sejam ouvidas como prece nem repetidas com 
fervor, 
Apenas respeitadas como a única coisa que resta a um homem inundado de 
sentimento. 
Porque metade de mim é o que eu ouço, 
mas a outra metade é o que calo. 
Que essa minha vontade de ir embora se transforme na calma e na paz que eu 
mereço, 
Que essa tensão que me corrói por dentro seja um dia recompensada. 

Porque metade de mim é o que eu penso, 
e a outra metade é um vulcão. 
Que o medo da solidão se afaste, que o convívio comigo mesmo se torne ao 
menos suportável 
Que o espelho reflita em meu rosto o doce sorriso que eu me lembro de ter 
dado na infância. 
Porque metade de mim é a lembrança do que fui, 
a outra metade eu não sei... 
Que não seja preciso mais do que uma simples alegria para me fazer aquietar o 
espírito. 
E que o teu silêncio me fale cada vez mais. 
Porque metade de mim é abrigo, mas a outra metade é cansaço. 
Que a arte nos aponte uma resposta, mesmo que ela não saiba, e que ninguém a 
tente 
Complicar porque é preciso simplicidade para fazê-la florescer. 
Porque metade de mim é a platéia e a outra metade, a canção. 
E que minha loucura seja perdoada. 
Porque metade de mim é amor e a outra metade... também. 
 
 



    

 

iii 

 
 
 

AGRADECIMENTOS 
 

 
Agradeço a Profa. Dra. Lázara Cordeiro, minha orientadora e exemplo de 

mulher, pelos ensinamentos científicos, idéias, vibração a cada passo deste trabalho e 

acima de tudo pelo prazeroso convívio e amizade. Com a licença de suas palavras, valeu 

parceira!!! 

 
 

Aos demais professores e aos funcionários do Depto. de Botânica que contribuíram 
para o desenvolvimento deste trabalho: 

 
À Coordenadora do Curso de Pós-Graduação em Biologia Vegetal, Profa. Dra. 

Leonor Patrícia C. Morelatto, pelo apoio nas questões burocráticas e contribuição para 

minha formação em suas disciplinas; 

Ao chefe do Departamento de Botânica Prof. Dr. Victor J. Mendes Cardoso pela 

utilização dos laboratórios e equipamentos, jardim experimental e por sua contribuição no 

meu exame de qualificação, juntamente com o Prof. Dr. Osvaldo Aulino da Silva, que 

muito tem me ensinado nas poucas vezes em que conversamos; 

Aos Profs. Marco Antonio de Assis e Reinaldo Monteiro deste depto., e aos Profs. 

Dr. Jorge Yoshio Tamashiro e Dra. Ana Maria Gourlart de Azevedo Tozzi, da Unicamp 

pelas identificações taxonômicas das plantas selecionadas para este trabalho; 

Aos técnicos e funcionários pelo envolvimento e ajuda ao longo destes anos, em 

especial ao José Cecílio Toledo (Zi) e ao Edward, do laboratório de fisiologia vegetal, 

pelos valorosos ensinamentos práticos e convivência, os quais foram fundamentais na 

execução deste trabalho. Também agradeço a ajuda de, Ari R. Pesce, Ruy, Valnice, 

Wenilton Luís Daltro e da secretária mais legal do departamento, Célia Maria Hebling pela 

disponibilidade e amizade. 

 

Às Instituições de fomento e pesquisa : 

 

 Ao Conselho Nacional de Pesquisa Científica e Tecnológica (CNPq) pela 

concessão da bolsa de mestrado; 



    

 

iv 

 À Fundação para o Desenvolvimento da UNESP (Fundunesp) pelo auxílio 

financeiro para a execução de parte do trabalho; 

 Ao Instituto de Pesquisas e Estudos Florestais –IPEF, Esalq/USP e ao Instituto 

Florestal de São Paulo – IF , pela doação de sementes; 

 Ao Centro de Pesquisas CENA na pessoa da Profa. Dra. Siu Mui Tsai, que além de 

disponibilizar equipamentos e dependências do laboratório para algumas análises sempre 

foi muito receptiva e transmitiu muitos ensinamentos. Aos técnicos Francisco e Wagner 

deste lab. por suas contribuições. 

 

Aos amigos: 

 Do departamento, de convívio diário ou esporádico que muito contribuíram com 

este trabalho diretamente, na ajuda prática do trabalho manual, nas dicas de uso do 

computador, dos sites interessantes da internet, dos programas estatísticos, no uso dos 

aparelhos do laboratório, etc. Também por compartilharem dos afazeres e prazos. 

Agradeço em especial a ajuda indireta de muitos, nas horas de descanso ou do café, onde 

muito se aprende da biologia e da vida num sentido mais humano, entretanto agradeço em 

especial a: 

 Mayra Teruya Eichemberg, por compartilhar de muitos momentos importantes em 

minha vida e por suas sábias colocações e Victor Fernandes Oliveira de Miranda, um 

exemplo de profissionalismo e amizade, por me ajudar sempre com muito prazer. 

Obrigada! 

À Tiago Pires Marques que me auxiliou nas etapas iniciais deste trabalho; 

 Às grandes amigas: Simone Peixoto Brito e Simeire Manarim; 

E aos amigos mais próximos: Elias, Denise, Luiza, Mauricio, Eduardo (Bob), 

Bibiana, João, Mantovani, Gil e Marcelinho, pela agradável convivência. 

 E para finalizar, agradeço em especial Eduardo Gross, uma pessoa admirável em 

todos os sentidos e que me faz querer ser cada dia melhor. Obrigado pela ajuda mais que 

direta neste trabalho, por compartilhar sonhos e por me ensinar o amor. 

 

 

 

 

 



    

 

v 

 
Fixação de nitrogênio e micorrização em leguminosas de mata ciliar 

 

Camila Maistro Patreze 

Orientadora: Lázara Cordeiro 

 

 

RESUMO 

 
 

Algumas leguminosas formam simbiose mutualística com bactérias fixadoras de 

nitrogênio e com fungos micorrízicos arbusculares. Conseqüentemente, estas plantas 

podem crescer mais rapidamente, além de enriquecer o solo com nitrogênio, fósforo e 

outros nutrientes. O presente trabalho analisou a nodulação, colonização micorrízica, 

crescimento e desenvolvimento iniciais de Anadenanthera colubrina (Vell. Conc.) Brenan. 

(angico-branco); Mimosa bimucronata (DC.) Kuntze (espinho-de-maricá); Parapiptadenia 

rigida (Benth.) Brenan (angico-vermelho); Enterolobium contortisiliquum (Vell. Conc.) 

Morong (tamboril); Inga laurina (Sw.) Willd. (ingá); Platypodium elegans Vogel 

(jacarandá-banana) e Lonchocarpus muehlbergianus Hassl (embira-de-sapo), usando solo 

de mata ciliar não esterilizado, adubação mineral e inoculação de rizóbio e micorriza, em 

viveiro. Todas as espécies foram colonizadas por fungos micorrízicos (inoculados e 

nativos) e nodularam. Somente P. rigida e P. elegans não apresentaram nodulação 

espontânea. A adição de nitrogênio mineral inibiu o número e a massa seca de nódulos, a 

atividade da nitrogenase e o teor de leg-hemoglobina em todas as espécies. A adição de 

fósforo mineral diminuiu a colonização micorrízica somente em A. colubrina e M. 

bimucronata. A inoculação dos fungos não afetou o crescimento das plantas e não 

favoreceu a absorção de fósforo; entretanto, a inoculação de rizóbio favoreceu a nodulação 

de A. colubrina e a nodulação e crescimento de M .bimucronata, L. muehlbergianus e E. 



    

 

vi 

contortisiliquum em tratamentos inoculados apenas com rizóbio ou conjuntamente com 

fungo micorrízico. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



    

 

vii 

Nitrogen fixation and mycorrhizal in legumes from riparian forest 

 

Camila Maistro Patreze 

Orientadora: Lázara Cordeiro 

 

ABSTRACT 

 

Some legumes develop mutualistic symbiosis with nitrogen-fixing bacteria, as rhizobia and 

arbuscular mycorrhizal fungus. As a result, these plants can grow more rapidly and also to 

amend the soil with nitrogen, phosphorus and others nutrients. The present study examined 

the nodulation, mycorrhizal colonization, initial growth and development of seven species: 

Anadenanthera colubrina (Vell. Conc.) Brenan. (angico-branco); Mimosa bimucronata 

(DC.) Kuntze (espinho-de-maricá); Parapiptadenia rigida (Benth.) Brenan (angico- 

vermelho); Enterolobium contortisiliquum (Vell. Conc.) Morong (tamboril); Inga laurina 

(Sw.) Willd. (ingá); Platypodium elegans Vogel (jacarandá-banana) and Lonchocarpus 

muehlbergianus Hassl (embira-de-sapo), using riparian forest soil non-sterilized, mineral 

fertilization and inoculation of rhizobia and mycorrhiza, in the nursery. All species were 

colonized by mycorrhizal fungus (inoculated and native) and were nodulated. Only P. 

rigida and P. elegans did not show spontaneous nodulation. The mineral nitrogen added 

inhibited nodules number and dry weight, nitrogenase activity and leghemoglobin content 

in all species. The mineral phosphorus added diminished the mycorrhizal colonization only 

in A. colubrina and M. bimucronata. Fungi inoculation did not affect the plants growth and 

neither support the P uptake; however, rhizobial inoculation supported the nodulation of A. 

colubrina and the nodulation and growth of M. bimucronata, L. muehlbergianus and E. 

contortisiliquum in inoculated treatments only with rhizobia or associated with mycorrhizal 

fungusinoculation.



    

 

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1. INTRODUÇÃO 

 

1.1. Considerações sobre as matas ciliares e as leguminosas 

 

As matas ciliares ou florestas ripárias são importantes facilitadoras dos fluxos 

biológicos, reduzem a erosão, estabilizam as margens dos rios e exercem função protetora 

sobre os recursos naturais bióticos e abióticos. Esta vegetação apresenta ampla distribuição 

pelo território nacional e grande heterogeneidade florística (Rodrigues & Nave 2000). No 

entanto, apesar das matas ciliares terem sido incluídas como áreas de preservação 

permanente (Lei n. 4771/65 do Código Florestal Brasileiro), elas vem sendo degradadas 

e/ou destruídas por interesses que, muitas vezes, desrespeitam o ambiente e as relações 

ecológicas. 

A família Leguminosae representa uma parcela significativa na composição 

florística de vários ecossistemas, inclusive nas matas ciliares. Gibbs & Leitão Filho (1978) 

encontraram 10 representantes desta família em uma área de floresta de galeria próxima de 

Mogi Guaçu - SP, tendo sido esta a família que apresentou maior número de espécies. 

Bernacci et al. (1998) constataram uma segunda maior riqueza de espécies pertencentes à 

subfamília Fabaceae em 15 fragmentos florestais ripários da Bacia do Jacaré- Pepira – SP  



    

 

2

e, se considerarmos também Mimosaceae e Caesalpiniaceae como subfamílias da família 

Leguminosae (Polhill & Raven, 1981), esta família apresentará maior riqueza. Segundo 

Leitão Filho (1982), o estrato superior das matas ciliares apresenta uma clara dominância 

de espécies da família Leguminosae. 

