JXB Advance Access published online on October 8, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm181
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© 2007 The Author(s).
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RESEARCH PAPER |
Infection process and the interaction of rice roots with rhizobia
1ARC Centre of Excellence for Integrative Legume Research, Genomic Interactions Group, Research School of Biological Sciences, GPO Box 475, Canberra, ACT 2601, Australia
* To whom correspondence should be addressed. E-mail: rolfe{at}rsbs.anu.edu.au
Received 27 May 2007; Revised 9 July 2007 Accepted 13 July 2007
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Abstract |
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Most rhizobial strains inhibit rice root growth in the presence of calcium or potassium nitrates, but not ammonium nitrate. Certain rhizobial strains, however, such as strain R4, do not inhibit rice growth and can enter rice roots and multiply in the intercellular spaces. By using the green fluorescent protein (GFP) as a visual marker, it was found that Rhizobium became intimately associated with rice seedling roots within 24–48 h. During this initial period it was observed that strain R4 could cause structural changes resembling infection threads within the rice root hairs. Generally, the sites of the emerging lateral roots provide a temporary entry point for rhizobia, either by root hair entry or crack entry. All tested GFP-labelled Rhizobium strains infected the root hairs near the base of growing lateral roots. This study suggests that some strains may have the ability to infect rice root tissues via root hairs located at the emerging lateral roots and to spread extensively throughout the rice root.
Key words: Green fluorescent protein, infection, non-legumes, Rhizobium, Rhizobium–rice association, rice growth inhibition, short lateral roots
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Rice growth studies by Perrine and colleagues (Perrine et al., 2001, 2004, 2005; Perrine-Walker et al., 2007) demonstrated that the sequenced model micro-symbiont, Sinorhizobium meliloti 1021 could intimately infect and affect the growth of rice. By using GFP-labelled strains, plasmid-cured, and deleted derivatives of the wild-type strain Sm1021, it was determined that the pSymA plasmid of S. meliloti Sm1021 was largely responsible for rice growth inhibition (Perrine et al., 2004, 2005). Moreover, the pSymA-borne genes related to nitrate, nitrite, and/or IAA metabolism were most probably involved in rice growth inhibition (Perrine et al., 2004, 2005). The use of plasmid-cured derivatives of characterized wild-type Rhizobium strains showed that inhibition and stimulation of rice growth and development by rhizobia was plasmid-associated (Perrine et al., 2001), and it also highlighted the complex interaction between rhizobia, the growth medium, and rice plants (Perrine et al., 2001). Recently, a study of the plasmid profiles of Rhizobium etli strains, isolated from the maize rhizospheres, maize roots, and inside stem tissues found several different types of rhizobia (Rosenblueth and Martinez-Romero, 2004). Some of these strains from maize were more competitive at associating with maize roots than other strains and so, in traditional areas where maize is intercropped with beans, there appears to have been a selection of maize endophytes similar to the rice rhizobia endophytes (Yanni et al., 1997). Clearly, in soils and paddy fields there are a diverse group of micro-organisms (Liesak et al., 2000) which may be stimulatory, inhibitory, or have no effect on plant growth.
The rhizobia are also able to induce the formation of morphologically defined structures called nodules on legume roots. The rhizobia within these nodules can reduce atmospheric nitrogen to ammonia, which the plant can use for growth (Rolfe and Gresshoff, 1988). Agricultural scientists have long wanted to understand this symbiotic process, be able to manipulate it, and then transfer the system to the cereals and other non-leguminous plants. In the study of Rhizobium infection of Trifolium repens L. by Rhizobium leguminosarum bv. trifolii, Kodama (1992), recorded the events on videotape over a period of 9 d. The experiments recorded three alternative infection/entry processes, via root hairs, via wounds (cracks), and via the intact epidermis. The author concluded that, in addition to the generally described infection thread entry, there was a small percentage of what was called pseudo-infections. Studies by Chi et al. (2005) concluded that much remains unknown about the infection and colonization processes of the Rhizobium–rice association, particularly the primary portals of bacterial entry into the root. Many colonization studies with the non-legume Parasponia have shown that Rhizobium can enter the root tissue by crack entry via the erosion of epidermal cells (Bender et al., 1987; Rolfe and Gresshoff, 1988). Yet, in the case of the non-legume rice, colonization studies had produced evidence that Rhizobium could enter through cracks in the rice epidermis and the fissures created during the emergence of lateral roots (Reddy et al., 1997; Webster et al., 1997; Yanni et al., 1997; Prayitno et al., 1999). Prayitno et al. (1999) used Green Fluorescent Protein (GFP)-labelled rhizobia to demonstrate not only colonization of rice root tissue, but that certain strains of R. leguminosarum bv. trifolii could also multiply and migrate inside the growing lateral roots. The GFP detection via fluorescent microscopy was easy, inexpensive, and non-destructive, however, even after these studies the mechanism by which Rhizobium can enter rice root tissues was still not precisely known.
