Mycorrhizal colonisation and arbuscular content in AMF-rice japonica and indica rice cultivars
To assess the symbiotic compatibility between the three AMF genotypes (RIN, RIR, FM) and the six rice cultivars (listed in Table 1), we analysed the mycorrhization rates in the 18 combinations. We focused on the global fungal colonisation rate and the percentage of visible arbuscules in the mycorrhizal roots (see Material & Methods). The use of mycorrhizal index of global mycorrhization and arbuscular intensity allowed us to quantify the interaction between AMF and rice. The mean of each mycorrhizal index for each combination are presented in Fig. 1 and in Additional file 2: Table S2.
We tested whether rice cultivar and AMF genotype have an impact on rice’s global mycorrhization and arbuscular intensity index, with a two-way ANOVA test. Both indexes are statistically significantly affected by either rice cultivar or AMF genotype (Two-Way ANOVA, F = 66.91593, p < 2*10− 6; F = 4.9315, p < 0.01).
All the rice cultivars tested were root colonised by each AMF genotype (Fig. 1, Additional file 2: Table S2). Each fungal organ (hyphal structures, spores, vesicles and arbuscules) was clearly visible on each combination as shown in Fig. 2 and Additional file 3: Fig. S1. The global intensity of mycorrhization ranged from 27.20% (Phka Rumduol with RIN) to 83,90% (Nipponbare with RIR). Independently of the fungal inoculation, indica rice varieties have the less intense symbiotic percentage, ranging from 27.20% (Phka Rumduol - RIN) to 46.8% (IR64 - RIN) (Additional file 2: Table S2). The percentage of mycorrhization of the japonica cultivars ranges from 43.20% (Kitaake with FM) to 83.90% (Nipponbare with RIR). Nipponbare is the most intensely mycorrhized cultivar with 79%, 77.90% and 83.90% for FM, RIN and RIR inoculation, respectively (Additional file 2: Table S2).
The arbuscular percentage of the mycorrhizal roots ranged from 3.22% (Azucena with FM) to 49.40% (Nipponbare with RIR) (Additional file 2: Table S2). Japonica rice cultivars showed the highest percentage of visible arbuscules in the mycorrhized system compared to indica rice, with RIN and (Fig. 1). On the other hand, each rice genotype in interaction with FM formed almost no arbuscules, with a maximum of 5.24% in Zhonghua 11 (Additional file 2: Table S2).
Growth response of rice cultivars to AMF colonisation
The phenotypic response of rice to AMF inoculation and symbiosis establishment was assessed by growth measurements. Maximum height, fresh and dry shoot weight and fresh root weight were measured for each combination (n = 20) and are shown as boxplots in Fig. 3. All the corresponding measured values are listed in Additional file 2: Table S2.
Throughout the dataset, an increase, decrease or no significant effect of AMF inoculation can be observed. We observed a significant decrease in the height of IR64 during the interaction with FM or RIN (-18.81% and − 14.12% respectively, Fig. 3A and Additional file 2: Table S2). For the RIR genotype, we measured a significant increase both on root and shoot weights on Phka Rumduol, Kitaake, Zhonghua 11 and Nipponbare while non-significant on Azucena. The effect on rice’s dry weight wasn’t as significant as on height or fresh weight, but was still a good proxy for the beneficial effect of AMF on rice growth (Fig. 3).
Under our growth conditions, we observed different growth rates depending on the rice cultivar. Uninoculated Kitaake was the smallest rice cultivar both in size (24.65 cm) and weight (0.19 g, 0.11 g, 0.09 g on average for fresh shoot, root and dry shoot weight, respectively; Additional file 2: Table S2). Still, mycorrhization of Kitaake induced a clear improvement in growth: the highest dry weight being obtained in association with RIN (0.30 g, Fig. 3D, Additional file 2: Table S2). This combination showed the greatest positive effect on rice’s growth on all variables: 36% taller, 259%, 270% and 221% heavier on biomass of fresh shoots and roots, and dry shoots, respectively (Additional file 2: Table S2).
Globally, FM inoculation affected rice growth non-significantly (Nipponbare, Phka Rumduol, Zhonghua 11) or negatively (Azucena, IR64), in both height and weight (Fig. 3). The only significant positive interaction was with Kitaake: 20% taller, 163%, 177%, 125% heavier on its fresh shoot & root and dry shoot weights, respectively (Fig. 3, Additional file 2: Table S2). The effect of RIN inoculation was contrasting: beneficial on Kitaake and Zhonghua 11, negative on IR64 and Phka Rumduol or non-significant on Azucena and Nipponbare (Fig. 3). RIR was the AMF genotype that induce the most positive effects among all rice cultivars: +35 and + 20% leaf height with Kitaake and Phka Rumduol, respectively (Fig. 3A); +182 and 103% fresh shoot weight with Kitaake and Zhonghua 11, respectively (Fig. 3B) and + 196 and + 149% fresh root weight with Kitaake and Phka Rumduol, respectively (Fig. 3C).
Our results show that the effect of AMF inoculation on rice growth depends on both rice cultivars and AMF genotypes: ranging from negative, neutral to beneficial outcomes across the 18 combinations under study.
Mycorrhiza-induced resistance
The potential of each fungal inoculum to induce systemic resistance in the leaves of each rice cultivar during a shoot phytopathogen infection was investigated. Rice plants were infected by leaf-clipping with Xanthomonas oryzae pv oryzae (Xoo) strain PXO99 and the extent of chlorosis and necrosis was recorded 14 days later. The results are shown as boxplots in Fig. 4 and all the corresponding measured values are listed in Additional file 2: Table S2.