Plantas dessa família podem crescer mais rapidamente e ter importante papel na 

recuperação de áreas florestais, uma vez que elas são capazes de se associar a bactérias 

conhecidas como rizóbios e também a fungos micorrízicos, relacionados ao processo de 

fixação do nitrogênio atmosférico e à maior absorção de fósforo e alguns nutrientes, 

respectivamente. Estas associações garantem um suprimento adequado de nutrientes para a 

planta, além de aumentar o potencial de enriquecimento do solo. 

 

1.2. A simbiose rizóbio-leguminosa 

 

A associação entre rizóbios e leguminosas traz benefícios para ambos os simbiontes 

e é visualizada comumente pela presença de nódulos na superfície das raízes. A nodulação, 

quer seja expontânea ou através da inoculação de estirpes do rizóbio, pode apresentar 

diferentes graus de eficiência na fixação biológica de nitrogênio (FBN). A FBN é a 

incorporação de dinitrogênio (N2) à planta por meio de sua transformação em amônia 

realizada por diversos seres vivos, entre os quais, o rizóbio. A nodulação não parece 

freqüente na subfamília Caesalpinioideae (23%), mas é muito freqüente em Mimosoideae 

(90%) e Papilionoideae (97% das espécies estudadas) (Roggy & Prévost, 1999). Apesar 

disso, 40% dos 643 gêneros de Leguminosae ainda não foram estudados quanto à 

capacidade de nodular e fixar nitrogênio (Sprent, 2001). 

A eficiência do processo de fixação de nitrogênio pode ser analisada observando-se 

os efeitos dessa associação na planta hospedeira, tais como no crescimento da parte aérea e 



    

 

3

do sistema radicular, no aumento da área foliar, teor de nitrogênio, do número de nódulos e 

na disposição destes no sistema radicular, entre outros. Além disso, o teor de leg-

hemoglobina (Lb), coloração dos nódulos e atividade da enzima nitrogenase podem 

evidenciar o potencial de fixação.  

A enzima nitrogenase é responsável pela catálise do nitrogênio atmosférico (N2) e 

sua transformação em amônia (NH3), que corresponde à fixação do nitrogênio 

propriamente dita. Essa atividade é regulada por nodulinas, como a Lb, produzidas pela 

planta em associação. A Lb tem por função transportar oxigênio para o bacteróide na zona 

central do nódulo, devido à alta sensibilidade que a enzima nitrogenase apresenta em 

relação ao oxigênio, apesar do rizóbio ser aeróbico. Na região central do nódulo a 

concentração de oxigênio é baixa, e conseqüentemente não inativa o complexo nitrogenase 

(Bruijn et al., 1994). 

A associação com rizóbio ocorre na natureza geralmente quando há deficiência de 

nitrogênio no solo, pois o processo de fixação biológica é altamente energético. A 

aplicação de nitrogênio combinado na forma de nitrato ou uréia pode inibir a infecção das 

raízes por rizóbio ou inibir a nodulação e também a atividade da enzima nitrogenase. O 

nitrato, na forma KNO3, foi inibitório para o crescimento e nodulação de Anadenanthera 

colubrina e Anadenanthera peregrina nos experimentos conduzidos por Mendonça & 

Schiavinato (1996).  

Entretanto alguns pesquisadores enfatizam que a aplicação de nitrogênio 

combinado é requerida para maximizar a produção ou ainda que, em concentrações baixas, 

doses iniciais de nitrogênio combinado podem estimular a fixação de nitrogênio (Becker et 

al. 1991), principalmente em espécies anuais. Estes autores observaram em Sesbania 

rostrata que o aumento na quantidade de N mineral inibiu a nitrogenase (ARA) e diminuiu 

o número de nódulos nas raízes, mas não afetou em nódulos caulinares e, no campo, uma 



    

 

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dose inicial de 30 Kg ha-1 estimulou a FBN, o crescimento e a acumulação de nitrogênio. 

Desta maneira, a inibição ou o estímulo do processo de FBN pela presença de nitrogênio 

no solo dependem do requerimento nutricional da planta, da concentração e formas de 

nitrogênio disponíveis no solo. 

Há uma certa especificidade entre o rizóbio e a planta hospedeira que depende de 

propriedades intrínsecas dos simbiontes, compatibilidade genética e da influência 

ambiental. Os principais fatores ambientais que ocorrem nos trópicos e influenciam a FBN 

são alta temperatura, déficit hídrico e acidez do solo, a qual influencia no balanço 

nutricional do mesmo, favorecendo o aumento na concentração ou a deficiência de 

determinados nutrientes. Assim, a toxidez por Al provocada pela acidez do solo pode 

provocar a deficiência de P e Mo e o aumento do pH aumenta a disponibilidade de Ca para 

a planta e para a  bactéria (Hungria & Vargas, 2000). 

 

1.3. Micorrizas vesículo-arbusculares (MVA) 

 

Micorriza é um termo geral para designar as associações mutualísticas entre certos 

fungos do solo e as raízes da maioria das espécies vegetais. Existem vários tipos de 

micorrizas, sendo as MVA as de maior importância ecológica (Smith & Read, 1997). 

Nessa associação morfológica e fisiológica, a planta beneficia-se pelo aumento da absorção 

de água e nutrientes, principalmente o fósforo, proporcionado pelas hifas fúngicas, que 

funcionam como uma extensão do sistema radicular (Lopes et al. 1983), enquanto fornece 

ao fungo fotoassimilados permitindo que ele complete seu ciclo, o que só ocorre na 

presença da planta hospedeira (Smith & Read, 1997). Além disso, estas associações são 

conhecidas por aumentar a resistência das plantas a estresses ambientais e a patógenos 

(Hirsh & Kapulnik, 1998). 



    

 

5

O fungo funciona como um “competidor” por produtos elaborados pela planta, pois 

ele aumenta a respiração das raízes e causa acúmulo de carbono no citoplasma das células 

corticais. Para compensar essas perdas, plantas colonizadas exibem maiores taxas 

fotossintéticas. O balanço entre o efeito benéfico do fungo na maior absorção de nutrientes 

e a utilização dos produtos da planta geralmente resulta em aumento no crescimento das 

plantas colonizadas, mas pode também resultar em redução no caso de plantas crescendo 

em altos níveis de P disponível (Lopes et al. 1983). Entretanto, a fertilização com N ou P 

pode aumentar o crescimento dos fungos micorrízicos onde há uma limitação inicial de 

nutrientes, diminuir o crescimento micorrízico onde as plantas são limitadas em termos 

nutricionais mas os fungos não o são e pode não ter efeito no crescimento dos fungos 

quando nenhum organismo é limitado em termos nutricionais (Treseder & Allen, 2002). 

Os fungos MVA são atualmente classificados na ordem Glomales, nos gêneros 

Glomus, Acaulospora, Scutellospora, Gigaspora, Paraglomus e Archaeospora (Morton & 

Redecker, 2001) e são conhecidos por ocorrerem na maioria das plantas tropicais, sendo 

uma parte integral do sistema radicular de quase todas as plantas nos trópicos, não somente 

sob condições naturais mas também em condições de cultivo (Michelsen, 1992) e 

apresentar, geralmente, baixa especificidade (Zhu et al. 2000).  

Os fungos são simbiontes obrigatórios que na ausência de uma planta hospedeira 

estão presentes no solo como esporos multinucleados envolvidos por uma parede celular  

ou como fragmentos de hifas em raízes mortas ou secas. A germinação do esporo, 

proliferação e ramificação das hifas são estimuladas por sinais moleculares sintetizados  e 

secretados pela raiz. Essas moléculas são geralmente flavonóides, várias das quais também 

servem como sinais nas simbioses rizóbio-leguminosas (Hirsch & Kapulnik, 1998). 

Embora seja possível quantificar a colonização micorrízica, é necessário levar em 

consideração as interações fisiológicas entre os fungos e as plantas hospedeiras e também 



    

 

6

medidas da viabilidade dos fungos uma vez que a biomassa nem sempre reflete a eficiência 

das associações simbióticas em termos de aumento de crescimento da planta hospedeira 

(Zhao et al. 1997) 

 

1.4. As relações rizóbio-micorriza-leguminosas 

 

Os fungos MVA também podem aumentar a performance de leguminosas 

inoculadas por rizóbio (Barea et al. 1992, Abd-Alla et al. 2000), sendo portanto benéfica a 

dupla inoculação de rizóbio e esses fungos. A dupla inoculação melhora a aquisição de 

nutrientes, ajuda as plantas a se estabelecerem no campo e confere resistência ao estresse 

hídrico (Herrera et al., 1993). A presença de fungos micorrízicos e o aumento na fixação 

de N2 também podem estar relacionados ao fato de que plantas micorrizadas podem ter 

altas concentrações de P, Zn, e Cu, os quais influenciam a nodulação e fixação de N2. 

(Redente & Reeves, 1981). 

Existem muitos estudos envolvendo a dupla inoculação em plantas de interesse 

agronômico (Ross & Harper, 1970; Mosse, 1977; Abbott & Robson, 1977; Redente & 

Reeves, 1981; Barea & Azcon-Aguilar, 1983, Ames & Bethlenfalvay (1987), Vejsadová et 

al., 1992, Abd-Alla, 2000; Goss & Varennes, 2002), sendo que a maior parte deles envolve 

a soja. Os trabalhos de dupla inoculação em espécies arbóreas florestais são geralmente 

conduzidos em viveiro, enfocando a posterior utilização das plantas em sistemas 

agroflorestais ou objetivando a recuperação de áreas degradadas.   

No campo, Bakarr et al. (1996), ao examinar as raízes de espécies arbóreas em uma 

floresta plantada e um sítio de reflorestamento na África, encontraram 7, de 11 espécies de 

Mimosoideae e 2, de 3 espécies de Papilionoideae com dupla infecção por rizóbio e 

micorriza. Marques et al. (2001) trabalhando com Centrolobium tomentosum (araribá) 



    

 

7

cultivado em mata ciliar observaram que a dupla inoculação com estirpes selecionadas de 

rizóbio e fungos micorrízicos melhoraram o crescimento dessa leguminosa nativa da 

Floresta Atlântica Brasileira.  

Gonçalves (2000) estudou o crescimento de Dalbergia nigra (jacarandá-da-bahia) 

inoculada com rizóbio e fungos micorrízicos em área ciliar e constatou que os níveis de 

nitrogênio foliar da espécie estudada consolidaram a efetiva contribuição dos inoculantes 

para o enriquecimento do solo, especialmente com nitrogênio, favorecendo o crescimento 

das espécies consorciadas. Entretanto, Santiago et al. (2002) observaram que a co-

inoculação de uma estirpe de rizóbio e fungo micorrízico não aumentaram o peso seco 

desta mesma espécie vegetal, conteúdo de nitrogênio e fósforo ou colonização micorrízica 

em solo de floresta Atlântica e solo de eucalipto.  O tempo de análise dos dados pode não 

ter sido suficiente para a observação dos efeitos dos simbiontes no crescimento do 

jacarandá-da-bahia, uma vez que essa espécie apresenta crescimento lento e os dados 

analisados por esses autores foram em plantas de até 12  meses de  idade.  

