Campo et al. Rice (2020) 13:42 https://doi.org/10.1186/s12284-020-00402-7 ORIGINAL ARTICLE Open Access Effect of Root Colonization by Arbuscular Mycorrhizal Fungi on Growth, Productivity and Blast Resistance in Rice Sonia Campo1* , Héctor Martín-Cardoso1, Marta Olivé1, Eva Pla2, Mar Catala-Forner2, Maite Martínez-Eixarch3 and Blanca San Segundo1,4 Abstract Background: Arbuscular mycorrhizal (AM) fungi form symbiotic associations with roots in most land plants. AM symbiosis provides benefits to host plants by improving nutrition and fitness. AM symbiosis has also been associated with increased resistance to pathogen infection in several plant species. In rice, the effects of AM symbiosis is less studied, probably because rice is mostly cultivated in wetland areas, and plants in such ecosystems have traditionally been considered as non-mycorrhizal. In this study, we investigated the effect of AM inoculation on performance of elite rice cultivars (Oryza sativa, japonica subspecies) under greenhouse and field conditions, focusing on growth, resistance to the rice blast fungus Magnaporthe oryzae and productivity. Results: The response to inoculation with either Funneliformis mosseae or Rhizophagus irregularis was evaluated in a panel of 12 rice cultivars. Root colonization was confirmed in all rice varieties. Under controlled greenhouse conditions, R. irregularis showed higher levels of root colonization than F. mosseae. Compared to non-inoculated plants, the AM-inoculated plants had higher Pi content in leaves. Varietal differences were observed in the growth response of rice cultivars to inoculation with an AM fungus, which were also dependent on the identity of the fungus. Thus, positive, negligible, and negative responses to AM inoculation were observed among rice varieties. Inoculation with F. mosseae or R. irregularis also conferred protection to the rice blast fungus, but the level of mycorrhiza-induced blast resistance varied among host genotypes. Rice seedlings (Loto and Gines varieties) were pre-inoculated with R. irregularis, transplanted into flooded fields, and grown until maturity. A significant increase in grain yield was observed in mycorrhizal plants compared with non-mycorrhizal plants, which was related to an increase in the number of panicles. Conclusion: Results here presented support that rice plants benefit from the AM symbiosis while illustrating the potential of using AM fungi to improve productivity and blast resistance in cultivated rice. Differences observed in the mycorrhizal responsiveness among the different rice cultivars in terms of growth promotion and blast resistance indicate that evaluation of benefits received by the AM symbiosis needs to be carefully evaluated on a case-by-case basis for efficient exploitation of AM fungi in rice cultivation. Keywords: Arbuscular mycorrhiza, Funneliformis mosseae, Japonica, Magnaporthe oryzae, Resistance, Rhizophagus irregularis, Rice, Symbiosis, Yield * Correspondence: sonia.campo@cragenomica.es 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del Vallés), Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Campo et al. Rice (2020) 13:42 Page 2 of 14 Background years to introduce an R gene into a rice variety, even Arbuscular mycorrhizal fungi (AMF) are obligate bio- longer when pyramiding several R genes to generate new trophs that establish mutualistic associations with roots varieties with broader blast resistance. For these reasons, of most terrestrial plants, including many crops (Smith durable resistance to the rice blast fungus remains chal- and Read 2008; Parniske 2008; Bonfante and Genre lenging, and the control of rice blast depends on the use 2010; MacLean et al. 2017; Choi et al. 2018). Root of fungicides. colonization by AM fungi improves the uptake of min- Evidence supports the natural colonization of rice by eral nutrients in the host plant, mainly phosphorus and AM fungi in rice fields. Root colonization by AM fungi nitrogen, in exchange for photosynthetically fixed car- was reported in European rice varieties grown in aerobic bon. Along with this, root colonization by AM fungi has conditions (Vallino et al. 2009). Here, all the checked been shown to improve nutrition, ultimately helping in rice varieties were colonized by AM fungi, the majority plant growth and development. Another benefit con- of AM fungal taxa being assigned to the Rhizophagus ferred by the AM symbiosis in several plant species is irregularis and Funneliformis mosseae (previously known improved resistance to biotic and abiotic stress (Fritz as Glomus intraradices and Glomus mosseae, respect- et al. 2006; Pozo and Azcón-Aguilar 2007; Campos- ively) (Stockinger et al. 2009; Schüßler and Walker Soriano et al. 2012; Nair et al. 2015; Cornejo et al. 2017; 2010). In other studies, rice plants grown in different lo- Wang et al. 2018; Rivero et al. 2018). Nevertheless, the cations in the southern United States exhibited natural benefits conferred by the AM symbiosis to the host plant colonization under non-flooded conditions (Bernaola vary depending on the identity of both the host plant et al. 2018a). (species and variety) and the AM fungal species (Sikes Although it is well known that rice can establish sym- et al. 2009; Fernández et al. 2014; Taylor et al. 2015; biotic associations with AM fungi, our current know- Wang et al. 2016; Sawers et al. 2017; Watts-Williams ledge on the possible beneficial effects of the AM et al. 2019). Furthermore, AM symbiosis is not always symbiosis in rice is still scarce. Most probably, this lack advantageous, as the effects can be