Effect of segregation and genetic exchange on arbuscular mycorrhizal fungi in colonization of roots

Arbuscular mycorrhizal fungi (AMF) are abundant soil organisms and form symbioses with roots of the majority of terrestrial plants (Smith & Read, 2008). The symbiosis with AMF can promote plant productivity and diversity, and tolerance to pathogens and to herbivores (Newsham et al., 1995; van der Heijden et al., 1998; Bennett et al., 2006; Bennett & Bever, 2007). The hyphae produced by spores are coenocytic, harbouring many nuclei in a common cytoplasm. Moreover, genetic differences among co-occurring nuclei have been observed and this explains the high intra-individual genetic diversity found in AMF (Pringle et al., 2000; Clapp et al., 2001; Kuhn et al., 2001; Rodriguez et al., 2004; Hijri & Sanders, 2005). Two processes related to the within-individual genetic variation in AMF have been recently demonstrated in the species Glomus intraradices and can affect the nucleotype content of an AMF in a very short time span (Croll et al., 2009; Angelard et al., 2010). First, genetic exchange between two genetically different AMF lines can lead to new spores having a mixture of parental nucleotypes (Croll et al., 2009). Second, one mother spore can produce new spores with different nucleotype contents as a result of the segregation of nucleotypes at spore formation (Angelard et al., 2010). It has recently been shown that progeny obtained through genetic exchange and segregation (called crossed AMF and segregated AMF, respectively) can differentially alter plant growth and plant gene transcription compared with their parents or other progeny (Angelard et al., 2010). The plant genes studied were previously shown to be specifically expressed in the symbiosis at both early and late stages of fungal development (Gutjahr et al., 2008). Additionally, the alterations in plant growth caused by different AMF lines were not fixed in that they differed among different plant species (Angelard et al., 2010). So far, the effects of genetic exchange on fungi have been conducted using an in vitro system, and crossed lines can have different phenotypes compared with their parents or other progeny (Croll et al., 2009). Croll et al. (2009) only measured extra-radical hyphae and spores produced outside the roots in in vitro cultured fungal lines. However, nothing is currently known about the effect of segregation on the fungal phenotypes, or about how genetic exchange and segregation affect the colonization and development of the fungus inside plant roots. G. intraradices form three structures inside plant roots, namely hyphae, arbuscules (where the plant and the fungus exchange nutrients) and vesicles (propagules and storage organs) (Smith & Read, 2008). In the present study, we determined the effect of genetic exchange and segregation in G. intraradices on the development and growth of the fungus in plant roots. Our analysis was based on the glasshouse experiments described by Angelard et al. (2010), where the author investigated the effects of these two mechanisms on plant growth and plant gene transcription. Angelard et al. (2010) inoculated two plant species, Plantago lanceolata and Oryza sativa, with AMF lines of G. intraradices in two independent experiments. In the genetic-exchange experiment, plants were inoculated with parental and crossed lines. The segregated lines were obtained by cultivating separately single spores from crossed lines, and both the crossed and segregated lines were used in the segregation experiment of Angelard et al. (2010). Ten replicates were made in individual pots for each treatment (a combination of one AMF line with one plant species) in individual pots. Here, we determined the fungal growth of all of these AMF lines by measuring the proportion of hyphae, arbuscules and vesicles formed inside plant roots using the method of McGonigle et al. (1990). The methods are fully described in the Supporting Information Notes S1. Based on previous results, we hypothesized that crossed and segregated AMF lines would have different fungal traits inside plant roots compared with their parents, even at an early stage of colonization. Moreover, we hypothesized that the phenotypic changes among AMF lines would differ depending on the plant species. Our results show that both genetic exchange and segregation can result in fungi that colonize plants differently compared with their parents or other offspring (1, 2). For example, the hyphal colonization of the crossed line Sc2 on P. lanceolata was significantly different compared with the hyphal colonization of both parents (Fig. 1). Also, the crossed line, Sb, exhibited vesicular colonization on P. lanceolata that was significantly different from that in either of the parents (Fig. 1). Several segregated lines also had different phenotypes compared with other segregated lines and compared with their respective crossed lines (see, for example, the colonization of the segregated line S4c inside roots of P. lanceolata, Fig. 2). Therefore, the main conclusion of our study was that the two processes by which AMF can genetically change, namely genetic exchange and segregation, affect how the fungus develops inside the host. Fungal colonization in the genetic exchange experiment. Mean hyphal, arbuscular and vesicular colonization of Oryza sativa and Plantago lanceolata by parental lines (closed columns) and crossed lines (open columns). Arrows indicate the phenotypic (white arrows) and genetic (black arrows) similarity between crossed and parental lines (only shown when fungal colonization was different among arbuscular mycorrhizal fungi (AMF) lines). For example, vesicular colonization in P. lanceolata by crossed lines S1, S3 and S5 was most similar to colonization by parental line C2. Conversely, those crossed lines were genetically more similar to parental line C3 than to parental line C2 (as determined by amplified fragment length polymorphism analysis (AFLP); Angelard et al., 2010). Error bars represent the SD, and different letters above bars indicate a significant difference (P < 0.05) according to the Tukey–Kramer Honestly Significant Difference (HSD) test. Fungal colonization in the segregation experiment. Mean hyphal, arbuscular and vesicular colonization of Oryza sativa and Plantago lanceolata by crossed lines (closed columns) and segregated lines (open columns). Error bars represent the SD, and different letters above bars indicate a significant difference (P < 0.05) according to the Tukey–Kramer Honestly Significant Difference (HSD) test. In