Em condições de viveiro, Rocha (1995) observou em plantas de Acacia mangium 

que a nodulação foi favorecida pela pré-colonização com fungos micorrízicos, 

independentemente das doses de P testadas, apesar das plantas não terem sido inoculadas 

com rizóbio. Outras espécies de Acacia foram estudadas por De La Cruz  et al.  (1988) 

Khan & Uniyal (1999) e Munro et al. (1999). Khan & Uniyal (1999) observaram maior 

crescimento em termos de biomassa seca nos tratamentos com dupla inoculação seguido da 

inoculação simples com micorriza em A. nilotica, e, Munro et al. (1999), verificaram que a 

nodulação foi baixa em todos os tratamentos de A. tortilis, embora tenham sido inoculados 

com rizóbio, mas a micorrização melhorou a infecção micorrízica e o crescimento das 

plântulas em solo não esterilizado. Esses últimos autores sugerem que os métodos de 

inoculação por eles aplicados podem ser adaptados a condições de viveiro a baixo custo. 



    

 

8

Burity et al. (2000) verificaram que mudas de Mimosa caesalpiniifolia com dupla 

inoculação apresentaram valores significativos no crescimento, área foliar, atividade da 

enzima nitrogenase e porcentagem de colonização radicular, independentemente do nível 

de P usado. Khan et al. (2001) investigaram o crescimento de Dalbergia sissoo e 

verificaram que a inoculação apenas com micorriza pode reduzir a dependência de D. 

sissoo da fertilização. Entretanto, todos os tratamentos com combinações entre rizóbio e 

micorriza não diferiram dos três tratamentos com diferentes doses do fertilizante testado. 

Ingleby et al (2001) concluíram que a inoculação micorrízica de Calliandra calothyrsus 

tem o potencial de aumentar o crescimento e a nodulação das plântulas em casa de 

vegetação.   

Além destes estudos, há as revisões de Hirsch & Kapulnik (1998), sobre a 

transmissão de sinais em associações micorrízicas em comparação com a simbiose 

Rhizobium – leguminosas, onde é discutido que alguns sinais moleculares como 

flavonóides produzidos pela raiz são conservados em ambas as simbioses e de Provorov et 

al. (2002), sobre desenvolvimento da genética e evolução de estruturas simbióticas em 

nódulos fixadores de nitrogênio e micorrizas arbusculares.  

Pode-se contudo perceber que ainda existem muitas espécies a serem testadas 

quanto a dupla inoculação, o que requer uma “simbiose” entre os estudos de taxonomia, 

anatomia, fisiologia, genética, evolução e ecologia desses microrganismos e das relações 

destes com o sistema radicular das plantas hospedeiras nas mais diversas condições 

ambientais. 

 

 

 

 



    

 

9

 

 

 

 

 

2. OBJETIVOS 

 

2.1 Objetivos gerais 

Estudar o potencial de fixação de nitrogênio e micorrização de 7 espécies de 

leguminosas que ocorrem em matas ciliares brasileiras e fornecer subsídios para utilização 

dessas espécies em plantios de recuperação de áreas alteradas, sendo elas: 

Mimosoideae (Mimosae): Anadenanthera colubrina (Vell.Conc.) Brenan. (angico-branco); 

Mimosa bimucronata (DC.) Kuntze (espinho-de-maricá); 

Parapiptadenia rigida (Benth.) Brenan (angico-vermelho);  

Mimosoideae (Ingeae): Enterolobium contortisiliquum (Vell. Conc.) Morong (tamboril); 

Inga laurina (Sw.) Willd. (ingá); 

Papilionoideae (Tephrosieae): Platypodium elegans Vogel (jacarandá-banana); 

Lonchocarpus muehlbergianus Hassl (embira-de-sapo). 

 

 

 

 

 

 

 



    

 

10

 

 

 

 

 

 

 

 

2.2 Objetivos específicos 

1- Verificar se a fertilização com adição de N mineral inibe a nodulação das plantas; 

2- Verificar se a fertilização com P inibe a colonização micorrízica; 

3- Verificar a ocorrência de nodulação e colonização micorrízica em tratamentos não 

inoculados com rizóbio ou micorriza; 

4- Verificar se a colonização micorrízica é favorecida pelas inoculações simples ou 

dupla de rizóbio e fungos MVA; 

5- Verificar o crescimento das plantas no tratamento com deficiência em N e 

inoculação com rizóbio ou com rizóbio e micorriza, comparado ao crescimento das 

plantas do mesmo tratamento, porém não inoculadas; 

6- Verificar o crescimento das plantas no tratamento com deficiência em P e 

inoculação com micorriza ou com rizóbio e micorriza, comparado ao crescimento 

das plantas do mesmo tratamento, porém não inoculadas; 

7- Verificar se a nodulação, atividade da nitrogenase e os teores de leg-hemoglobina 

são aumentados em tratamentos com inoculação de rizóbio. 

 

 



    

 

11

 

 

 

 

 

 

 

 

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12

 

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p. 23-54, 1998. 

 

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SILVA, M. L. R. B.. Efetividade da inoculação com rizóbio e fungos micorrízicos 

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of three legume trees inoculated  with VA mycorrhizal fungi and Rhizobium. Plant and 

Soil, v.108, n.111-15. 

 

GIBBS, P. E., H. F. LEITÃO FILHO & ABBOTT, R. J. Aplication of the point-centred 

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e decomposição de sua liteira nas margens do Rio Doce. 2000. 105f. Dissertação 

(Mestrado em Ecologia, Conservação e Manejo de Vida Silvestre) - Universidade Federal 

de Minas Gerais, Belo Horizonte. 

 

GOSS, M. J. & DE VARENNES, A. Soil disturbance reduces the efficacy of mycorrhizal 

associations for early soybean growth  and N2 fixation. Soil Biology & Biochemistry, v. 

34, p. 1167-1173, 2002. 

 



    

 

13

HERRERA, M. A.; SALAMANCA, C. P.; BAREA, J. M. Inoculation of woody legumes 

with selected arbuscular mycorrhizal fungi and rhizobia to recover desertified 

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133, 1993. 

 

HIRSCH, A.M. & KAPULNIK, Y. Signal Transduction Pathways in Mycorrhizal 

Associations : Comparisions with the Rhizobium-Legume Symbiosis. Fungal Genetics 

and Biology, v. 23, p. 205-212, 1998. 

 

HUNGRIA, M. & VARGAS, M.A.T. Environmental factors affecting N2 fixation in grain 

legumes in the tropics, with an emphasis on Brazil. Field Crops Research, v. 65, p.151-

164, 2000. 

 

INGLEBY, K.; FAHMER, A.; WILSON, J. NEWTON, A. C.; MASON, P. A. & SMITH, 

R. I. Interactions between mycorrhizal colonisation, nodulation and growth of Calliandra 

calothyrsus seedlings supplied with different concentrations of Phosphorus solution. 

Symbiosis, v.30,p.15-28, 2001. 

 

KHAN, S. N. & UNIYAL, K. Growth response of two forest tree species to VAM and 

Rhizobium inoculations. Indian Forester, v.125, n.11, 1125-1128, 1999. 

 

KHAN, S.N., UNIYAL, K. & PANDEY, R. Growth response of Dalbergia sissoo to AM 

and Rhizobium inoculations and fertilization in nursery. Indian Forester, v.127, n.8, p. 

906-909, 2001. 

 

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Brasileira de Ciência do Solo, v.7, n.1,p.1-19, 1983. 

 



    

 

14

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woody legume (Centrolobium tomentosum) with rhizobia and mycorrhizal fungi in south-

eastern Brazil. Agroforestry Systems, v. 52, p. 107-117, 2001. 

 

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MICHELSEN, A. Mycorrhiza and root nodulation in tree seedlings from five nurseries in 

Ethiopia and Somalia. Forest Ecology and Management, v.48, p. 335-344, 1992. 

 

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MUNRO, R.C.; WILSON, J.; JEFWA, J. & MBUTHIA, R.W. A low-cost method of 

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15

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16

 

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17

 

 

 

 

CAPÍTULO 1  

 

 

 

 

 

Nitrogen-fixing and vesicular-arbuscular mycorrhizal symbioses in 

tropical tree of tribe Mimoseae (Leguminosae-Mimosoideae) 

 

 

 

(Trabalho a ser enviado para a revista Plant and Soil) 

 

 

 

 

 

 



    

 

18

 

 

 

 

 

 

 

Symbioses in tropical tree of Mimoseae 

 

Nitrogen-fixing and vesicular-arbuscular mycorrhizal symbioses in tropical tree of 

tribe Mimoseae (Leguminosae-Mimosoideae) 

 

Camila Maistro Patreze1,2 & Lázara Cordeiro1 

 

1Instituto de Biociências, Departamento de Botânica, P.O.Box 199,  Universidade 

Estadual Paulista,  SE-13506 900 Rio Claro, SP, Brazil. 2Corresponding author* 

 

 

 

 

 

                                                 
* Fax No.: (19) 3534 0009 
E-mail: cpatreze@rc.unesp.br 



    

 

19

 

 

Abstract 

Response to mineral fertilization and dual inoculation with rhizobia and arbuscular 

mycorrhiza fungi (AMF) were studied in nursery conditions in Anadenanthera colubrina, 

Mimosa bimucronata and Parapiptadenia rigida legume native trees from Brazilian 

riparian forests. Each species was submitted to seven treatments, being fertilization with N 

and P added, with P without N and with N without P and inoculated or not with rhizobia 

(r), mycorrhiza (m) or both (rm), respectively: NP, P, P+r, P+rm, N, N+m and N+rm. 

Results showed that P uptake by symbionts was not sufficient to sustain good growth 

plants of three species. Native fungi infected these three hosts and AMF inoculations not 

enhanced the mycorrhizal colonization. Also, the P treatments affected negatively the 

AMF colonization in A. colubrina and M. bimucronata, but not in P. rigida. The absence 

of mineral N in A. colubrina and P. rigida plants not fertilized with this element limited the 

growth them. This probably mineral nitrogen deficiency in M. bimucronata was supplied 

by biological nitrogen fixation. Spontaneous nodulation occurred in A. colubrina and M. 

bimucronata. In N treatments, this element inhibited the nodulation. A. colubrina and M. 

bimucronata plants of P+r treatment and P+r and P+rm treatments, respectively, had 

increased in nodules number, nitrogenase activity and leghemoglobin content, being that 

the growth and development of M. bimucronata seedlings were positively influenced by 

nodulation. Thus, extents of rhizobial and mycorrhizal symbiosis in this specie upon 

nursery conditions can amend post-planting success. 

 

Key words  



    

 

20

Biological nitrogen fixation, Mimoseae, mycorrhiza, rhizobia, riparian forest, symbiosis. 