positive, negative, or of knowledge is because the traditional rice farming sys- neutral to the host plant (Tawaraya 2003; Grace et al. tems involve growing rice in flooded (paddy) fields, and 2009; Sawers et al. 2017; Watts-Williams et al. 2019). plants growing in aquatic environments were previously Environmental factors, such as soil properties, nutrient considered to be non-mycorrhizal. At present, however, availability, or agricultural management practices, can this scenario has drastically changed, and the occurrence also affect the association of crop plants with AM fungi. of AM fungi in roots of aquatic plants is well recognized Hence, although AM fungi have been proposed as an al- (Lumini et al. 2011; Ruíz-Sánchez et al. 2011; Wang ternative production practice to promote yield in crops, et al. 2011; Kohout et al. 2012; D’Souza 2016; Moora root colonization by AM fungi might not always be fa- et al. 2016). vorable for the host plant. The AM fungus R. irregularis is one of the most wide- Legume species easily establish and benefits from AM spread AM fungal species in the world. Evidence also symbiosis. Accordingly the legume species Medicago support that Rhizophagus irregularis can grow and truncatula and Lotus japonicus have been widely used in colonize rice plants in flooded soil while maintaining its studies on the AM symbiosis. Major cereal crops are also functional capacities (Vallino et al. 2014). In other stud- hosts for AM fungi, and the beneficial effects of the AM ies, the application of AMF at the nursery stage was symbiosis are documented in maize, sorghum, oat, millet found to increase yield by 14–21% in the wetland rice and wheat (Koide et al. 1988; Sawers et al. 2008, 2017, cultivar Nipponbare (Solaiman and Hirata 1997). In wet- 2018; Beltrano and Ronco 2008; Ceasar et al. 2014; land rice Prakash, grain yield increased by 35–62% upon Watts-Williams et al. 2019). inoculation with Acaulospora sp., Glomus fasciculatum, Rice is one of the most important cereal crops in the or G. mosseae (Secilia and Bagyaraj 1994). On the other world and a staple for more than half of the global popu- hand, we previously reported that root colonization by lation. Rice production systems include flooded and up- the AM fungus R. irregularis enhances resistance to the land cultural systems, with a large predominance for the blast fungus M. oryzae in the rice cultivar Senia (O. former because higher yields are obtained on flooded sativa, ssp. japonica) (Campos-Soriano et al. 2012). areas. However, rice production is severely threatened by Whether the AM symbiosis positively impacts blast re- the blast disease caused by the fungal pathogen Magna- sistance deserves further investigation. A better under- porthe oryzae (Wilson and Talbot 2009). Traditional re- standing of the impact of root colonization by AM fungi sistance (R) genes may confer blast resistance, but on rice yield and disease resistance will facilitate agricul- resistance conferred by R genes often breaks down in a tural exploitation of the AM symbiosis in rice. few years due to the high variability and fast-evolving The aim of this research is two-fold. Firstly, to investi- populations of this fungus. Moreover, it takes several gate the effect of inoculation with AM fungi (R. Campo et al. Rice (2020) 13:42 Page 3 of 14 irregularis, F. mosseae) in a panel of temperate rice culti- observations revealed the presence of all the events re- vars (japonica genotypes), focusing on plant growth and lated to fungal development in plants that had been in- blast resistance. Secondly, to investigate whether the AM oculated with either F. mosseae or R. irregularis. They symbiosis can improve grain yield in rice cultivars grown were: intraradical and extraradical hyphae, arbuscules at under flooded conditions in experimental fields. different morphological stages of formation, and vesicles (Fig. 3a). The overall colonization of rice roots inocu- Results lated with F. mosseae ranged from 46.1% (Gines) to Effect of AMF Inoculation on Growth of Rice Plants 17.8% (TN67) (Fig. 3b), whereas R. irregularis We conducted experiments to assess the phenotypic re- colonization was higher, ranging from 58.1% (Puntal) to sponse to inoculation by an AM fungus, Funneliformis 33.4% (TN67) (Fig. 3b). However, the percentage of mosseae and Rhizophagus irregularis, in a panel of tem- arbuscules in root fragments colonized by F. mosseae or perate japonica rice cultivars (see Additional file 1: R. irregularis was similar (Fig. 3c). Loto was the rice var- Figure S1a for the experimental design). Depending on iety with the maximum arbuscule abundance (F. mos- the rice variety, positive, neutral, or negative effects seae, 50.1%; R. irregularis, 55.4%) (Fig. 3c). could be observed in response to AMF inoculation, On the other hand, it is well known that the AM sym- which were evident as early as 3 weeks post-inoculation biosis activates carotenoid biosynthesis in several plant (wpi) with the AM fungus. Except for Galileo, inocula- species (Strack and Fester 2006). Increased pigmentation tion with F. mosseae stimulated growth in all the rice in roots represents a visual marker for AM symbiosis es- cultivars assayed in this study (Fig. 1). Inoculation with tablishment, including rice (Campos-Soriano et al. R. irregularis also stimulated growth in seven out of the 2010). In most species, AM fungi colonize the elongation 12 varieties assayed, namely Bomba, JSendra, Loto, zone of roots (Mathesius 2003; Gutjahr et al. 2009). As TN67, Guara, Maratelli, and Puntal (Fig. 1). Contrary to expected, a higher yellow-orange