In this paper, the early stages of the Rhizobium association with rice plants have been investigated. Examination was then made of the infection in rice by GFP-labelled Rhizobium strains R4, ANU843, and E4 and compared to infection in berseem clover.
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Materials and methods |
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Bacterial strains and plasmids
Bacterial strains used in this study, R4, E4, ANU843 (control), and the GFP-labelled isolates of R4 and E4 (Table 1) are described by Perrine et al. (2005) and Perrine-Walker et al. (2007) (Table 1). Routine subculturing of rhizobia was done on BMM or TY plates at 28 °C. E. coli strains were subcultured on LB or TY plates at 37 °C. Antibiotics were incorporated into plates whenever rhizobia and E. coli strains carried a marker (Perrine et al., 2005, Perrine-Walker et al., 2007).
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Fluorescent labelling of Rhizobium strain ANU843
A GFP-encoding plasmid (pHC60) (Cheng and Walker, 1998) was introduced from a donor E. coli into the recipient Rhizobium strain, ANU843, by the triparental mating technique of Sinclair and Holloway (1982) as described in Perrine et al. (2001) (Table 1). The pHC60 plasmid is a spontaneous mutant of plasmid pHC41 carrying an insert that expresses GFP (GFP-S65Y) and containing an RK2 stabilization fragment; Tcr (Cheng and Walker, 1998).
Plant seed sterilization and germination
Seeds of berseem clover (Trifolium alexandrum L.) were soaked in 100% ethanol for 2 min then rinsed in sterile water. The seeds were surface-sterilized in 25% hypochlorite for 15 min. After being washed three times with sterile water, the seeds were germinated on BMM agar plates. Berseem clover seeds were directly incubated at 29 °C for 4 d. The plates were positioned vertically to allow the roots to grow down.
Rice seeds of cultivar Pelde and Calrose were de-husked, surface-sterilized, and germinated as described in Prayitno et al. (1999). The infection experiments described here are for cv. Pelde although cv. Calrose (data not shown) was essentially the same.
Nodulation test and plant growth conditions
To test the ability of Rhizobium strains to form nodules, T. alexandrum L. seedlings were inoculated with 5 µl of bacterial suspension (OD600 nm = 0.1) from overnight BMM liquid culture at 29 °C. Negative control plants were inoculated with 5 µl of sterile water or BMM liquid medium. A piece of folded aluminium foil was put on the edge of the Petri dishes to allow for better gas exchange and to prevent ethylene build-up. To reduce the dehydration of agar medium, the bottom of the Petri dishes was sealed with NescofilmTM. The plates were covered with a piece of brown paper to reduce the light intensity on the roots.
Following Rhizobium inoculation, the plates containing T. alexandrum L. seedlings were grown vertically in a growth cabinet set at 22 °C (16 h day) and 18 °C (8 h night) and photon flux density of 140 µmol m–2 s–1 as described in Mathesius et al. (2001). Plants were grown for 3–4 weeks before nodules were counted and nodules sectioned to view under light and fluorescent microscope to observe GFP-labelled bacteria.