 

Introduction 

 

The Mimoseae tribe (Leguminosae- Mimosoideae) comprises several genera as 

Anadenanthera, Mimosa and Parapiptadenia which are common in lowland tropical 

rainforests, especially near rivers and lakes (Polhill et al., 1981). Anadenanthera colubrina 

(Vell.) Brenam, Mimosa bimucronata (DC) O. Kuntze and Parapiptadenia rigida (Benth) 

Brenan are legume trees which have been sampled in various floristic surveys of riparian 

forests in Brazil. A. colubrina was sampled by Nilsson (1989), Silva et al. (1992), Vilela et 

al. (1995), Santarelli (1996); Bernacci et al. (1998), Dias et al. (1998) and Sampaio et al. 

(2000). M. bimucronata was founded in floristic studies of Santarelli (1996) and Metzger el 

al. (1998) and P. rigida was sampled by Nilsson (1989) and Dias et al. (1998). 

The most of Mimoseae plants, including the species cited above, are capable of 

fixing dinitrogen in association with root nodule bacteria (rhizobia) and increases 

phosphorus contents and others nutrients by association with arbuscular mycorrhizal fungi 

(AMF). Nodulation is very frequent in the subfamily Mimosoideae (90% of studied 

species) (Sprent, 1995; according to the taxonomy of Polhill et al., 1981) and the AMF are 

of widespread occurrence (Gerdemann, 1968; Trufem, 1990) beyond to represent the 

natural status of most plant species (Siqueira et al., 1998). Also, AMF symbiosis may 

improve defense herbivores, enhance the acquisition of water (Herrera et al., 1993) and 

increase a plant resistance to pathogens (Moora and Zobel, 1998). 

Many previous studies, mainly on cultivated plants, have shown that dual 

inoculation of legumes with rhizobia and AMF increases plant growth (Ross and Harper, 

1970; Mosse et al., 1976; Redente and Reeves, 1981; Abd-Alla, 2000). In West Africa 



    

 

21

were found dual infections involving AMF and bacterial nodules in seven forest and 

plantation of tropical legume trees of Mimosoideae (Bakarr et al., 1996). In Brazil, Franco 

and Faria (1997) have leaded studies of nodulated and mycorrhizal legume trees to 

revegetate poor or depleted soils with the goal to restarte their fertility. Little information is 

available about symbiotic relationships of dual inoculation in Brazilian legume native tree 

whereas we require ecophysiological knowing of these species in nursery conditions. 

Burity et al. (2000) recorded that the plants growth and nodulation of M. caesalphiniifolia 

were improved by mycorrhiza. 

Information on nodulation or only reporting the capacity of nodulation in A. 

colubrina (Mendonça and Schiavinato, 1996), M. bimucronata (Campello, 1996) and P. 

rigida (Lechtova-Trnka, 1931; Rothschild, 1970; Corby, 1988) contributes to studies of 

both rhizobia and AMF inoculations, which were not reported for these species yet. In 

addition, they contribute with important information about the use of these species as 

revegetation on drastically disturbed lands. 

The aim of our study was to investigate nitrogen-fixing and arbuscular mycorrhizal 

symbioses of three Mimoseae species that occur in Brazilian riparian forest. We submitted 

A. colubrina, M. bimucronata and P. rigida to treatments of mineral fertilization and 

inoculation with rhizobia and AMF in nursery conditions in order to aid the choice of 

species to recover riparian forests. 

 

Materials and methods  

 

Rhizobia inoculum 

Rhizobia isolates were obtained from root nodules collected from Anadenanthera 

peregrina (L.) Speg and Mimosa bimucronata plants growing in the field of Unesp 



    

 

22

(Universidade Estadual Paulista “Júlio de Mesquita Filho”)- Rio Claro, SP, Brazil (22º44’S 

and 47º33’W, 610 m a.s.l.) and riparian forest of Corumbataí, SP, Brazil (22º 20’S and 

47º40’W, 604 m a.s.l.), respectively. The isolates were grown on yeast extract mannitol 

(YEM) agar at 28ºC and stored in the Rhizobial Bank of Unesp Rio Claro, SP Brazil with 

numbers IBRC 199 and 200 for A. peregrina and IBRC 201 and 204 for M. bimucronata 

isolates. Aerial parts of these plants were pressed and catalogued in the Bioscience Institute 

Herbarium –UNESP as a number HRCB-34330 and 34501, respectively.  

 

Source and germination of seeds 

Seeds of A. colubrina were supplied by IPEF (Instituto de Pesquisas e Estudos 

Florestais) da Esalq/USP (Escola Superior de Agricultura Luiz de Queiroz/Universidade de 

São Paulo) and seeds of P. rigida, supplied by IF (Instituto Florestal de São Paulo). Seeds 

of M. bimucronata and the soil used as substrate were collected in the riparian forest of 

Corumbataí, SP, Brazil (22º 20’S and 47º40’W, 604 m a.s.l.). All seeds had their surface 

sterilized and were germinated in 4L plastic pots containing a non-sterilized mixture (2:1) 

of soil (Table 1) and vermiculite. M. bimucronata required dormancy break treatment with 

imbibitions in boiling water for 30 seconds. 

 

Table 1 Chemical analysis of substrate used in the experiment before the applications of mineral nutrients 

and inoculations. 

N P Resina M.O. pH K Ca Mg H+Al Al SB CTC V B Cu Fe Mn Zn S 
ppm mg.dm-3 g.dm3 CaCl2 mmolc.dm-3 % mg.dm-3 
1000 7 6 5.5 1 12 4 10 7 17 27 63 0.01 0.7 23 8.7 0.4 9 

 

Plant growth 

All pots received the following basal nutrients prior to sowing (in mg. Kg-1 

substrate): K (60), CaCO3 (80), MgCO3 (40), S (30), B (1), Zn (2), Cu (2), Fe (4), Mn (20), 



    

 

23

Mo (4) and three fertilizations treatments: a full mineral fertilization contained N (40) and 

P2O5 (80) (NP); fertilization with P2O5 (80) without N (P) and fertilization with N (40) 

without P (N). These treatments varied in relation to inoculation of microsymbionts: 

inoculated or not with rhizobia (r), mycorrhiza (m) or both (rm). In this way, the 

experiment comprised seven treatments with ten replicates: NP, P, P+r, P+rm, N, N+m and 

N+rm. For plants from P treatments, we added a start dose of 3.8 mM of N. All plants were 

grown in a greenhouse under natural daylight in randomized blocks. Additional nutrients 

(10 mL of solution) were added to the surface of each pot every 30 days, according to 

fertilization treatments above cited. 

For rhizobial treatments, seeds were left to soak in a turbid suspension (100 mL) of 

mixture of isolates IBRC 199 and 200 to inoculate A. colubrina and P. rigida and IBRC 

201 and 204 to M. bimucronata during 1 hour. Reinoculation (10 mL of the same mixture) 

was made near the roots of seedlings 30 days later. For the AMF inoculation, pieces of A. 

peregrina var. falcata roots with ±1cm of length collected from Corumbataí cerrado 

reserve (22º15’S and 47º00’W, 810 m a.s.l.) were mixed on each pot (0.4g) surface nearly 

seedlings at twenty-eight days of sowing. 

 

Measurements 

Height growth of plants was recorded each fifteen days, starting at 30 days of 

sowing, being measured ten plants per treatment until 120 days and five plants per 

treatment from 120 days until the end of the experiment, at 255 days of sowing. 

Five plants per treatment were harvested at 120 and 255 days of sowing. Leaf area 

was measured by CI-202 Area Meter (CID, Inc). Roots, stems and leaves were separated, 

dried to constant weight at 70ºC, weighed and analyzed the nutrient content of the shoot 

and substrate. Root nodules were weighted, counted and sieved with sieves of mesh to 



    

 

24

separate: larger than 4mm, between 2-4mm and smaller than 2mm of diameter size. 

Nitrogenase activity of root nodules of two plants per treatment was assayed by acetylene 

reduction activity (ARA) according to Hardy et al. (1968) and nodules were stored in the 

refrigerator at 10ºC to leghemoglobin content analysis, following Becana et al. (1986). The 

nodules morphology was classified according to Corby (1981). Standard methods were 

followed for staining of roots (Philips and Hayman 1970), and the quantification of AMF 

infection in roots was estimated using the gridline intersect method (Giovannetti and 

Mosse, 1980) under a stereomicroscope (40x). 

 

Statistical analysis 

Data were analyzed separately using ANOVA. Dunn’s test was applied to compare 

means at P ≤ 0.05 using the program BioEstat 2.0 (Ayres et al., 2000). Principal 

component analysis (PCA) was performed for all data by PCOrd program, version 4.0 for 

Windows. Variables were log transformed or arc square root transformed in order to 

equalize variance. Coefficients correlation r < 0,05 in the first axis were eliminated of 

analysis. 

 

Results 

 

Growth of plants and AMF colonization 

Although seedlings were grown under similar nursery conditions, their growth 

indicated specific responses. M. bimucronata presented the best growth, as measured by 

height, dry biomass and leaf area (Table 2), mainly at 255 days of sowing. The growth 

parameters were positively affected by full mineral fertilization (NP) in the three species 

(Table 2). Plants not fertilized with P and N had their growth limited, except M. 



    

 

25

bimucronata in the P treatments, which mineral nitrogen deficiency was probably supplied 

by biological fixation with rhizobia. 



    

 

27

25 

 

Table 2: Response of A. colubrina, M. bimucronata and P. rigida to mineral fertilization and inoculation treatments at 120 and 255 days 

of sowing. Mean of five plants per treatment. N and P content were measured in the shoot (leaf plus stem). 

a Mean of ten plants per treatment. 
 b Mean of two plants per treatment. 