pigmentation was ob- this, inoculation with R. irregularis had a negative effect served in the elongation region of roots of plants that on the growth of Gleva and Gines, whereas Selenio, have been inoculated with either F. mosseae or R. irregu- Gigante Vercelli, and Galileo seemed to respond to in- laris, further supporting that the AM symbiosis is well oculation by R. irregularis only slightly (Fig. 1). Growth established (Fig. 3d). stimulation in the AM-responsive varieties was more evident 10 weeks after inoculation (Additional file 1: Fig- Effects of Inoculation with an AM Fungus on Grain Yield ure S1b). in the Field Altogether, this study revealed a differential growth re- We investigated whether grain yield was affected in AM- sponse to AM inoculation among rice cultivars, including inoculated rice varieties grown under field conditions. positive, negligible, and even negative growth responses. For this, the Loto and Gines cultivars were inoculated The growth response to AM inoculation among rice var- with the AM fungus R. irregularis and grown for 3 weeks ieties varied depending on the host genotype and the iden- under controlled greenhouse conditions to allow the es- tity of the AM fungus, F. mosseae or R. irregularis. tablishment of the AM symbiosis. Then, AM-inoculated and non-inoculated plants were transplanted into freshly Accumulation of Inorganic Phosphate in Leaves of AM- flooded paddy fields as described in Material and Inoculated Rice Varieties Methods (end of May, 2016), and allowed to grow for Root colonization by AM fungi improves plant nutrition the whole season (see Additional file 2: Figure S2 for the by increasing the availability and translocation of nutri- experimental design). The panicles were harvested ents, specifically phosphate (Pi). To assess the function- manually, and yield parameters were recorded. ality of the AM symbiosis, we analyzed the Pi content in Interestingly, yields of Loto and Gines plants inocu- leaves of mock-inoculated and AM-inoculated rice culti- lated with R. irregularis were 41.61% and 28.68% greater vars at 4 weeks after inoculation with either F. mosseae than non-inoculated plants, respectively (Fig. 4a). Also, or R. irregularis (see Additional file 1: Figure S1a for the the number of panicles per plant increased by 30.13% experimental design). Compared with the non- and 14.77% in mycorrhizal Loto and Gines rice varieties, inoculated plants, inoculation with one or another AMF respectively (Fig. 4b). However, the number of grains in increased the Pi level in leaves of all the rice varieties each panicle did not differ significantly between mycor- here assayed (Fig. 2). rhizal and non-mycorrhizal plants (only a slight increase in the number of seeds/panicle could be observed in Root Colonization by AM Fungi in Cultivated Rice mycorrhizal plants) (Fig. 4c). When measuring grain Varieties weight, no statistical differences were observed between To verify root colonization, we stained the roots of AM- non-inoculated and R. irregularis-inoculated plants from inoculated plants with cotton blue. Microscopic each variety (Fig. 4d). These results demonstrated that Campo et al. Rice (2020) 13:42 Page 4 of 14 Fig. 1 Growth of AM-inoculated rice varieties. Rice cultivars (temperate japonica genotypes) were inoculated with either F. mosseae or R. irregularis. Three weeks after inoculation with the AM fungus, plant height was measured (see Additional file 1: Figure S1a for the experimental design). Values in boxplots represent biological replicates (n = 12). Error bars indicate the first and third quartiles. The horizontal line within the box represents the median value (i.e., 50th percentile). Outliers are represented. Two independent experiments were carried out with similar results. Asterisks denote statistical differences (* P < 0.05, ANOVA test) inoculation with the AM fungus R. irregularis in the levels of root colonization by AM fungi, which together Loto and Gines rice varieties enhances grain production with a preferential colonization of large lateral roots in in the field, mainly by increasing the number of panicles. rice, might explain difficulties encountered for quantifi- These results also demonstrated that rice maintains cation of root colonization in field-grown rice plants. functional AM symbiosis when grown under flooded conditions. In this respect, it has been reported that in Resistance to Infection by the Rice Blast Fungus M. oryzae rice roots, large lateral roots mostly support AM in Mycorrhizal Rice Plants colonization, whereas fine lateral roots are not suscep- In this study, we examined the resistance of mycorrhizal tible to colonization by AM fungi (Gutjahr et al. 2009, rice plants to infection by the foliar pathogen M. oryzae. 2015; Vallino et al. 2014; Fiorilli et al. 2015). Although For this, the different rice varieties in the panel were in- root colonization was confirmed in AM-inoculated rice oculated with either F. mosseae or R. irregularis, or non- plants grown under controlled conditions, in our hands, inoculated and allowed to continue growth (controlled analysis of root colonization in field-grown rice plants greenhouse conditions). At 3 weeks after AM inocula- was proven to be difficult using standard staining tech- tion, the rice seedlings were challenged with M. oryzae niques (e.g. cotton-blue staining). It is also true that, spores (see Additional file 3: Figure S3 for the experi- compared with legume species, rice roots reach lower mental design). Disease symptoms were evaluated at Campo et al. Rice (2020) 13:42 Page 5 of 14 Fig. 2 Pi content