Effect of arbuscular mycorrhizal fungi on the acclimatizationof micropropagated banana plantlets
Accepted: 24 May 1999
Abstract The effect of arbuscular mycorrhizal fungi(AMF) on micropropagated banana plantlets was eval-uated during the acclimatization period. Plants wereinoculated with Acaulospora scrobiculata, Glomus ela-rum or Glomus etunicatum. After cultivation in agreenhouse for 3 months, height, leaf area, fresh weightand dry matter of root and shoots, levei of AMF colo-nization, nutrient levei, photosynthesis and transpira-tion rate, water potential and stomatal conductancewere measured. The number of AMF spores producedin each treatment was also determined. Plantlets inocu-lated with AMF had greater height, leaf area and freshweight of shoots and roots, as well as higher rates ofphotosynthesis and transpiration than controls. Plantsinoculated with Glomus were superior in most of theevaluated parameters.
Key words Musa spp .. Endomycorrhiza . Glomales .Acclimatization
Introduction
The vegetative micropropagation of fruit, ornamentaland forest species is the main tool for producing geneti-cally homogeneous plants in a good phytosanitary con-dition and in large numbers. The transfer to the green-house of plants cultivated in vitro is one of the mostimportant steps in the structural and physiological ad-aptation during preparation of plantlets. This phase,known as acclimatization, is the beginning of the auto-trophic existence of the plant, with the initiation of the
• physiological processes necessary for survival. During
A.M. Yano-Melo (13J) . L.c. MaiaDepartamento de Micologia, CCB,Universidade Federal de Pernambuco,50670-420, Recife-PE, Brazile-mail: [email protected]
0.1. Saggin' Jr. . J.M. Lima-Filho' N.F. MeioEmbrapa Serni-Árido, CX. Postal 23,56300,000, Petrolina-PE, Brazil
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this time, plantlets must increase absorption of waterand minerais and the photosynthetic rate (Grattapagliaand Machado 1990).
Micropropagated plantlets are free of diseases, butthey also lack arbuscular mycorrhizal fungi (AMF).AMF are known to increase the vigor of plants by in-creasing absorption of water and mineral nutrients, es-pecially phosphorus (P). Moreover, AMF can protecthost plants from root pathogens and mitigate the ef-fects of extreme variation in temperature, pH and wa-ter stress (Dixon and Marx 1987; Siqueira 1994).
Successful inoculation of AMF at the beginning ofthe acclimatization period (Granger et aI. 1983; Brazan-ti et aI. 1992; Guillemin et aI. 1995) or even during invitro cultivation (Mathur and Vyas 1995) has been de-monstrated. The beneficial effect of the symbiosisformed with the root system of plants from tissue cul-ture manifests itself in the development of vigorousplants with high photosynthetic and transpiration rates,improved absorption of nutrients and water and in-creased stress tolerance (Azcón-Aguilar et aI. 1997;Jaizme-Vega et aI. 1997).
lnoculation of micropropagated plants with AMFduring initial growth ex vitro may contribute to highcolonization rates through positive mycorrhizal symbio-sis effects on the activity of the rootmeristem. This hy-pothesis is supported by results of Berta et aI. (1995),who demonstrated that AMF association altered thebranching pattern of roots of Prunus cerasifera. Theinoculum type used in the acclimatization is important.Fortuna et aI. (1992) recommended the use of infectiveand efficient species of AMF which promote rapid in-crease in plant growth. These authors also demon-strated, while comparing the efficiency of two AMFspecies in promoting growth of micropropagated P. cer-asifera, that the infectivity of the fungi influenced theireffectiveness. Greater fresh and dry matter and heightincreases were found with plants inoculated with Glo-mus mosseae than with G. coronatum, but at the end ofthe experiment both groups of plants showed similargrowth.
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The aim of our work was to evaluate the effects ofthe inoculation of three native AMF species isolatedfrom irrigated banana plantations of the Brazilian semi-arid region on growth, nutrition and physiology of ba-nana plantlets developed in vitro.