Means followed by the same letter are not significantly different by ANOVA, test Dunn at P<0.05 level of significance. Lower case letters compare treatments within 
of the one species and upper case letters compare species for the same treatments. No such analyses were indicated for AMF, N and P of shoots. 

 c not determined 

Treatments
Area leaf Height Biomass AMF N P Area leaf Height Biomass AMF N P 

(cm2) (cm)
a

(g) (%)
b

(mg.g-1) (mg.g-1) (cm2) (cm) (g) (%)
b

(mg.g-1) (mg.g-1)

NP 208.4 aA 54.9 aA 7.4 aA 1.7 23.0 2 259.2 aA 94 aB 35.3 aA 0.0 19 2.9
P 41.9 bcA 41.9 abA 4.1 abA 1.3 14.0 2.8 25.3 bcB 51.6 cB 5.7 bB 1.5 17.5 5.6
P+r 33.4 bcA 26.7 bcA 1.5 bcA 2.8 20.0 3.8 59.5 bcB 54.8 bcB 7.1 bB 4.5 15.5 4.2
P+rm 25.1 cAB 15.2 dB 0.5 cB 3.6 20.0 5.4 18.8 cB 55.8 bcB 5.4 bB 2.0 14.5 3.8
N 38.5 bcA 20.2 cdA 0.8 cB 8.1 25.0 1.2 192.1 aA 71.4 abB 11.2 abA 13.5 18 0.8
N+m 28.7 bcB 24.3 cB 0.8 cB 1.4 26.5 0.9 128.4 abA 71 abcB 5.6 bB 14.0 22 0.7
N+rm 66.3 abA 27.8 bA 1.3 bcA 12.4 29.0 1 195.2 aA 76.2 abB 7.2 bB 10.8 18.5 0.7

NP 195.3 aA 64.6 aA 11.8 aA 32.2 39.5 1.9 530.9 aA 138.2 abA 46.7 aA 46.6 15.5 1.9
P 139.7 aA 53.5 abA 6.8 abA 22.9 11.5 2.1 295.4 abA 161.8 aA 40.8 aA 23.2 10.5 1.6
P+r 78.6 aA 33.6 cdA 2.5 bcA 46.3 21.0 2.8 452.1 aA 143.2 abA 55.6 aA 12.8 17 1.6
P+rm 70.7 aA 28.8 dA 2.4 bcA 43.8 22.0 2.7 225.2 abA 127.4 abcA 38.2 abA 17.3 16.5 1.5
N 74 aA 29.1 dA 2.9 bcA 50.5 14.5 1.0 158.7 abcA 100.8 cA 15.1 cA 70.3 17 0.5
N+m 102.9 aA 39.5 bcA 2.8 bcA 1.9 22.0 0.7 131.3 bA 127.4 abcA 17.4 bcA 68.4 15 0.4
N+rm 67.3 aA 31.5 cdA 1.6 cA 9.6 27.5 1.1 61 cB 114.8 bcA 15.0 cA 70.3 16.5 0.4

NP 94.8 aA 12.6 abB 1.6 aB 21.6 22.5 1.8 197.1 aA 40.6 aC 18.02 aB 25.4 23 2.6
P 32.9 bcA 14.3 abB 0.5 aB 12.1 22.5 3.6 53.5 aB 28.4 abB 3.7 cdB 47.0 19 6
P+r 18.6 bcA 8.8 cdB 0.5 aA 13.1 14.5 3.7 53.2 aB 24.2 bcC 2.1 dC 45.6 19.5 4.4
P+rm 16.2 cB 7.2 dC 0.3 aB 35.0 c 4.6 29.8 aB 24 bcC 2.5 dB 58.6 22 6.5
N 55.7 abA 11.7 bcB 0.6 aB 10.7 28.0 1.8 142.6 aA 37.2 abC 8.5 bA 20.9 23.5 0.9
N+m 120.8 aA 16 aC 1.8 aAB 21.2 25.5 1.1 71.8 aA 23.2 bcC 4.3 cdB 22.4 27 0.8
N+rm 31.2 bcA 11.2 bcB 0.6 aA 5.0 26.5 0.7 72.9 bA 34.8 abC 6.1 bcB 32.0 23.5 0.8

Parapiptadenia rigida

At 120 days At 255 days

Anadenanthera colubrina

Mimosa bimucronata



    

 

27

M. bimucronata exhibited the highest values for root colonization by AMF (Table 

2), which ranged 70.3% of infected roots. A. colubrina and P. rigida had percentages 

ranging 14% and 58.6%, respectively. Typical hyphae and vesicles were observed in the 

preparations of all species. Native-born fungi were capable to infect these three host plants. 

When we compare the results in the same fertilization treatments, AMF inoculated 

treatments promoted similar values of infection of roots than those uninoculated. These 

results indicate that the AMF inoculation not enhanced the mycorrhizal colonization. 

Treatments with P added in A. colubrina and M. bimucronata, regardless of fungi 

inoculation, presented smaller AMF percents than N treatments, without P added. In P. 

rigida occurred the inverse situation. 

 

Biomass allocation  

A variation in resource allocation patterns was observed among species, as well as 

within species through time, but the treatments did not influence the biomass allocation in 

the three species studied. A. colubrina and M. bimuconata plants had more biomass of 

stem and leaves (Figure 1) while P. rigida produced more roots at 120 days of sowing. At 

255 days, A. colubrina and P. rigida increased biomass allocation to roots and M. 

bimucronata remained allocating stem and leaves biomass. 

 

Nodulation  

Morphological characteristics of the A. colubrina, M. bimucronata and P. rigida 

nodules result in its being classified as indeterminate and astragaloid, similarly to 

observations of Corby (1981) and Sprent (2001). 

Spontaneous nodulation from native soil born rhizobia occurred in A. colubrina and 

M. bimucronata but not in P. rigida (Table 3). In A. colubrina and P. rigida plants from all



    

 

28

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: Biomass production average (%) of roots, stems and leaves in A. colubrina (A), 

M. bimucronata (B) and P. rigida (C) plants cultiveted upon treatments of fertilization 

(NP, P and N) and inoculation of rhizobia (r), mycorrhiza (m) and the both (rm) at 120 and 

at 255 days of sowing. 

0%

50%

100%

leaves

stem

root

0%

50%

100%

0%

50%

100%

0%

50%

100%

0%

50%

100%

0%

50%

100%

NP P P+r P+rm N N+mN+rm
0%

50%

100%

NP P P+r P+rm N N+mN+rm

A

B

C

B
io

m
as

s 
(%

)

Treatments

At 120 days At 255 days 



    

 

29

Table 3: Nodule number per diameter size larger than 4, between 2 and 4 and smaller than 2 milimeters at 

120 and 255 days of sowing and nodule dry weight at 255 days of A. colubrina, M. bimucronata and P. 

rigida. Mean of five replicates, followed by the same letter in the colunn are not significantly different by 

ANOVA, test Dunn at p<0.05 level of significance. 

 

treatments with mineral N added, regardless of rhizobia inoculation, did not produced 

nodules. For M. bimucronata there were nodules in these treatments, but the nodules 

occurred in lower number (Table 3). Dual inoculation (rm) and single inoculation of 

rhizobia (r), associated with low N and P available in the P treatments improved the 

nodules number in M. bimucronata, P+r and P+rm treatments were statistically differ to 

the others, at 255 days of sowing (Table 3). The nitrogenase activity (ARA) and 

leghemoglobin contents in M. bimucronata nodules also were higher in these inoculated 

treatments (Figure 2). Nevertheless, dual inoculation (rm) did not increase nodules number 

(Table 3) in A. colubrina and P. rigida. Furthermore, A. colubrina roots presented 

significantly higher nodules number in the P+r than P+rm treatment. For this species, ARA 

presented similar response and leghemoglobin contents was similar between P treatments.  

 

Treatment >4 2 a 4 <2 Total >4 2 a 4 <2 Total >4 2 a 4 <2 Total

NP 0.8 3.6 1 5.4 b 3 7.2 9.6 19.8 b 0.08 0.04 0.01 0.13 a
P 2.2 3.4 0.6 6.2 b 5.6 9.2 25.2 40 b 0.16 0.03 0.01 0.20 a
P+r 2.6 42.8 63.6 109 a 12.8 36 75.8 124.8 a 0.19 0.18 0.05 0.42 a
P+rm 0.8 4 7.4 12.2 b 8.8 12 9 29.6 b 0.23 0.09 0 0.32 a

NP 33 118 99.4 250.6 a 99.2 311 279 688.4 b 0.92 0.90 0.25 2.07 b
P 73.4 117 64.4 254.8 a 149 246 155 550.2 b 2.95 1.07 0.18 4.20 a
P+r 17.2 66.8 47 131 ab 146 473 836 1455.4 a 2.35 1.94 0.76 5.05 a
P+rm 18 41.4 12.6 72 abc 41 367 1844 2252.2 a 1.06 1.35 1.61 4.02 a
N 1 7.2 18.4 26.6 bcd 2 30 161 193 bc 0 0.05 0.11 0.15 c
N+m 0 0.8 2 2.8 d 0.2 7.8 133 141.4 c 0 0.02 0.03 0.04 c
N+rm 0 0 7.8 7.8 cd 0 0.8 37 37.8 c 0 0 0.01 0.01 c

P+r 0.8 2.2 5.6 8.6 a 0 0.6 5.2 5.8 a 0 0 0 0a
P+rm 0 0.6 1.4 2 b 1 2.2 10 13.2 a 0.01 0.00 0.01 0.02 a

P. rigida

At 120 days At 255 days
Mean number of  nodule Dry weight of nodule (g)

A. colubrina

M. bimucronata



    

 

30

 

 

 

 

 

 

 

 

 

 

 

 
 

 

 

 

Figure 2: Acetylene reduction activity (ARA) and leghemoglobin content averages of A 

colubrina (A) and M. bimucronata (B) nodules upon fertilization treatments (NP, P and N) 

with inoculation of rhizobia (r), mycorrhiza (m) and the both (rm) at 120 and 255 days of 

sowing. Bar graph corresponds to ARA (in µmol C2H4 h-1 g-1 nodule fresh), line graph 

corresponds to leghemoglobin content (in mg Hb g-1 nodule fresh). 

 

Treatments 

0

0.02

0.04

0.06

0.08

0.1

A
R

A

0

0.45

0.9

Leghem
oglobin

0

700

1400

2100

A
R

A

0.3

0.45

0.6

Leghem
oglobin

A 
At 120 days At 255 days 

0

4

8

12

N
P P

P
+

r

P+
rm N

N
+

m

N
+r

m

A
R

A

0

0.45

0.9

Leghem
oglobin

0

30000

60000

90000

N
P P

P
+

r

P+
rm N

N
+

m

N
+r

m

A
R

A

0

0.6

1.2

L
e

g
h

e
m

o
g

lo
b

in

B 



    

 

31

The nodule dry weight of A. colubrina plants did not differ between the treatments. 

This probably occurred in spite of dry weight of nodules higher than 4mm (Table 3). For 

M. bimucronata nodules dry weight, had not difference between those formed with native 

rhizobia (P treatment) and inoculated treatments (P+r and P+rm). The ARA was relatively 

low in P. rigida, being measured at 255 days of sowing 128,58 and 292,23 (µmol of 

ethylene per g of fresh nodule per hour) at P+r and P+rm treatments, respectively. 

Leghemoglobin contents were not assessed of this specie because there were insufficient 

samples of nodule.  

Results from P contents in shoot (Table 2) and chemical analysis of the substrate at 

the last harvest (Table 4) showed low values of P in N treatments, which not had P added. 

There was apparent difference between the treatments on the N contents in leaves and in 

the substrate of the pots. 

 

Table 4: Chemical analysis of substrate of pots in relation to nitrogen (%), phosphorous (mg.dm-3) and K 

(mmolc.dm-3) of A. colubrina, M. bimucronata and P. rigida  at 255 days of sowing, cultived upon treatments 

of fertilization (NP, P and N) and inoculation of rhizobia (r), mycorrhiza (m) and the both (rm). 

 

Nutrient contents and PCA analysis 

We performed the PCA analysis of treatments and all data studied in order to see 

roughly, if the treatments would form groups and which factor(s) might explain the 

grouping. The PCA analysis simplifies the interpretation of complex results. Results of 

A colubrina M. bimucronata P. rigida
Treatments N P K N P K N P K
NP 0.05 56 1.9 0.07 51 2.5 0.05 34 1.5
P 0.07 108 1.4 0.05 85 2.2 0.05 64 1.6
P+r 0.05 84 0.9 0.07 54 1.3 0.05 73 1.4
P+rm 0.07 88 1.3 0.07 60 1 0.05 97 2.5
N 0.05 4 1.2 0.07 3 2.1 0.07 4 2.4
N+m 0.07 2 1.6 0.07 1 2.4 0.05 1 2.8
N+rm 0.07 3 2.1 0.07 1 2.2 0.07 1 2.9



    

 

32

nutrient contents of shoots and substrate were included in this analysis and each species 

was treated separately. The PCA plots showed that PC1 + PC2 axis explained 77,59; 88,47 

and 77,79 percent of total for A. colubrina, M. bimucronata and P. rigida, respectively 

(Figure 3). 