in leaves of AM-inoculated rice varieties. The panel of temperate japonica rice cultivars was inoculated with AM fungi (F. mosseae or R. irregularis). Pi content was determined from leaf fragments collected from plants four weeks after inoculation with AMF (see Additional file 1: Figure S1a for the experimental design). Data are mean ± SE (n = 5, each biological replicate is a pool of 2 individual leaves). Asterisks denote statistical differences (*p < 0.05, ANOVA test; AMF-inoculated vs non-inoculated). Fm, Funneliformis mosseae; Ri, Rhizophagus irregularis different times after inoculation with M. oryzae spores. blast disease, as it is the case for the rice variety Visual inspection of mycorrhizal and non-mycorrhizal Maratelli. plants revealed a positive impact of AMF inoculation on blast resistance in most of the rice varieties here assayed, Discussion which was further confirmed by quantification of the leaf In this study, we report the effect of AM fungal inocula- area showing blast lesions (Fig. 5). In particular, Guara, tion on growth responses and blast resistance in a panel Bomba, Puntal, TN67, JSendra, and Gines that had been of elite rice varieties (japonica subspecies). A commer- inoculated with either F. mosseae or R. irregularis devel- cial mycorrhizal inoculum of F. mosseae or R. irregularis oped less and smaller blast lesions compared to the cor- was used in these studies. Growth responses and blast responding non-mycorrhizal plants (Fig. 5, upper panel). resistance were evaluated in plants grown under con- A reduction in disease symptoms was also observed in trolled greenhouse conditions. Importantly, the benefi- AM-inoculated Gleva, Loto, and Galileo plants, but dif- cial effects of AM inoculation of rice seedlings prevailed ferences were not statistically significant. until harvest in rice plants growing in the field under In the case of Gigante Vercelli, this variety is known to flooding conditions. Here, pre-inoculation with R. irregu- show a basal level of blast resistance (Urso et al. 2016). laris results in an increase in grain yield in mycorrhizal In agreement with this, in our study, the non- Loto and Gines plants compared with non-inoculated mycorrhizal Gigante Vercelli plants developed small nec- plants. rotic spots early during infection, but these necrotic Rice seedlings that had been inoculated with either F. spots did not progress with time. The mycorrhizal mosseae or R. irregularis grew better compared to non- Gigante Vercelli plant, however, neither exhibited nec- inoculated seedlings in most rice cultivars, a rotic areas in response to M. oryzae infection nor devel- phenomenon generally known as positive Mycorrhizal oped the typical blast lesions (Fig. 5). We also noticed Growth response (MGR) (Johnson and Graham 2013). It that the AM symbiosis increased blast susceptibility in is generally assumed that the mycorrhiza-induced stimu- Maratelli, a rice variety for which a high degree of sus- lation of plant growth is the consequence of improved ceptibility to M. oryzae infection has been described nutrient uptake, generally phosphorus, as the AMF hy- (e.g., in the absence of mycorrhizal root colonization) phal network allows access to a large soil surface area in (Couch et al. 2005) (Fig. 5). the mycorrhizal root. This results in an increased phos- Overall, results obtained in this study revealed that the phorus uptake through a mycorrhizal pathway of Pi ac- AM symbiosis confers protection to infection by the rice quisition (Smith and Smith 2011). However, our results blast fungus, the magnitude of reduction of disease demonstrated that there are rice varieties in which in- symptoms being dependent on the rice variety. However, oculation with one or another AM fungus results in the AM symbiosis might also have a negative effect on growth retardation suggesting genotype-specific re- sponses to AM fungi in rice. Growth inhibition in Campo et al. Rice (2020) 13:42 Page 6 of 14 Fig. 3 AM-colonization of temperate rice japonica rice cultivars. Roots from AM-inoculated temperate japonica rice cultivars were collected and analyzed (10 weeks post-inoculation). a Cotton blue staining of rice roots colonized by F. mosseae (Fm) or R. irregularis (Ri). The typical fungal structures were observed, such as extraradical hyphae (eh), intracellular hyphae (ih), arbuscules (a), vesicles (v), and spores (sp). Scale bar = 100 μm. b-c Estimation of AM fungal colonization according to the Trouvelot method b F%, frequency of mycorrhiza in the root system, and c a%, arbuscule abundance in mycorrhizal parts of root fragments. d Appearance of roots of rice plants inoculated with AM fungi. Representative pictures are presented. Increased pigmentation is observed in AMF-roots (grey bar) response to AM inoculation (negative MGR) has been differential growth response cannot be explained solely previously reported in different plant species (Grace by a higher supply of Pi in mycorrhizal rice plants. In et al. 2009; Verbruggen et al. 2012). This negative effect line with this, it has been reported that plant Pi concen- is usually attributed to carbon losses from the host plant tration does not always increase with negative or positive to the AM fungus with no subsequent gain in plant fit- MGR (Smith and Smith 2011). For example, significant ness from increased nutrient supply provided by the fun- differences in Pi uptake are found in Medicago trunca- gus (Grace et al. 2009; Johnson et al. 2015), particularly tula colonized by three different AMF, all of them con- under high Pi conditions (Peng et al. 1993). Another hy- ferring