addition to the effect of the AMF line reported here, we found a host species effect on the growth of AMF (Tables 1 and 2). Moreover, this host effect was not the same for the different growth traits measured. Indeed, for both genetic exchange and segregation experiments, significant results show that AMF made overall more arbuscules and hyphae inside roots of O. sativa than inside roots of P. lanceolata, while we found the opposite for vesicles. Additionally, the changes in the different fungal growth traits among AMF lines were affected by the plant species, but these responses were not the same in each AMF line (shown by a significant AMF line by plant species interaction; Tables 1, 2 and 1, 2). For example, in the segregation experiment, arbuscular colonization of the segregated line S4b was higher inside roots of O. sativa than inside roots of P. lanceolata, whereas the difference in arbuscular colonization between the two hosts was not as large for the crossed line S4 (Fig. 2). Intriguingly, in the genetic-exchange experiment, these AMF line by plant species interactions can result in crossed lines growing similarly to one parent in one host, but growing similarly to the other parent in the other host. This is represented in Fig. 1 by the different direction of the white arrows showing the phenotypic similarity between parental and crossed lines. For example, most crossed lines between C3 and D1 made similar amounts of arbuscular colonization as the parental line D1 in O. sativa, but arbuscular colonization in P. lanceolata was similar to that in the other parent C3 (Fig. 1). This emphasizes that the crossed lines indeed grew in symbiosis in a different way to the parents. Overall, these results stress the importance of the genetics of the fungus on the potential interactions that can occur in symbiosis with different host species. From the studies of Croll et al. (2009) and Angelard et al. (2010), we know that all the crossed AMF lines used here were genetically more related to the parental line C3 (represented by the black arrows in Fig. 1). However, depending on both the trait measured and the host species, the crossed lines did not necessarily grow like the parental line C3 (shown by the different direction of the white and black arrows). The relationship between the genetic relatedness and the phenotypes of the segregated lines compared with their parent are more complex and difficult to analyse. Indeed, the variations in phenotypes among segregated lines originating from the same parent were mostly larger and occurred more frequently than variation among crossed lines. A potential explanation could be that genetic exchange is a less random phenomenon than segregation because of the constraints that can emerge from mixing genetically different nuclei. Consequently, the panel of new progeny obtained with different symbiotic characteristics would be larger through segregation than through genetic exchange. Combined with the results of Angelard et al. (2010) on the dry weight of the plants, we found significant, positive correlations between rice dry weight and arbuscular colonization, for both the genetic exchange and the segregation experiments (Pearson correlation coefficient r = 0.46, P < 0.001 and r = 0.17, P = 0.027, respectively). However, we found significant negative correlations between the dry weight of P. lanceolata and arbuscular colonization, for both the genetic exchange and the segregation experiments (r = −0.22, P = 0.047 and r = −0.42, P < 0.001, respectively). The correlations were similar with the other fungal colonization traits (data not shown). These results stress again the importance of the interaction between host species and AMF. However, the measures of the fungal colonization and the plant dry weight were made only once, at the end of the experiment. A time course of fungal colonization would be more accurate in order to make conclusions about the potential effect of fungal colonization on plant growth (or vice versa) as, indeed, such interactions could change through time. Angelard et al. (2010) conducted two independent experiments (genetic exchange and segregation experiments). Several controlled (the amount of soil and the amount of watering) and uncontrolled (such as temperature and humidity) parameters were not the same between the two experiments. This can explain the quantitative differences in colonization between the experiments found here. However, the parameters were standardized within each experiment with considerable replication, and statistical analyses have been performed, allowing us to compare the treatments within each experiment and to state accurately which treatments had a significant effect. Nevertheless, it is interesting to note that similar patterns (concerning fungal growth and the correlation between plant and fungal growth) have been found for both experiments. A previous study has shown that genetic exchange in AMF can lead to progeny having different phenotypes (spore and hyphal density) in in vitro culture systems compared with their parents and compared with each other (Croll et al., 2009). Here, we investigated the effect of both genetic exchange and segregation in AMF on the development and growth of the fungus in roots of non-transformed plants in glasshouse conditions. Our results show that the two processes can alter the pattern of development of fungus in the roots. Moreover, the fungal development was also affected by different plant species. We previously knew that genetic exchange and segregation can lead to progeny that differentially alter plant growth (Angelard et al., 2010). Combined with those results, our findings suggest that specific interactions could occur between different plant species and different AMF genotypes, and that the specificities could appear in the initial weeks of the establishment of the symbiosis. Genetic exchange and segregation could be two mechanisms, owing to the particular genetic structure of AMF, that can create new progeny with different symbiotic effects in a very short time span and that can adapt rapidly to different environmental conditions, such as different plant species. This work was supported by grants from the Swiss National Science Foundation (grant numbers 31000AO-105790/1 and 31003A-127371). Notes S1 Supporting methods provide a full description of the materials and methods used in the study. Table S1 Results of the Shapiro–Wilk tests (W) performed to test the null hypothesis that the data were normally distributed Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.