Mineral fertilization and nodulation influenced grouping in the three species. P 

treatments were grouping in the left and N treatments formed a group of the right of the 

axis. PC 1 can be seen as representing the nitrogen fixation capacity of A. colubrina and M. 

bimucronata. In P. rigida the axis PC 1 can be attributed to mineral fertilization, since that 

the nodulation was negligent. Because the vectors representing data nodulation pointed to 

the left of orig., the treatments on the left responded more positively to rhizobial 

inoculation. Vectors representing AMF colonization and K level pointed to the right of 

orig. for A. colubrina and M. bimucronata plants. This fact indicates that these features 

were increased in plants growth in N treatment (without P). In contrast, AMF percents 

pointed to P treatment (left) in P. rigida. 

Mn and B were positively correlated with mycorrhizal treatments in A. colubrina; 

Zn in M. bimucronata and S, Cu and Fe in P. rigida (Figure 3). 

 

Discussion 

 
The results showed that M. bimucronata plants grew better than A. colubrina and P. 

rigida and also exhibited the highest values for root colonization by AMF. Also, M. 

bimucronata was very nodulated, with rhizobia inoculation increasing the rhizobial 

efficiency. Native rhizobia were capable to promote high levels of infection in this plant  

 



    

 

33

 

Figure 3: Principal component analysis (PCA) to mineral fertilization treatments (NP, P and N) with  

inoculation of rhizobia (r), mycorrhiza (m) or the both (rm). Leaf area (la), height, biomass, nodule number 

(nn), dry weight nodules (dwn), acetylene reduction activity (ARA), leghemoglobin content (LegHb) and 

mycorrhization percentage (AMF) were performed for (A) A. colubrina, (B) M. bimucronata and (C) P. 

rigida. s: substrate; l: leaf,  macroelements: N, P, K, Ca, S and microelemnts: B, Mn, Zn, Cu and Fe. 

 

NP

P

P+r P+rm

N

N+m

N+rm

N l

P l
K l

MglBl

Fel

Mnl
Ps

pHs

Ks

Cas

Mgs

Bs

Mns

la

nn

dwn

ARA

Axis 1

A
xi

s 
2

N P

P

P+r

P+rm
N

N+m
N+rm

P l

K l

Znl A M F

height

biomass la

nn

dwn

ARA

LegHb

Axis 1

A
xi

s 
2

N P

P

P + r

P+rm

N

N + m

N+rm

P l

C a l

S l
Cul

Fe l

Mnl

Ps C a s

Cus

Mns
Ss

A M F

al

nn A R A

Axis 1

A
xi

s 
2

A 

B 

C 



    

 

34

probably because the soil used in the experiment was collected nearly to M. bimucronata 

nodulated plants population on the riparian forest of Corumbatai. This species is typically 

pioneer and produce small seeds, about 105.000 seeds Kg-1 (Lorenzi, 1998). Some authors 

reports that small-seeded species are seedlings that had high relative growth rate (Gross 

and Smith, 1991, Siqueira et al., 1998). These former authors recorded that under nutrient 

stress conditions, mycorrhizal effects on initial seedling growth are greater in small seeded 

species. The small seeds and therefore the low cotyledon reserves can have influenced the 

initial biomass allocation of seedlings to leaves and stems than roots in this species. A. 

colubrina and P. rigida present larger seeds in comparison to M. bimucronata, about 

15.600 and 38.600 seeds.kg-1 (Lorenzi, 1992), respectively.  

In this experiment we did not observe effect of inoculation of AMF in the root 

colonization and P uptake. Probably the fertilization treatments influenced both inoculated 

and native fungi. Both A. colubrina and M. bimucronata plants presented low AMF 

colonization in P treatments. This result is according to currently accepted literature that 

nutritionally adequade or high P supply tends to reduce colonization (Smith and Read 

1997, Siqueira et al., 1998), with the magnitude of the effect varying between plant species 

and also sensitive to change in environmental parameters (Smith and Read 1997). In 

opposition, Paron et al. 1997 observed benefic growth responses in Trema micrantha (L.) 

Blume inoculated by AMF and with 100 mg.kg-1 of P added. Similar response seems to 

have occurred in P. rigida in our experiment, whereas that higher percentage of 

mycorrhizal colonization in P treatments was noted. The substrate P level, in P treatments, 

for this species resulted in about 64 to 97 mg.dm-3 (see Table 4). Burity et al. (2000) noted 

that addition of P level enhanced AMF root colonization in M. caesalpiniifolia in both 20 

and 40 Kg.ha-1 of P2O5. Also, P addition (50 to 200 mg.dm-3) did not affect the 

colonization of Dalbergia nigra (Chaves et al., 1995). P level of 20 mg.kg-1 in the substrate 



    

 

35

was not inhibitory in 26 tree species studied by Carneiro et al. (1996). These variable 

responses to P level in different species leads to requirements of more experiments on 

legume trees to recognize the optimal P level to fungi colonization for each species of 

fungi and host plant. 

In A. colubrina, the AMF colonization was relatively low (0-14%). Carneiro et al., 

(1998) founded 20-49% for A. falcata and 1-19% for A. peregrina in nursery conditions. A 

part of nutritional factors, this low infection can be associated to specificity between host 

plant and fungi. In the other hand, our values of mycorrizal colonization of M. 

bimucronata (12.8 to70.3 %) and P. rigida (20.9 to 58.6 %) were similar to reported by 

Siqueira (1998), who founded AMF colonization in 8 pioneer species being generally high 

(mean of 60%), and by Frioni et al. (1999) in roots segments of Mimosa spp. (60%) and P. 

rigida (50%) in the field. However, the uptake of P by AMF in N treatments (without P 

added) was not sufficient to sustain a good growth plants.  

Information on the association of host plant with specific AMF is ambiguous: while 

some species of AMF have a wide distribution among host plants, others have been 

founded in rhizospheres of a single host plant (Carrenho et al., 2002). In P. rigida, for 

instance, Trufem (1990) observed the occurrence of only two Glomus species. 

Nutrient contents by PCA analysis showed positive correlation between K, Mn, B, 

S, Zn, Cu and Fe and AMF percentage. Carneiro et al. (1996) observed higher contents of 

S and Mn in Cassia rosa plants mycorrhized. According to Redente and Reeves (1981), 

plants colonized by mycorrhiza can have higher concentrations of Zn and Cu. 

A. colubrina and P. rigida were inoculated with rhizobia isolated from A. 

peregrina. This can have limited the infection or the satisfactory development of this 

symbiosis, mainly in P. rigida, which nodulation was negligent. Poor nodulation in the 

nursery condition due host specificity was reported in P. rigida by Frioni et al. (1998). 



    

 

36

The high capacity of nodulation in A. colubrina and M. bimucronata plants of P 

treatments can be related to absence of N, once that its presence has a negative influence 

on the nodulation and nitrogen fixation, or P nutrition enough, an essential element to 

enhance biological nitrogen fixation. The increasing of mineral nitrogen contents 

diminished the nodule number and inhibited the nitrogenase, as measured by ARA, in 

Sesbania rostrata, but the start of 30 Kg (N).ha-1 affected positively the biological nitrogen 

fixation (Becker et al. 1991). Mendonça and Schiavinato (1996) founded higher dry mass 

of Anadenanthera colubrina e A. peregrina in soil with 20 mg of (NH4)2SO4 than 

treatments without N. To found the adequate N dose to encourage the nodulation without 

stimulating its inhibition are others findings that we need to research in ours legume tree. 

Uliassi and Ruess (2002) observed that P fertilization increased total nodule dry 

biomass in Alnus teniufolia. Also, P and K contents increased the biological nitrogen 

fixation and nitrogenase activity (ARA) in Sesbania rostrata (Becker et al. 1991). Frioni et 

al. (1998) reported that environmental constrains, such as P deficiency could explain the 

failure in nodulation of P. rigida. Nitrogenase activity (ARA) for this species was 24.200 

µmol of ethylene h-1 g-1 fresh nodule h-1 by Frioni et al. (1998), who considered low 

activity compared with those of crop legumes, as already recorded by many authors. In 

opposition, M. bimucronata presented here about 8.0.104 µmol of ethylene h-1 g-1 fresh 

nodule h-1 at 255 days of sowing. Although low values of ARA that we founded to P. 

rigida and A. colubrina can be due to many factors, such as P deficiency, ineffective 

rhizobia, we believe that high rates of N2 fixation are less essential for perennial than 

annual species, according to Sprent (1994). We also need consider the size and 

morphology of nodules when are discussing the activity and potential efficiency of them. 

In spite of have been founded some nodules higher than 4 mm of size, with elevate values 

of dry weight in uninoculated treatments of A. colubrina and M. bimucronata, these 



    

 

37

treatments not presented high ARA or leghemoglobin content in the occasion of 

measurement. 

When trees are being grown in nurseries, the methods of cultivation and extent of 

rhizobial and mycorrhizal symbiosis can affect post-planting success, particularly when the 

trees are destined for disturbed lands or regeneration of riparian forest. In this way, we 

suggest that M. bimucronata may be more adapted to N-limited environments, mainly 

when dual inoculation (rhizobia and mycorrhiza) are performed in nursery. Although 

physiological aspects of seedling growth requirements still remain as a great gap in our 

knowledge and inoculation of rhizobia and mycorrhiza technique still not have been a 

practice commom, we can indicate the dual inoculation in this species in nursery and its 

use to reforestation and to recover riparian forests. 

 

Acknowledgements 

This study was funded by CNPQ (Conselho Nacional de Desenvolvimento 

Científico e Tecnológico) and partly funded by Fundunesp (Fundação para o 

Desenvolvimento da UNESP). We thank to Siu Mui Tsai from CENA-USP for assistance 

in nitrogenase analysis. We are also grateful to Eduardo Gross who provided critical 

comments of this manuscript. 

 

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nativas do Brasil. Vol.2. Editora Plantarum, Nova Odessa. 352p. 

 

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607-611. 

 

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Pepira (SP). Revista Brasil. Bot. 21(3), 321-330. 

 

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arbuscular mtcorrhiza. IX. Interactions between VA mycorrhiza, rock phosphate and 

symbiotic nitrogen fixation. New Phytol. 76, 331-342.  

 

Nilsson T T 1989 Levantamento do potencial econômico da mata ciliar e sugestões quanto 

ao seu aproveitamento racional. In Simpósio Sobre Mata Ciliar. Ed. L.M. Barbosa. pp 112-

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34. Royal Botanic Gardens, England. 

 

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Bot. Bras. 9(1), 87-100. 