positive MGR (Lendenmann et al. 2011). Also, pothesis is that the suppression of the direct Pi uptake negative MGR with increased Pi content is observed in pathway in the host is not compensated by the mycor- Glomus intraradices-inoculated barley plants (Grace rhizal Pi uptake pathway (Smith and Smith 2011; Smith et al. 2009). The genetic basis of variations in AM- et al. 2011). Most probably, P acquisition in the various induced growth responses among host plant genotypes rice genotypes relies on the interplay between the direct is still not well understood. and mycorrhizal P uptake pathways, the contribution of Although rice can have blast in all growth stages, blast each pathway being dependent on the host genotype, the incidence gradually decreases with the aging of plants as fungal species and Pi availability. However, our results they develop adult plant resistance to leaf blast (Kim indicated that Pi level increased in varieties positively et al. 1987). Accordingly, in this work, the rice cultivars responding to AM inoculation, as well in non-responsive were screened for seedling blast resistance. Results ob- or negatively responsive cultivars. On this basis, a tained demonstrated that root colonization by R. Campo et al. Rice (2020) 13:42 Page 7 of 14 Fig. 4 (See legend on next page.) Campo et al. Rice (2020) 13:42 Page 8 of 14 (See figure on previous page.) Fig. 4 Effect of inoculation with R. irregularis on grain yield in field-grown rice plants. Rice plants (Loto and Gines) were grown in Ebro Delta (May–October 2016). For each variety, three independent plots (1 to 3) were designed for each condition (mycorrhizal: Ri, colored in green; non-mycorrhizal: -, colored in black) (see Additional file 2: Figure S2 for the experimental design). Grain yield parameters were measured. Twelve plants per condition in each parcel were analyzed and pooled. a Grain yield (grams) per plant. b Number of panicles in each plant. c Number of grains per panicle. d Grain weight. Measurements were done for 100 grains from plants from each variety and condition. Error bars represent the standard deviation for each condition irregularis or F. mosseae results in blast resistance to M. blast resistance in mycorrhizal rice plants must be evalu- oryzae infection in most of the japonica rice cultivars ated on a case-by-case basis. assayed, namely Guara, Bomba, Puntal, TN67, JSendra, On the other hand, it is well known that rice plants Gines, Gleva, Loto, Gigante Vercelli, and Galileo. Blast maintain better growth and produce higher yields when resistance in mycorrhizal plants occurs regardless of the grown in flooded conditions (paddy fields, anaerobic level of root colonization in the various rice varieties. In conditions), compared to plants grown in dry soil (aer- other studies, different levels of Glomus versiforme obic conditions). Results obtained in this work demon- colonization were observed in sorghum genotypes which strated that pre-inoculation with R. irregularis results in conferred either beneficial or detrimental effects to the a significant increase in grain yield in Loto and Gines host plant, regardless of the level of root colonization plants that were grown in the field under flooded condi- (Grace et al. 2009). Several pieces of evidence support tions. Here, it is worth mentioning that despite extensive that root colonization by AM fungi confers disease re- research on the effect of AM inoculation in plant species sistance in several plant species, but most of these stud- growing in dry soil, the benefits received by AM symbi- ies focused on resistance to root pathogens (Azcón- osis in plants under flooded conditions, in particular Aguilar and Barea 1997; Sikes et al. 2009; Jacott et al. rice, remain less explored. Previously, it was reported 2017; Spagnoletti et al. 2020). Different results are found that root colonization by AM fungi is reduced in rice in the literature on the impact of the AM symbiosis on plants grown under permanent flooded conditions foliar pathogens which might well depend on the iden- (Lumini et al. 2011; Vallino et al. 2014). Evidence also tity of the AM fungus, the host plant, and the interacting support that the functional capacities of the AM symbi- organism (Fritz et al. 2006; Pozo and Azcón-Aguilar osis are not affected by flooding (Maiti et al. 2011; Val- 2007; Jung et al. 2012; Pozo et al. 2013; Jacott et al. lino et al. 2014). Based on these findings, in this work, 2017). Along with this, increased resistance, as well as the rice seedlings were inoculated with the AM fungus increased susceptibility to leaf pathogens, were described and grown for 21 days under aerobic conditions prior to in mycorrhizal plants (Liu et al. 2007; Pozo and Azcón- transplant into flooded fields. That the rice seedlings Aguilar 2007; Fiorilli et al. 2011; Song et al. 2015; benefited from mycorrhizal colonization before trans- Sanchez-Bel et al. 2016; Chen et al. 2019). In other stud- planting was supported by the observed stimulation of ies, mycorrhizal rice plants were reported to be more plant growth and increased Pi content. Transplanted, susceptible to insect pests and infection by the sheath AM-inoculated and non-inoculated rice plants, were blight fungus Rhizoctonia solani (Bernaola et al. 2018b; then grown to maturity in paddy fields using conven- Bernaola and Stout 2019). Also in rice, we previously re- tional rice cultivation systems. Our results suggest that ported that inoculation with R. irregularis in the rice cul- AMF inoculation at the seedling stage was beneficial for tivar Senia reduces blast disease