Materiais and methods
Plant material and soil properties
Mieropropagated banana plantlets(Musa spp. ev. Paeovan) wereobtained from the Laboratory of Bioteehnology, Embrapa Semi-Árido, Petrolina-PE, Brazil. The plantlets formed roots in vitrousing MS liquid eulture medium (Murashige and Skoog 1962) andwere later transferred to plastie eups (500 ml eapacity) filled witha fumigated (Bromo flora Methyl Bromide, Bromine CompoundsLtd., Beer Sheva, Israel) substratum: soil, sand, and organic mat-ter (1: 1: 1). The properties of the soil before mixture with sandwere 3.2 g organic matter kg :' soil, 0.84 mg P drn " soil (MehlichI), pH 5.1 (soil: water 1: 2.5).The inoculum (approximately 400spores per eontainer) was plaeed 5 em below the soil surfaee incontact with the banana plantlet roots and was covered with fumi-gated substratum. A filtrate of the inoculum soil was added to alitreatments to standardize microbiota. Plants were maintained inthe greenhouse under a 12-h light period of 800-1300lux,25 ± 4 °C and 70-90% relative humidity.
lnoeulum source
Spores ofAcau/ospora scrobiculata (isolate AcSe PE 002) andC/o-mus etunicatum (isolate GlEt PE 004) were isolated frorn the rhi-zosphere of banana plantations in the Submédio São FraneiscoValley, Petrolina, Pernambueo, while inoculum ofC/omus clarum.(isolate UFPE08) was obtained frorn the eulture eolleetion of theEmbrapa Agrobiologia. Seropédica, Rio de Janeiro. The isolateswere eultivated in a greenhouse for 4 months in 3-1 eontainersfilled with a mixture 01' soil, sand and vermieulite (2: I :1) andplanted with Panicum miliacium. The plants were irrigated withnutrient solution (Hewitt 1966) on alternate days and with dis-tilled water once a week.
Data eolleetion
Three months after inoeulation, pereent root colonization, num-ber of spores, shoot and root fresh and dry weights, plant height,and leaf area were measured. Physiologieal parameters sueh asphotosynthesis, transpiration, stomatal eonduetance, and waterpotential were also examined. Spores were extraeted from the soilby water and suerose centrifugation (Jenkins 1964). Roots werestained with 0.05% trypan blue (Phillips and Haymann 1970) andpereent eolonization was estimated by the gridline-intersect meth-
Table 1 Means of growth parameters and myeorrhizal coloniza-tion of banana plantlets (Musa spp. cv. Pacovan) inoeulated ornot with arbuseular myeorrhizal fungi (AMF) and the number ofAMF spores after 3 months acclimatization. Values followed by
od (Giovannetti and Mosse 1980). Plant height was measured di-rectly and leaf area determined by a Leaf Area Meter Li 3 100(LI-Cor Ine. Lineon, Neb., USA). Photosynthesis, transpirationand stomatal conductance rates were measured with a portablephotosynthesis system Li-6200 (LI-Cor) eoupled to a 250-ml as-similation ehamber. For determination of water potential, leafdiscs (1 em diameter) were eolleeted and transferred to psyehom-etric ehambers (C-52) connected to a mierovoltmeter (HR 33TWeseor).
Experimental design
The following treatments were established: (1) Without inocula-tion (2) inoeulated with Acaulospora scrobiculata (3) with Glo-mus clarum (4) with C. etunicatum. The experiment was random-ized with five replicates. For analysis of variance the data on per-eent eolonization and number of spores were arcsin transformedas the square roots of (x/100) and (x + 2.5), and statistieal differ-enees arnong means evaluated by Tukey's Test using the SASprogram (SAS Institute).
Results
Plant growth
After 3 months of acclimatization, statistically signifi-cant differences in plant height, leaf area, fresh and drymatter of shoot, and fresh weight of roots between ino-culated and non-inoculated plants were recorded (Ta-ble 1).