    

 

45

 

 

CAPÍTULO 2 
 

 

 

 

Nodulation, mycorrhizal colonization and growth of Enterolobium 

contortisiliquum and Inga laurina seedlings in the nursery 

 

 

 

 

(Trabalho a ser enviado para a revista Forest Ecology and Management) 

 

 

 

 

 

 

 

 

 

 



    

 

46

 

 

Nodulation, mycorrhizal colonization and growth of Enterolobium contortisiliquum 

and Inga laurina seedlings in the nursery 

 

 

 

 

 

CAMILA MAISTRO PATREZE1,2 and LÁZARA CORDEIRO1 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                 
1 Universidade Estadual Paulista, IB, Departamento de Botânica, Caixa Postal 199, 13506-900 

Rio Claro, SP, Brasil. 
2 Corresponding author: cpatreze@rc.unesp.br 



    

 

47

Abstract  

Effects of dual inoculation with rhizobia and arbuscular mycorrhiza fungi (AMF) on 

nodulation, mycorrhizal colonization and initial growth of E. contortisiliquum and I. 

laurina were examined in unsterile soil and fertilized with addition of N, P or both NP, 

inoculated or not by rhizobia (r), mycorrhiza (m) or dual inoculation (rm), following seven 

treatments: NP, P, P+r, P+rm, N, N+m and N+rm. Growth and nodulation of E. 

contortisiliquum and I. laurina plants were affected by P deficiency. On the other hand, P 

level added not diminished the mycorrhizal colonization. Seedlings of almost all treatments 

were colonized by mycorrhizal fungus. P deficiency in N treatments influenced the size of 

nodules, which were in general smaller than 4 mm and nodules activity, measured by 

acetylene reduction activity (ARA) and leghemoglobin content also were inhibited. Our 

results showed that dual inoculation increased the nodulation and mycorrhizal colonization 

of these plants in nursery with extension of the growth of E. contortisiliquum host, when 

associated to P fertilization. This suggest that the capacity of E. contortisiliquum to 

associate with two soil microorganisms (rhizobia and mycorrhiza) can be a strategy for 

their establishment in soils with low nitrogen levels. 

 

Keywords – Nodulation; Rhizobia; Mycorrhiza; Enterolobium contortisiliquum; Inga 

laurina 

 

 

 

 

 

 



    

 

48

1. Introduction 
 

 
 Enterolobium contortisiliquum (Vell.) Morong and Inga laurina (Sw.) Willd are 

woody leguminous trees with potential for reforestation on drastically disturbed lands, due 

to their ability to develop symbiotic associations with rhizobia and/or arbuscular 

mycorrhizal fungus (AMF). These plants differ in relation to the characteristics of 

succession. E. contortisiliquum, a species with 20-30 m tall at maturity (Engel & Parrota 

2001), is classified as pioneer or initial secondary, according to Resolution of SMA- 

Secretaria do Meio Ambiente nº21, 21/11/2001 and it has been used as enrichment 

planting, due to the great survival rate and growth height (Montagnini et al. 1997), and in 

the agroforestry systems for soil improvement (Eibl et al., 2000) and it is a tolerant species 

in heavy metal contaminated soil (Trannin et al., 2001). This tree species has been sampled 

in several Brazilian riparian forests (Catharino, 1989; Nilsson, 1989 and Bernacci et al., 

1998); its nodulation ability is well known (Stefan, 1906; Lechtova-Trnka, 1931; 

Rothschild, 1970; Corby, 1988; Oliveira, 1997; Milnitsky et al., 1997; Barberi et al., 1998; 

Frioni et al., 1998, Eibl et al., 2000) and AMF colonization also was already reported 

(Frioni et al.1999). 

I. laurina is classified as latest secondary or climax, according to Resolution of 

SMA- Secretaria do Meio Ambiente nº21, 21/11/2001. This species is generally founded in 

riparian forests primarily in seasonally flooded areas (Romagnolo & Souza 2000), but also 

has long been used as a shade tree and green manure in coffee and cacao plantations with 

good potential as agroforestry species (Tilki & Fisher, 1998). I. laurina was previously 

reported to be a N2 fixing tree (Halliday & Nakao, 1982) and I. fagifolia (L.) Benth., 

currently accepted as nomenclatural synonym, was reported to nodulation by the first time 

by Faria et al. (1987); however these studies were not on the physiology of nodulation to 



    

 

49

this species. AMF colonization in I. laurina has not been reported previously, although 

some Inga species had been studied in this aspect. In this way, our work also aims the 

capacity of I. laurina plants to develop mycorrhizal association (AMF) in nursery 

conditions. 

 The importance of associations with rhizobia and mycorrhiza in forests is well 

recognized, but the effectiveness of these associations change according to plant, rhizobia 

and fungus species involved and the environmental conditions. Thus, different plant 

species exhibit varied ability to establish mycorrhizal associations (Siqueira et al., 1998). 

Relatively few leguminous trees have been tested for their nodulation or nitrogen fixation 

ability (Sprent, 2001) and few is known about how we can manage symbiotic fungal and 

rhizobial associations more effectively (Marques et al., 2001). 

The purpose of the present study was to evaluate the effects of dual inoculation 

with rhizobia and mycorrhizal fungi on nodulation, mycorrhizal colonization and growth, 

of E. contortisiliquum and I. laurina under nursery conditions. 

 

2. Materials and methods  

 
Seeds of E. contortisiliquum were supplied by IPEF (Instituto de Pesquisas e 

Estudos Florestais) – Esalq/USP (Escola Superior de Agricultura Luiz de Queiroz/ 

Universidade de São Paulo). Seeds of I. laurina were collected in the field of Unesp 

(Universidade Estadual Paulista “Júlio de Mesquita Filho”) - Rio Claro, SP, Brazil 

(22º44’S and 47º33’W, 610 m a.s.l.). E. contortisiliquum seeds were scarified to dormancy 

break prior to planting. I. laurina seeds were storage in trays with vermiculite and ABA 

solution (10-4M) in the proportion 2:1 in volume in the cold chamber with 10±1ºC and 



    

 

50

85%±5% of relative humidity (Barbedo & Cicero, 2000) during twenty-four days, since the 

lost viability of Inga species occur rapidly under normal environmental conditions.  

Two rhizobia strains were previously isolated from nodules of E. contortisiliquum 

plants, collected from Corumbataí region (22º 08’S and 47º40’W, 684 m a.s.l), and two 

strains were isolated from nodules of Inga spp., collected in the riparian forest of 

Corumbataí, SP, Brazil (22º 20’S and 47º40’W, 604 m a.s.l.). The rhizobia cultures were 

grown on YEM (yeast extract mannitol agar) at 28ºC and were stored in the Rhizobial 

Bank of Unesp Rio Claro, SP Brazil (IBRC). For rhizobia inoculated treatments, seeds of 

E. contortisiliquum were left to soak in a turbid suspension (100 mL) of a mixture of two 

strains, IBRC-206 and 207 from E. contortisiliquum, during 1 h before the planting and 

pre-germinated seedlings of I. laurina received 10 mL of a mixture of two strains, IBRC- 

195 and 197 from Inga spp., directly on the surface of pots, one week after planting. To 

ensure the rhizobia infection, plants of both species were subsequently re-inoculated 30 

days after sowing. For the AMF inoculation, pieces of A. peregrina var. falcata roots with 

±1cm of length collected from Corumbataí cerrado reserve (22º15’S and 47º00’W, 810 m 

a.s.l.) were mixed on each pot (0.4g) surface nearly seedlings at twenty-eight days of 

sowing. 

Soil and vermiculite (2:1) not sterilized were performed as substrate of plastic pots 

of 4 L. The soil from riparian forest of Corumbataí, SP, Brazil (22º 20’S and 47º40’W, 604 

m a.s.l.) contained (in mmolc.dm-3): K, 1; Ca, 12; Mg, 4; H+Al, 10; Al, 7; P, 7mg.dm-3 and 

N, 1000ppm. Micronutrients (in mg.dm-3): B, 0.01; Cu, 0.07; Fe, 23; Mn, 8.7; Zn, 0.4 and 

S, 9, with pH 5.5 (Centro de Ciências Agrárias, Depto. de Recursos Naturais e Proteção 

Ambiental, Universidade Federal de São Carlos). Basal nutrients were added in the 

substrate prior to sowing (in mg. Kg-1 substrate): K (60), CaCO3 (80), MgCO3 (40), S (30), 

B (1), Zn (2), Cu (2), Fe (4), Mn (20), Mo (4). Beyond of these basal nutrients, were added 



    

 

51

nitrogen and phosphorus, according to three different lots: NP, fertilization with addition of 

N (40) and P2O5 (80); P, with phosphorus P2O5 (80) and a start dose of 3.8 mM of N; and 

N, with nitrogen N (40) but without phosphorus. So, these lots were submitted to 

inoculations with rhizobia (r), mycorrhiza (m) and both (rm), resulting in seven treatments: 

NP; P; P+r; P+rm; N; N+m; N+ rm. Ten plants per treatment were grown in a greenhouse 

under natural daylight in randomised blocks. After 120 days of sowing, plants were 

fertilized with additional nutrients (10 mL of solution) every 30 days, following initial 

fertilization treatments. 

The height of the plants was recorded each two weeks, starting at 30 days of 

sowing, in an average of ten plants per treatment until 120 days, thereafter they were 

averages of five plants. Five plants per treatment were harvested at 120 and 255 days, with 

intact root system for evaluation of nodulation and nitrogenase activity. Leaf area was 

measured by CI-202 Area Meter (CID, Inc). Roots, stems and leaves were separated, dried 

to constant weight at 70ºC and weighted. Chemical analyses of shoots (roots plus stems) 

were performed in both harvests, and chemical analyses of substrate were determined in 

the beginning and end of experiment (Centro de Ciências Agrárias, Depto. de Recursos 

Naturais e Proteção Ambiental, Universidade Federal de São Carlos). 

Nodules were weighted, counted, sieved with sieves mesh and they were classified 

per size: larger than 4 mm, between 2-4 mm and smaller than 2 mm. The nodules 

morphology was classified according to Corby (1981). Nitrogenase activity was assayed by 

acetylene reduction activity (ARA) according to Hardy et al. (1968) in root nodules of two 

plants per treatment. Nodules of these same pots were stored in the refrigerator at 10ºC to 

leghemoglobin content analysis, following Becana et al. (1986) with absortion at 540nm. 



    

 

52

For AMF colonization, roots were stained (Philips & Hayman, 1970) and the 

percentage of infected roots was estimated using the gridline intersect method (Giovannetti 

& Mosse, 1980) under a stereomicroscope (40x).  

Data were analyzed separately by ANOVA and means were compared by Duncan’s 

test, at P ≤ 0.05, using the Statistica for Windows (StatSoft, Inc. 2000).  

 

3. Results 

 
In general, E. contortisiliquum plants fertilized with N, but not with P had the 

lowest growth parameters in both the harvests (Table 1). Plants of treatment P+r grew 

similarly to the NP treatment plants and also presented similar values of leaf, stem and root 

dry mass, but it was significantly differ to the others treatments (Table 1). The highest 

root/shoot ratios were in the last harvest of the N without P treatments (Table 1). 