symptoms (Campos- cultivated rice varieties after transplanting to flooded Soriano et al. 2012). conditions in terms of productivity. Recent studies demonstrated that AM colonization in A unique characteristic of rice roots is the presence of roots of wild rice (Oryza rufipogon) was significantly large air spaces in mature roots, or aerenchyma, which higher than that in cultivated rice, the AM-inoculated provide an efficient air passage from shoots to roots wild rice being also more resistant to M. oryzae infection (Colmer 2003; Rebouillat et al. 2009). As oxygen is pro- than AM-inoculated cultivated rice (Tian et al. 2019). It vided by the aerenchyma in rice roots, the AM fungi that was proposed that the beneficial effects of the AM sym- had entered into rice roots before flooding would remain biosis might have been lost or reduced during rice do- viable in the roots in flooding conditions. Thus, although mestication. Results here presented indicate that flooding reduces the initiation of colonization (Lumini mycorrhizal-induced resistance (MIR) to infection et al. 2011; Vallino et al. 2014), once the fungus is estab- occurs in elite japonica varieties, thus, confirming the lished in the roots, it is able to maintain a functional re- potential of the AM symbiosis to improve blast resist- lationship with the host plant in flooded conditions. ance in cultivated rice. However, as there were also spe- This would explain why AM fungi are commonly cific rice varieties in which AM inoculation increased present in rice roots (Secilia and Bagyaraj 1994; Solai- blast susceptibility (e.g., Maratelli), the effectiveness on man and Hirata 1997; Zhang et al. 2016). Campo et al. Rice (2020) 13:42 Page 9 of 14 Fig. 5 Resistance to M. oryzae infection of AM-inoculated rice. Rice seedlings (one-week-old) were inoculated with an AM fungus: F. mosseae or R. irregularis. Three weeks later, the rice seedlings were infected with M. oryzae spores (see Additional file 3: Figure S3 for the experimental design). Pictures show blast symptoms in each variety at the following times after M. oryzae infection: 4 dpi, Loto and Guara; 5 dpi, Puntal, TN67, Gleva and Maratelli; 6 dpi, JSendra, Gigante Vercelli, Gines, Galileo and Bomba, and 7dpi, Selenio. Three independent experiments were carried out with similar results. Box plots represent the percentage of leaf area affected by blast lesions as determined by image analysis. Values obtained for each biological replicate were plotted (n = 10). Error bars represent the first and third quartiles. The horizontal line within the box represents the median value (i.e., 50th percentile). Asterisks denote statistical differences (*p < 0.05, ANOVA test). Outliers are included. Representative images of M. oryzae-infected leaves from mycorrhizal and non-mycorrhizal plants are shown Campo et al. Rice (2020) 13:42 Page 10 of 14 Although results here presented support that the of growth, blast resistance, and productivity is crucial to mycorrhizal symbiosis increases rice yield in Loto and exploit the AM symbiotic association in sustainable rice Gines varieties grown in paddy fields, this beneficial ef- farming through reducing inputs that have environmen- fect might depend on the stage of the fungus-root asso- tally negative impacts (i.e., fertilizers and pesticides). ciation when flooding occurred. From the perspective of Due to the observed differential responses among rice practical application, the rational use of AM fungi in rice cultivars, the evaluation of potential benefits received farming requires further investigation on how flooding from the AM symbiosis needs be done on a case-by-case might affect the establishment and functionality of the basis and in different environmental conditions. mycorrhizal association in rice. It will also be of interest to investigate whether seed inoculation with AM formu- Materials and Methods lations, followed by direct seeding onto dry soil, is effect- Plant Material ive for the development of sustainable rice production Rice temperate japonica rice (Oryza sativa) cultivars were systems while reducing water consumption. used in this study (named in the text as panel). They in- In summary, our study demonstrated the potential of cluded cultivars grown in Spain (Guara, J. Sendra, Puntal, using AM fungi for increased yield in the elite rice culti- Gleva, and Bomba), Italy (Loto, Gigante Vercelli, Maratelli, vars Loto and Gines using conventional farming systems Galileo, and Selenio) and France (Gines) (Courtois et al. in paddy fields. Further studies would be needed to de- 2012; Reig-Valiente et al. 2016). A Taiwanese cultivar termine the extent to which the AM symbiosis improves Tainung 67 (TN67) closely related to Japanese and Korean production in other rice cultivars and whether inocula- temperate japonica varieties (Kim et al. 2018) was also in- tions with an AM fungus could also be one way to pro- cluded in this panel. Rice plants were grown in the green- tect rice plants from the blast disease in the field. house under controlled conditions (14 h/10 h day/night Modern rice farming largely depends on the input of fer- cycle, 28 °C/25 °C, and 60% humidity). tilizers and pesticides to obtain maximum yields and to reduce losses due to pathogen infection. As a conse- Inoculation of AM Fungi and Growth of AMF-Inoculated quence, environmental problems have arisen in different Seedlings rice-growing areas due to excessive and inappropriate Rice seeds were dehusked, surface-sterilized twice with 5% use of fertilizers and pesticides. Results here presented sodium hypochlorite for 