Leaf are a and height of inoculated plants were ap-proximately 57% and 32% higher, respectively, thannon-inoculated plants. Dry matter of shoots increased45-64% in mycorrhizal plants. Banana plantlets inocu-lated with G. clarum showed an increment of around45% in the fresh weights of shoots and roots over non-inoculated plants. Root percent colonization and sporenumber were not significantly different among thetreatments (Table 1).
Physiological aspects
Banana plantlets inoculated with A. scrobiculata and G.etunicatum reached the highest photosynthetic rates,being 45% more efficient than the non-inoculatedplants.
the same letter are not signifieantly different (P<0.05) (DW dryweighr, FW fresh weight, AcSc Acaulospora scrobiculata, CIC!Glomus clarum, CIE! Glomus etunicatums
Non-inoculated 7.518 b 0.976a 2.850 b 0.376 b 60.3 b 11.2 b O b ObAcSc 1O.372ab 0.976a 3.964ab 0.544a 91.8a 14.8a 63a 37aClc/ 11.660b 1.274a 4.420a 0.602a 96.5a 15.3a 346a 33aeu. 10.342ab 0.982a 4.240a 0.616a 95.0a 15.0a 112a 30a
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Table 2 Nutrient contents of roots and shoots of micropropagated banana plantlets. Values followed by the same letter are not sig-nificantly different (P<O.05)
There was an increase in stomatal conductance inplants inoculated with AMF and an increase in thetranspiration rate of 60-86%. These increases were es-pecially clear in banana plantlets inoculated with A.scrobiculata. In contrast, the water potential of bananaplantlets inoculated with A. scrobiculata was not af-fected and was marginally increased in plantlets inocu-lated with Glomus isolates (data not shown).
Nutrient absorption
The nutrient contents of the root systems of AMF-ino-culated banana plantlets only differed significantlyfrom the control for copper (Cu), sodium (Na) andmagnesium (Mg) (Table 2). The Cu content was in-creased most in banana plantlets inoculated with A.scrobiculata. The Na content of plantlets inoculatedwith Glomus species was 70-95% higher than that ofthe control plantlets (Table 2), while for Mg a highercontent was found in the control than in plants inocu-lated with G. etunicatum.
Differences in the contents of K, Ca, Mg, Mn and Znwere found in shoots of micropropagated banana plant-lets (Table 2). Mycorrhizal plantlets had higher con-tents of Zn and Mn than the control. Conversely, thecontents of K, Ca and Mg were higher in the control(Table 2) than in plants inoculated with G. etunica-tum.
Fe and Na accumulated to higher levels in roots thanin shoots. In contrast, most of the absorbed Mn wastransferred to the shoots (Table 2).
Discussion
Plant height, leaf area and dry matter of shoots of ino-culated banana plantlets were significantly higher thanthose of control plants. These results reinforce thoseobtained by Monteiro et aI. (1991), who presentedheight as the parameter showing the most significantdifferences in cultures of banana (variety Mysore) ino-
culated with G. clarum. Lin and Chang (1987) also ob-tained increased height, diameter of the pseudostem,and dry matter weight of banana plantlets inoculatedwith species of Glomus, 4 months after transplant fromin vitro culture to the greenhouse.
Thus Glomus species are apparently effective in pro-moting growth of banana plantlets. Declerck et aI.(1994) reported that G. mosseae was more effectivethan G. geosporum at improving growth of micropropa-gated banana plants. Later, Declerck et al. (1995) ob-served that promotion of growth varied among bananacultivars and that plants inoculated with G. macrocar-pum grew more than those inoculated with G. mosseae.The growth stimulation was associated with increasedphosphorus in shoots. Results obtained in our experi-ments show that G. clarum as well as the native G. etu-nicatum and A.scrobiculata are effective in promotinggrowth of banana plantlets during the acclimatizationperiod, with little difference in the effectiveness of thethree species.
AMF colonization did not differ among the inocu-lated treatments in this study. Declerck et aI. (1995)found differences in colonization of banana cultivars byAMF isolates. According to Douds et al. (1998), thephysiological response of a plant is the resultant of in-teractions between environment, plant, and fungus ge-notype. It is worthwhile to point out that increases ingrowth rate are not always related to colonization(Guillemin et aI. 1992), but may be influenced by otherfactors, such as extension of the external mycelium andtransport of nutrients from the soil to the host.