The effect of AMF inoculation on the chlorosis and necrosis symptoms of rice induced by Xoo PXO99 differed greatly between combinations. Only two combinations, both with RIN, showed a significant increase in leaf symptoms: Azucena on chlorosis (+ 69%) and Phka Rumduol on necrosis (+ 106%). Regarding the bio-protective effects of AMF, chlorosis symptoms were significantly reduced on IR64 in combination with FM (-24%) and RIR (-26%), on Zhonghua 11 in combination with FM (-44%), RIN (-28%) and RIR (-29%), and on Nipponbare with FM (-40%) and RIN (-34%) (Fig. 4 and Additional file 2: Table S2). For necrosis, only Zhonghua 11 and Nipponbare showed significant reductions of symptoms. These reductions in the size of necrosis were observed with the three AMF genotypes: -65%, -66% and − 64% for Zhonghua 11 with FM, RIN and RIR respectively; and − 78%, -87% and − 64% for Nipponbare with FM, RIN and RIR, respectively (Fig. 4 and Additional file 2: Table S2).
RT-qPCR analysis of growth and immunity molecular marker genes in contrasting rice-AMF combinations
We observed contrasted patterns of symbiotic compatibility among our AMF-cultivar combinations. In order to link these observed differences with the expression level of leaf marker genes, we selected two rice cultivars with contrasting AMF responses: Nipponbare and IR64. The first is a japonica model cultivar and was the most intensely mycorrhized, regardless of the AMF genotype, with the interaction having non-significant to beneficial effects on its growth and tolerance to Xoo infection (Fig. 1, Fig. 3 and Fig. 4). The latter is an indica model cultivar, that was significantly less mycorrhized, with non-significant to negative effects of the AMF interaction on its growth, but with beneficial effects on its tolerance to Xoo infection (Fig. 1, Fig. 3 and Fig. 4). We selected 22 markers genes of mycorrhization, development, nutrient homeostasis, hormonal balances and defence and their expression was normalised to that of EF1a reference gene. The list of marker genes, their function and the primers used in this study are listed in Table 2. A summary of the statistical comparison of gene expression for each combination, for both Nipponbare and IR64, is provided in Additional file 4: Table S3.
The expression of two AMF colonisation marker genes, OsAM3 coding for a putative LysM domain containing protein, and OsPT11 coding for an inorganic phosphate transporter, known to be induced in roots (Gutjahr et al., 2008) was recorded in leaves. In Nipponbare leaves, OsPT11 expression was significantly repressed whatever the AMF genotype, whereas no variation was observed for OsAM3 (Fig. 5). In IR64 leaves, the expression of these two genes wasn't significantly affected by AMF. (Fig. 5, Additional file 4: Table S3).
The expression of two cellular growth marker genes, OsYABBY6 and OsXTH17, was recorded. A non-significant induction of OsXTH17 expression was observed for Nipponbare in interaction with RIN (p = 0.055), while that of OsYABBY6 is repressed with FM (p = 0.009) (Fig. 5, Additional file 4: Table S3). In IR64, their expression was not significantly affected independently of the AMF genotype (Fig. 5, Additional file 4: Table S3).
To assess the effect of mycorrhization on mineral homeostasis in leaves, one iron transporter (OsIRO2), one nitrate-reductase (OsNIA1) and four Pi transporters (OsMGD2, OsPAP23, OsPT11 and OsSPX3) were selected. The expression of almost all mineral marker genes was significantly reduced in Nipponbare leaves (OsNIA1, OsMGD2, OsPAP23 with RIN, OsSPX3 with either AMF genotype), except for a non-significant strong induction of OsIRO2 expression when associated with RIN (p = 0,09) (Fig. 5, Additional file 4: Table S3). In leaves of IR64, the expression of OsMGD2 and OsPAP23 was not affected. The expression of the other mineral marker genes was reduced. This repression was significant only for OsSPX3 in RIN-mycorrhized leaves, and for OsNIA1 and OsIRO2 in FM-mycorrhized leaves (Fig. 5, Additional file 4: Table S3).
Mycorrhization is known to affect the hormonal balance in mycorrhized plants. Its effect on the expression of jasmonate (JA: OsJAMyb, OsJAZ6, OsLOX4), ethylene (ET: OsACS1) and salicylic acid (SA: OsNPR1, OsWRKY45) pathways was investigated. Overall, jasmonate- and ethylene-related genes expression was not significantly repressed in Nipponbare and IR64 leaves (Fig. 5, Additional file 4: Table S3). SA-related genes were not significantly repressed, except for OsNPR1 in RIN mycorrhized-IR64 leaves (Fig. 5, Additional file 4: Table S3).
The expression of defence-related genes was recorded to assess how mycorrhization affects the defence response in healthy leaves. Globally, defence genes appeared to be more induced in mycorrhized IR64 leaves than in Nipponbare leaves (Fig. 5). In Nipponbare leaves, we observed i) a significant repression of OsPR5 expression, irrespective of the AMF species, ii) a significant down-regulation of OsPAL4 expression with FM, iii) a non-significant down-regulation with RIN (Additional file 4: Table S3). In leaves of IR64, OsPBZ1 and phytoalexin biosynthesis-related genes (OsDXS3 and OsTGAP1) were not significantly induced in plant associated with AMF. mycorrhized with RIR, the expression of OsWRKY30 is significantly induced but not the one of OsMPK10 (p-value = 0.03 and 0.07, respectively). (Fig. 5, Additional file 4: Table S3).