15 min, and washed with sterile open the possibility of using the AM symbiosis in rice water minimum five times. Seeds were germinated on cultivation, thus providing new opportunities to enhance petri dishes with sterile water for 7 days. Then, germinat- rice yield and to promote sustainable agriculture. ing seedlings were transplanted to 150 ml-cones (20.5 cm; 2 plants/cone) containing a mix of 63.3% quartz sand Conclusion (0.3–0.8 mm), 31.6% soil (turface and vermiculite 2:1), and In this study, we report the influence of inoculation with 5% of either a granular inoculum of Funneliformis mosseae arbuscular mycorrhizal fungi on growth and blast resist- (formerly Glomus mosseae; FR140) or Rhizophagus intrar- ance of japonica rice cultivars. Twelve elite rice varieties adices (formerly Glomus intraradices; FR121), both com- were examined. All rice varieties were susceptible to root mercially available (MycAgro; Bretenière, France; http:// colonization by AM fungi: F. mosseae or R. irregularis. www.mycagrolab.com/). This granular inoculum is com- However, a substantial variation occurred in the plant posed of mineral inert solid particles (clay, zeolite) and growth response to AM inoculation among the different propagules of AM fungi (e.g., spores, mycelium and cultivars, this differential response being dependent on mycorrhizal root pieces) at a concentration of minimum host genotype and identity of the fungus. Thus, positive, 10 propagules/gram of granular inoculum. No inoculum negative and neutral effects were observed on growth of was added to the substrate for the non-inoculated, control mycorrhizal rice plants upon inoculation with either F. plants. A plastic cover was used to maintain high humidity mosseae or R. irregularis. In most rice varieties, AM during a 9-day acclimatization period. Transplanted seed- symbiosis conferred protection to infection by the rice lings were bottom-watered and allowed to continue blast fungus Magnaporthe oryzae. Mycorrhiza-induced growth under controlled conditions (Additional file 4: Fig- resistance varied in the different rice genotypes. In the ure S4). After this period, the plastic cover was removed case of Maratelli, characterized as highly susceptible to and seedlings were top-fertilized with a modified Hoag- blast, AM inoculation was found to enhance blast sus- land half-strength solution (2.5 mM KNO3, 2.5 mM Ca ceptibility. Field experiments revealed that pre- (NO3)2·4H2O, 1 mM MgSO4·7H2O, 0.5 mM NH4NO3, inoculation with R. irregularis substantially increased 25 μM KH2PO4, 23.15 μM H3BO3, 4.55 μM MnCl2·4H2O, grain yield in Loto and Gines cultivars when grown 0.38 μM ZnSO4·7H2O, 0.1 μM CuSO4·5H2O, 0.14 μM under flooding conditions. Identifying the extent to Na2MoO4·2H2O, 26 μM Fe-EDDHA, pH 5.5) every 2 days which rice cultivars benefit from AM symbiosis in terms (15 ml solution/cone) (Sánchez-Sanuy et al. 2019). Campo et al. Rice (2020) 13:42 Page 11 of 14 Bottom-watering was continuously maintained. After conditions; Additional file 3: Figure S3). After 3 weeks, 3 weeks of AMF inoculation, seedlings were grown ac- seedlings of either AMF-inoculated or non-inoculated cording to the specific experiment (Additional file 4: were sprayed with a suspension of spores from the fun- Figure S4). gal pathogen M. oryzae (5 × 105 spores/ml; 0.4 ml/plant) using an aerograph (pressure, 2 atm) (Sánchez-Sanuy Field Experiments et al. 2019). The inoculated seedlings were maintained in Field experiments were carried out to evaluate the effect the dark overnight (at 90% humidity) and then allowed of inoculation with R. irregularis on grain yield. Experi- to continue growth under controlled conditions for the ments were carried out at the IRTA (Institute of Agri- required time (Additional file 3: Figure S3). The percent- food Research and Technology) Experimental Station, age of leaf area affected by blast lesions was determined Ebre Delta (Catalonia, NE Spain; May–October, 2016). using the ImageJ/Fiji v2.00 software (http://fiji.sc/Fiji). Each experiment included two conditions: R. irregularis- inoculated and non-inoculated rice plants. Inoculation Pi Content with R. irregularis and plant growth were carried out as Rice plants were AMF-inoculated or not, and grown described above (greenhouse, controlled conditions) under greenhouse conditions as described above (Add- (Additional file 2: Figure S2a). At 3 weeks after inocula- itional file 1: Figure S1a for the experimental design). The tion with the AM fungus, the rice seedlings were trans- youngest totally expanded leaves were harvested at 4 week planted in irrigated rice fields (Additional file 2: Figure after inoculation with one or another AM fungus. Five S2b). Three replications (plots) were performed, each biological replicates were analyzed, each one consisting in one containing non-inoculated and R. irregularis-inocu- a pool of two leaves obtained from two individual plants. lated plants for each variety (Loto, Gines). These plots Pi content of rice leaves was determined using a colori- were located in different experimental fields in which metric as previously described (Ames 1966). commercial varieties were also grown (Additional file 2: Figure S2b). For each rice variety and condition (R. irre- Statistical Analyses gularis-inoculated, non-inoculated), 12 seedlings were Means and standard errors were calculated using Micro- transplanted in each of the three plots with a 3-m spa- soft Excel. Significant differences among non-mycorrhizal cing. Thus, a total of 36 plants were grown for each var- and mycorrhizal plants were assessed using ANOVA test iety and condition. Rice was grown in flooded conditions (p-value ≤ 0·05). from 24 of May to 4 of October (2016). For evaluation of grain production, grains from the 12 plants of each Supplementary information individual plot, for each variety and condition were har- Supplementary information accompanies this paper at https://doi.org/10. vested and pooled. 