I. laurina plants fertilized with NP were significantly taller than the others (Table 

2), which were not statistically differ among them. More differences, although not 

statistically significant, can be observed between the treatments at 255 days than 120 days 

of sowing. 

All nodules were classified as indeterminate (Corby, 1988), being E. 

contortisiliquum astragaloid and I. laurina muconoid (Sprent, 2001). In general, plants of 

two species were well nodulated, mainly in the second harvest (Figure 1). For the two 

species the most part of nodules were smaller than 2 mm after 120 days and between 2 and 

4 mm of diameter size 255 days. 

 

 

 

 



    

 

53

Table 1 Growth of E. contortisiliquum in response to mineral fertilization (NP, P and N) and inoculation 

treatments with rhizobia (r), mycorrhiza (m) or both (rm) at 120 and 255 days of sowing.  

a Mean of five plants per treatment in all parameters, except to height after 120 days (ten replicates). 

Mean in lines with different letters are significantly different as determined by Duncan test at 5% 

significance level (P<0.05). 

 

 

 

 

 

 

 

 

 

 

 

 

 

Growth of
 E. contortisiliquum NP P P+r P+rm N N+m N+rm

After 120 days
  Height (cm) 33.6 a 20.3 b 20.9 b 15.0 c 12.5 c 14.6 c 14 c

  Leaf area (cm2) 261.1 ab 164 bc 284.4 a 141.9 bc 51.1 c 92 c 43.4 c
  Leaf dry mass (g) 3.30 a 1.68 b 2.34 a 1.72 b 0.49 c 0.64 c 0.33 c
  Stem dry mass (g) 4 a 1.47 bc 1.71 b 1.18 b 0.3 c 0.41 c 0.28 c
  Root dry mass (g) 2.85 a 1.56 ab 1.51 ab 1.1 ab 0.37 b 0.51 b 0.34 b
  Root/shoot ratio 0.39 0.49 0.37 0.38 0.47 0.49 0.56
After 255 days
  Height (cm) 143.6 a 114.2 ab 146.4 a 87.6 b 36.6 c 35.8 c 32.2 c
  Leaf area (cm2) 338.6 b 727.1 ab 1397.2 a 1121.7 a 204 b 266.3 b 116.4 b
  Leaf dry mass (g) 13.78 ab 9.41 b 20.15 a 12.44 b 1.96 c 1.97 c 1.48 c
  Stem dry mass (g) 33.68 a 15.71 b 34.47 a 18.14 b 3.03 c 2.53 c 1.91 c
  Root dry mass (g) 16.09 a 12.01 b 24.43 a 16.03 ab 3.81 b 5.01 b 2.71 b
  Root/shoot ratio (g) 0.34 0.48 0.45 0.52 0.76 1.11 0.80

Treatments of fertilization and  rhizobia (r) and mycorrhiza (m) inoculations



    

 

54

Table 2. Growth of I. laurina in response to mineral fertilization (NP, P and N) and inoculation treatments 

with rhizobia (r), mycorrhiza (m) or both (rm) at 120 and 255 days of sowing.  
 

a Mean of five plants per treatment in all parameters, except to height after 120 days (ten replicates). 

Mean in lines with different letters are significantly different as determined by Duncan test at 5% 

significance level (P<0.05). 

 

 

 

 

Growth of
I. laurina NP P P+r P+rm N N+m N+rm

After 120 days
  Height (cm) 17.1 a 11.5 b 6.9 c 6.15 c 8.3 c 9.2 bc 8.05 c

  Leaf area (cm2) 145.9 a 62.3 b 36.2 b 8.9 b 36.5 b 51.5 b 42.7 b
  Leaf dry mass (g) 1.41a 0.72 b 0.43 b 0.12 b 0.43 b 0.57 b 0.43 b
  Stem dry mass (g) 0.36 a 0.21 b 0.09 bc 0.03 c 0.11 bc 0.15 bc 0.10 bc
  Root dry mass (g) 0.70 a 0.53 ab 0.31 ab 0.05 b 0.29 ab 0.3 ab 0.26 ab
  Root/shoot ratio 0.40 0.58 0.60 0.36 0.54 0.41 0.50
After 255 days
  Height (cm) 78.2 a 39.6 b 26.4 cd 21 d 31 bcd 35.2 bc 29.6 bcd

  Leaf area (cm2) 503.4 a 132.6 b 139.9 b 182.9 b 379.4 ab 377.3 ab 280.6 ab
  Leaf dry mass (g) 15.30 a 5.10 b 2.31 c 2.01 c 4.18 b 4.84 b 4.10 b
  Stem dry mass (g) 9.81 a 2.16 b 0.85 cd 0.64 d 1.46 bcd 1.85 bc 1.97 b
  Root dry mass (g) 15.63 a 4.19 b 1.57 bc 0.87 c 4.05 bc 3.91 bc 3.63 bc
  Root/shoot ratio (g) 0.62 0.58 0.50 0.33 0.72 0.58 0.60

Treatments of fertilization and  rhizobia (r) and mycorrhiza (m) inoculations a



    

 

55

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Fig.1.  Nodule number average per diameter size (larger than 4 mm, between 2 to 4 mm and smaller than 2 

mm) of A, E. contortisiliquum and B, I. laurina plants in the substrate fertilized with NP, P and N and 

inoculated by rhizobia (r), mycorrhiza (m) or both (rm), at 120 and 255 days of sowing. Values are means of 

five plants per treatment. Nodules number total followed by the same letter are not significantly different by 

ANOVA, test Duncan at P<0,05 level of significance. 

 
 

 

 

 

 

 

 

 

bc c
bc

abc

ab

 a a

0

20

40

60

80

100

120

140

bbb

a

ab

ab

a

0

100

200

300

400

500

600

<2

2 a 4

>4

b b
b

a

a

 b b

0

5

10

15

20

25

30

35

NP P P+r P+rm N N+m N+rm

bc

c

b

a

c c

c

0

10

20

30

40

50

60

70

80

90

NP P P+r P+rm N N+m N+rm

N
od

ul
es

 n
um

be
r

Treatments 

A 

B 

At 120 days At 255 days 



    

 

56

Regardless of harvest, the number (Figure 2) and dry weight (Table 3) of nodules 

from two species were affected by P deficiency in N treatments. Also, the most of the 

nodules in these treatments were smaller than 2 mm of size (Figure 2) and the nodules 

activity, as measured by acetylene reduction activity (ARA) and leghemoglobin content 

(Figure 3) were inhibited. Nodules of E. contortisiliquum of the P+r treatment presented 

higher ARA at 255 days (Figure 3A). ARA of I. laurina nodules were not detected in the 

first harvest, despite of nodule presence, and leghemoglobin content were not shown 

because of not achieving of replicates. In the second harvest, the ARA was higher in 

uninoculated treatments (NP and P) and leghemoglobin was assayed on the NP, P, P+r and 

P+rm treatments (Figure 3B). 

 
Table 3 

Dry weight average of nodules (g) of A , E. contortisiliquum and B, I. laurina plants in the substrate fertilized 

with NP, P and N and inoculated by rhizobia (r), mycorrhiza (m) or both (rm), at 255 days of sowing. Mean 

of five plants per treatment, followed by the same letter are not significantly different by ANOVA, test 

Duncan at p<0.05. 

 

 
 

>4 mm 2 a 4 mm <2 mm Total
Treatments
NP 4.407 ab 0.811 ab 0.098 ab 5.317
P 2.207 bc 0.213 b 0.018 b 2.440
P+r 6.807 a 0.730 ab 0.038 ab 7.577
P+rm 3.533 abc 1.562 a 0.215 a 5.311
N 0.011 c 0.007 b 0.019
N+m 0.007 c 0.002 b 0.010
N+rm 0.001 c 0.001 b 0.004

NP 0.102 b 0.034 b 0.001 b 0.139
P 0.350 a 0.021 b 0.373
P+r 0.016 b 0.052 b 0.006 ab 0.075
P+rm 0.085 b 0.100 a 0.010 a 0.197
N 0.004 b 0.005
N+m 0.000
N+rm 0.001 b 0.012 b 0.015

Nodule dry weight (g)

E. contortisiliquum

Inga laurina



    

 

57

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Fig. 2. Acethylene reduction activity (µmol C2H4 g-1 nodule fresh h-1) and leghemoglobin content (mg Hb g -1 

nodule fresh) averages of A, E. contortisiliquum plants, at 120 and 255 days of sowing and B, I. laurina 

plants at 255 days, fertilized with NP, P or N and inoculated with rhizobia (r), mycorrhiza (m) and both (rm). 

 
Although the plants of the two species had been nodulated with native rhizobia, 

which were actives, demonstrated by ARA e leghemoglobin content mainly at 255 days 

(Figure 2), the nodulation of the two hosts were benefited by inoculation of rhizobia strains 

and mycorrhizal fungi, as can be seen in nodule number of P+rm treatment of Figure 1. 

Thus, dual inoculation associated with P fertilization increased nodulation in these two 

species. 

Despite of the N content of plants not have been tested statistically, we could note 

that treatments not fertilized with nitrogen (P treatments) presented N content similar those 

0

0.5

1

1.5

NP P P+r P+rm N N+mN+rm

um
ol

C
2H

4 
g-

1 
no

du
le

 f
re

sh
 h

-1

0

0.15

0.3

0.45

m
g

 H
b

 g
-1

 n
o

d
u

le
 fre

sh

0

5000

10000

15000

20000

25000

NP P P+r P+rm N N+m N+rm

um
ol

C
2H

4 
g-

1 
no

du
le

 f
re

sh
 h

-1

0

0.15

0.3

0.45

0.6

m
g

 H
b

 g
-1

 n
o

d
u

le
 fre

sh

0

500

1000

1500

NP P P+r P+rm N N+mN+rm

Treatments

um
ol

C
2H

4 
g-

1 
no

du
le

 f
re

sh
 h

-
1

0

0.15

0.3

0.45

0.6

m
g

 H
b

 g
-1

 n
o

d
u

le
 fre

sh

A 

B 

At 120 days At 255 days  

At 255 days 



    

 

58

plants of NP treatments, although for E. contortisiliquum these treatments had been lower 

than those fertilized only with N (Figure 3A). For I. laurina this difference were not 

apparently observed (Figure 3B). 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 
Fig.3. Percentage (%) of colonization by arbuscular mycorrhiza fungi (AMF), N and P contents (%) in shoots 

of A, E. contortisiliquum and B, I. laurina plants on the fertilization (NP, P and N) and inoculation with 

rhizobia (r), mycorrhiza (m) and both (rm) treatments, at 120 and 255 days after sowing. Mean of two plants 

for AMF colonization and a mixture of five plants per treatment for chemical analysis of shoots. 

 

I. laurina was colonized either by inoculated as native mycorrhizal fungi, 

demonstrating its mycorrhizal infection potential, still not reported in the literature. 

However, in general, mycorrhizal infections were low in both species (Figure 3).  

 

0

5

10

15

20

25

30

A
M

F
 

120 days

255 days

0

10

20

30

40

50

NP P P+r P+rm N N+m N+rm

A
M

F

0

1

2