Jaizme-Vega et aI. (1991) observed that root freshmatter of micropropagated banana plantlets (Musaacuminata Colla AAA, subgroup Cavendish) increasedsimultaneously with increase in root colonization by G.mosseae and G. fasciculatum. Although these experi-ments were over a longer time period than ours, rootcolonization was lower than we observed. Nevertheless,AMF inoculation led to similar increases in root freshweight in the two experiments. This difference in levelsof root colonization by AMF is probably attributable todifferences in mycorrhizal dependency among varietiesof banana and to abiotic factors.
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Increased leaf area observed by Allen et aI. (1981)and Harris et aI. (1985) was concomitant with an in-crease in photosynthetic rate of Bouteloua graeilis thatwas thought to be an effect of inoculation with G. fasei-eu/atum. In contrast, Freeden and Terry (1987) in re-porting results obtained with Glycine max inoculatedwith G. faseieulatum observed a similar increase in leafarea but not in photosynthetic rate. Furthermore, thisrelationship was not observed in our study, where meanphotosynthetic rates did not differ significantly betweentreatments.
Duan et aI. (1996) found higher stomatal conduc-tance in mycorrhizal plants than in controls and as-cribed this to increased water absorption capacity ofroots, which even in dry soils results simultaneously ina decrease in leaf resistance. Similar results were ob-tained in our experiments, although the soil was alwaysmoist.
We found that mycorrhizal plants had higher Cu lev-eis in their root systems than controls. Roots of mycorr-hizal banana plants contained 150% more Cu thanshoots, which suggests that although AMF increase theamount of Cu accumulated in the roots, they may alsoprotect shoots from an excess of heavy metaIs (Sylviaand Williams 1992).
Mn was translocated in large amounts to shoots.This effect is contrary to that observed in soybeans byBethlenfalvay and Franson (1989) and in most studieswith AMF (Nogueira 1996), but similar to that re-corded by Medeiros et aI. (1995) in experiments withsorghum. The results suggest that AMF effects on lev-eIs of MN in the aerial parts of plants may differ ac-cording to host species and fungal isola te.
That mycorrhizal plants showed reduced levels of K,Ca and Mg in shoots and Mn in roots, compared withnon-mycorrhizal plants, may be explained by the dilu-tion effect of the increased dry mass of mycorrhizalplants (Jarrel and Beverly 1981). This effect is commonin studies comparing levels of nutrients in plants withand without mycorrhizas.
Brazanti et aI. (1992) observed no differences in mi-neral contents of mycorrhizal and non-mycorrhizalplants, even when supplemented with P. Although, wefound no statistical differences in concentrations of P,Ca, and Zn in roots and P in shoots between inoculatedand non-inoculated plants, the total contents of theseelements were significantly higher in mycorrhizal thanin non-mycorrhizal plants. In general, elements withlow mobility in the soil, such as P, Cu and Zn, are ab-sorbed in higher amounts by mycorrhizal than by non-mycorrhizal plants (Stribley 1987; Colozzi-Filho andBalota 1994). In our work, the concentrations of Cuand Zn in roots and shoots, respectively, point to suchan effect.
Menge et aI. (1978) and Jaizme-Vega and Azcón(1991) considered inoculation with AMF a good strate-gy for successful plant transplantation because of im-proved water and nutrient absorption. In our experi-ments, inoculation with AMF increased growth of mi-
cropropagated banana plantlets during the acclimatiza-tion period, and this may benefit rates of photosynthe-sis and aiso nutrient transport by mass flow. Isolates ofboth na tive and introduced AMF improved the growthand the nutritional status of banana plantlets and thismay have importance for banana plantations.
Acknowledgements The authors thank Dr. David Sylvia for dis-cussions about the manuscript and the anonymous reviewers fortheir valuable suggestions. This work was supported financiallyby Embrapa Sem i-Árido and FACEPE. t.c M. and A.M.Y. M.acknowledge the fellowships provided by CNPq.
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