1186/s12284-020-00402-7. Additional file 1: Figure S1. Experimental design used in this study for Analysis of Root Colonization Growth and Pi analyses. Root samples were collected at 10 weeks post- Additional file 2: Figure S2. Experimental design used in this study for inoculation with AM fungi, extensively washed with ster- Field Experiments. ile water, and analyzed for AM colonization with 0.1% Additional file 3: Figure S3. Experimental design used in this study for cotton blue in acid lactic as previously described (Berruti Blast resistance assays. et al. 2013). Roots were cut then into 1 cm fragments Additional file 4: Figure S4. Comparison of the different experimental designs used in this study. and mounted onto microscope slides. Estimation of mycorrhizal colonization was done from 75 root frag- ments (Trouvelot 1986). AMF structures were examined Abbreviations AM: Arbuscular mycorrhiza; AMF: Arbuscular mycorrhizal fungus; dpi: Days from cotton blue-stained roots an Axiophot Zeiss micro- post-infection; wpi: Weeks post-inoculation; Pi: Inorganic phosphate scope equipped with a Digital color camera (DP70 Olympus) and 40X magnification. Acknowledgements We thank members of the laboratory for assistance in the field. Blast Resistance Assays Authors’ Contributions The fungus M. oryzae (strain Guy-11) was grown on Conceived and designed experiments: SC, BSS. Performed experiments: SC, HM-C, MO, EP, MC-F, MM-E; Analyzed data: SC, HM-C. Wrote and revised Complete Media Agar (CMA) supplemented with chlor- manuscript: SC and BSS. All authors read and approved the final manuscript. amphenicol (30 mg/L) in Petri dishes (9 cm) for 15 days at 28 °C. M. oryzae spores were prepared as described Funding (Campo et al. 2013). Inoculation with AMF, and growth This research was supported by the FACCE-JPI project GreenRice (Sustainable and environmental friendly rice cultivation systems in Europe, ref. 618105), of non-inoculated or AMF-inoculated rice plants was funded by the Instituto Nacional de Investigación y Tecnología Agraria y Ali- done as described above (greenhouse, controlled mentaria (INIA), and project RTI2018–101275-B-I00 funded by the “Ministerio Campo et al. Rice (2020) 13:42 Page 12 of 14 de Ciencia, Innovación y Universidades (MCIU)-Agencia Estatal de Investiga- infection. Mol Plant Pathol 13:579–592 https://doi.org/10.1111/J.1364-3703. ción (AEI)” and “Fondo Europeo de Desarrollo Regional (FEDER).. We also ac- 2011.00773.X knowledge support from the CERCA Programme (“Generalitat de Catalunya”), Ceasar SA, Hodge A, Baker A, Baldwin SA (2014) Phosphate concentration and and the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016– arbuscular mycorrhizal colonisation influence the growth, yield and 2019 (SEV-2015-0533). expression of twelve PHT1 family phosphate transporters in foxtail millet (Setaria italica). PLoS One 9:e108459 https://doi.org/10.1371/journal.pone. Availability of Data and Materials 0108459 All data generated or analyzed during this study are included in this Chen Q, Wu W-W, Qi S-S, Cheng H, Li Q, Ran Q, Dai Z-C, Du D-L, Egan S, Thomas published article and its Additional files. T (2019) Arbuscular mycorrhizal fungi improve the growth and disease resistance of the invasive plant Wedelia trilobata. J Appl Microbiol https://doi. Ethics Approval and Consent to Participate org/10.1111/jam.14415 Not applicable. Choi J, Summers W, Paszkowski U (2018) Mechanisms underlying establishment of arbuscular mycorrhizal symbioses. Annu Rev Phytopathol 56:135–160 Consent for Publication https://doi.org/10.1146/annurev-phyto-080516-035521 Not applicable. Colmer TD (2003) Aerenchyma and an inducible barrier to radial oxygen loss facilitate root aeration in upland, paddy and deep-water rice (Oryza sativa L.). Competing Interests Ann Bot 91:301–309 https://doi.org/10.1093/aob/mcf114 The authors declare that they have no competing interests. Cornejo P, Seguel A, Aguilera P, Meier S, Larsen J, Borie F (2017) Arbuscular mycorrhizal fungi improve tolerance of agricultural plants to cope abiotic Author details stress conditions. In: Plant-microbe interactions in agro-ecological 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, perspectives. Springer Singapore, Singapore, pp 55–80 Campus Universitat Autònoma de Barcelona (UAB), Bellaterra (Cerdanyola del Couch BC, Fudal I, Lebrun MH, Tharreau D, Valent B (2005) Origins of host- Vallés), Barcelona, Spain. 2IRTA Institute of Agrifood Research and specific populations of the blast pathogen Magnaporthe oryzae in crop Technology, Field crops, Amposta, Spain. 3IRTA Institute of Agrifood Research domestication with subsequent expansion of pandemic clones on rice and and Technology, Marine and Continental Waters, Sant Carles de la Ràpita, weeds of rice. Genetics 170:613 Spain. 4Consejo Superior de Investigaciones Científicas, Barcelona, Spain. Courtois B, Frouin J, Greco R, Bruschi G, Droc G, Hamelin C, Ruiz M, Clément G, Evrard J-C, van Coppenole S, Katsantonis D, Oliveira M, Negrão S, Matos C, Received: 6 March 2020 Accepted: 11 June 2020 Cavigiolo S, Lupotto E, Piffanelli P, Ahmadi N (2012) Genetic diversity and population structure in a European collection of rice. 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