The role of arbuscular mycorrhizal fungi (AMF) in the control of migratory endoparasitic nematodes is nowadays largely admitted. Most studies were conducted under greenhouse conditions and a few used in vitro cultures with transgenic root organs. Here, we reported, for the first time, on the interaction between an AMF, Rhizophagus irregularis MUCL 41833 and Radopholus similis in roots of banana plantlets grown under in vitro culture conditions. The banana plantlets were pre-mycorrhized in an extraradical mycelium network arising from a Medicago truncatula donor seedling, before transfer to an autotrophic in vitro cultivation system and subsequent nematode inoculation. Both microorganisms were able to complete their life cycle in the absence as well as in presence of each other. The total R. similis population (i.e., summed over the roots and growth medium) as well as the surface of root necrosis was significantly reduced by 60 and 56 %, respectively, in the AMF-colonized banana plantlets. By contrast, nematodes had no visible impact on root colonization (i.e., percentage of arbuscules, intraradical spores/vesicles, and hyphae) by AMF and on the number of spores and hyphal length produced in the medium. These results clearly demonstrated that pre-mycorrhized banana plants could outcompete R. similis, while root colonization was not affected by the nematodes. They underline the interest of the novel in vitro cultivation system as a promising tool to investigate the biochemical factors and molecular mechanisms involved in the bio-protection conferred by AMF to a major root pathogen of banana.
Bananas are susceptible to various plant-parasitic nematodes, including Radopholus similis. The control of this migratory endoparasite relies on the application of nematicides as well as on measures such as fallow, paring and hot water treatment of the corms, the use of resistant cultivars and the large-scale micropropagation of plantlets (Quénéhervé, 2009). The application of biocontrol organisms is another option considered nowadays as a potential alternative to decrease the damage caused by nematodes (FAO, 1997). Within the rhizosphere, arbuscular mycorrhizal fungi (AMF) are key microorganisms that form symbiotic associations with nearly 80% of plant species, including bananas. They improve plant nutrition and have also been reported to decrease nematode infestation. Elsen et al. (2001) demonstrated that Rhizophagus irregularis MUCL 41833 was able to decrease the reproduction capacity of R. similis in excised root organs of carrot {i.e., in root organ culture). More recently, Koffi et al. (2009) developed an in vitro culture system associating autotrophic micropropagated banana plantlets with R. irregularis MUCL 41833. With a closely related in vitro cultivation system, Koffi et al. (2012) were the first to investigate the impact ofR. irregularis MUCL 41833 on the resistance of cv. Grande Naine, a banana cultivar particularly sensitive to R. similis. They observed a decrease of 60 and 56% in the nematode population and surface of root necrosis, respectively, in the plantlets associated with the AMR In the present work, cv. Yangambi km5, a partially resistant cultivar to R. similis, was coupled with R. irregularis MUCL 41833 under strict in vitro culture conditions and challenged with R. similis. The effects of the AMF on the reproduction ratio of the nematodes as well as the impact of the latter on the AMF were studied.Tissue-cultured banana (Musa acuminata) plantlets, cv. Yangambi km5, were provided by the International Musa Germplasm Collection at INIBAP (KUL, Belgium). The plantlets were proliferated, regenerated and rooted as in Koffi et al. (2012). A mycelium donor plant (MDP) in vitro culture system was set up (Koffi et al., 2012), consisting of bi-compartmented Petri plates each with a root compartment (RC) and a hyphal compartment (HC). The two compartments were filled with 100 ml Modified Strullu-Romand (MSR) medium (Declerck et ai, 1998) without sucrose and vitamins, and supplemented with 10 ml r' MES buffer (MSRss -H MES) and solidified with 3 g r' Phytagel (Koffi et ai, 2012). Seeds of Medicago truncatula Gaertn. cv. Jemalong A17 (SARDI, Australia) were surface-sterilised and plantlets subsequently placed in the RC of each MDP in vitro culture system (for full details see Koffi et ai, 2012). Half of the plantlets were inoculated with 100 spores of/?, irregularis (+AMF treatment), the other half remaining free of AMF (-AME treatment). The Petri plates were placed in an incuba-'
Micropropagated banana plantlets represent a good planting material to establish new growing areas or replace old plantations. The plantlets are devoid of pathogens but also of beneficial root inhabitants (e.g., endophytes and arbuscular mycorrhizal fungi [AMF]) that may help plants withstand stresses. Mycorrhization of banana is usually conducted at the hardening/post-hardening phases. Only a few studies have reported their application in vitro, and none have investigated the subsequent impact on ex vitro acclimatization. Here, we report for the first time the effects of in vitro mycorrhization of banana plantlets on growth following transfer to soil. Banana plantlets were pre-mycorrhized under autotrophic conditions. During in vitro culture, heavy root colonization of the banana plantlets was observed. At 5 and 7 wk after transfer to a peatsand soil substrate, the root system remained highly colonized. The growth (pseudostem height) and biomass were significantly higher than those of control plants grown on media without AMF. The mean shoot dry weight of the premycorrhized plants at 5 wk after transplanting was 66.7 and 34.6% greater than those of non-mycorrhized plantlets grown on modified Strullu-Romand (MSR) and Murashige and Skoog (MS) media, respectively, and was also greater at week 7 (by 64.7 and 40%, respectively). In vitro mycorrhization under autotrophic culture conditions thus represents a promising tool for the pre-adaptation of micropropagated banana plantlets to ex vitro conditions.
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