Rice plant assay and rice plant growth conditions
Three-day-old rice seedlings, with root lengths ranging from 2.0 cm to 4.0 cm, were inoculated for 90 min in a 20 ml bacterial suspension as described in Perrine et al. (2001). The bacterial suspensions were made to an OD600 nm of 0.1 by suspending 3-d-old colonies from BMM plates into sterile water. The bacterial suspensions were diluted 1:20 in sterile water. This dilution was used to inoculate rice seedlings. Control plants were inoculated with sterile water for 90 min. The seedlings were then removed and transferred aseptically to Magenta jars containing 250 ml of growth medium, three or six seedlings per treatment per jar. Growth medium used was nitrogen-free liquid medium (Vincent, 1970) supplemented with 10 mM KNO3– (referred to as F10 medium). These conditions were designed to mimic a rice paddy field supplemented with nitrate-based fertilizer. Magenta jars were wrapped in a black plastic jacket to keep the roots in darkness. The plants were incubated in a growth chamber with a photon flux of 575–600 µmol m–2 s–1 using a 12 h 30/20 °C day/night cycle and 70% relative humidity. The rice plants were harvested at 7, 14, or 21 d after inoculation. Shoots were cut at the stem base and the dry mass was determined as described previously (Prayitno et al., 1999). Each experiment was repeated twice (unless otherwise stated) and each treatment was duplicated in a random block design.
Bacterial colonization studies in rice roots
Rhizobium strains, expressing the GFP marker, were screened for their ability to colonize the roots of rice grown in the glass slide system and in Magenta jars. For the study of bacterial entry into rice root over 7 d, the glass slide technique of Gage et al. (1996) was used with some modifications. The glass slide system involved the use of a glass slide with a cover slip using silicon sealant (Silastic® glue) instead of a dialysis membrane. The glass slide technique did not use agar that was overlaid on the glass slide as described by Gage et al. (1996). This allowed the liquid F10 medium to fill the space between the glass slide and the coverslip. Inoculated 3-d-old rice seedlings were placed between the glass slide and the glass cover slip, one seedling per glass slide. The slides were placed in Coplin jars (maximum of six slides) containing 90 ml of F10 liquid medium, the Coplin jars were covered with aluminium foil to keep the roots in the dark for the first 7 d. The jars were incubated under the same rice growth conditions mentioned above. Bacterial colonization was examined by placing the slide, holding the seedling, under a fluorescence microscope at time 0, 1, 2, 3, 6 4, 5, 6, and 7 d. After each examination, the slide was replaced in the Coplin jars and reincubated in the rice growth chamber.
For Magenta jar assays, rice seedlings of cv. Pelde were inoculated and transferred to Magenta jars containing F10 liquid medium and incubated in a growth cabinet using the rice growing conditions described above. Examination of inoculated rice roots was made at various times from 48 h on and a more precise analysis at 14 d and 21 d after inoculation bacterial colonization was assessed using fluorescence microscopy.
Fluorescence microscopy
A Nikon Optiphot inverted microscope stand (Nikon, Tokyo, Japan) with a 495 nm excitation 24 filter, a dichroic DM505 filter, and eyepiece-side absorption 515W filter slides was used for fluorescence microscopy. Images were taken on Fujichrome Provia 400 film (Fuji, Tokyo, Japan).
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Results |
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Colonization of young cv. Pelde rice seedlings in liquid F10 medium over 7 d by GFP-labelled R. leguminosarum bv. trifolii strains ANU843, E4, and R4
Earlier studies with strains R4, E4, and ANU843 had shown the bacteria to clump along the developing rice root, reside along grooves of the main roots and form micro-colonies on the root surfaces, often at the junction of a lateral root (Prayitno et al., 1999). Two sets of experiments were pursued to examine the early events of Rhizobium association with rice seedling roots. When strain R4 was inoculated onto seedlings grown in liquid medium under the glass slide assay, it was observed to enter into lateral roots of the rice seedlings and to form long lines of intercellularly dividing cells in the growing roots (Prayitno et al., 1999). In order to investigate strain R4 entry into rice roots, a more detailed examination of the Rhizobium-rice association was made over 7 d post-inoculation and the numbers of root hairs with associating and infecting rhizobia were recorded and estimated (see Materials and methods). Prayitno et al. (1999) had reported observing strain R4(GFP)-labelled cells attaching to rice root hairs after 2 h.
Ninety minutes after inoculation, GFP-labelled Rhizobium strains ANU843, E4, and R4 were observed on the surface of the first main root. At this stage no motility or clusters of GFP-1-labelled bacteria were observed in liquid culture or on the rice root surface. After 24 h, however, GFP-labelled rhizobia had adhered to the surface of the roots. Rice seedlings at 2 d had between 1 and 4 emerging lateral roots. Clusters of GFP-labelled rhizobia were observed on the surface of the rice roots. Motile GFP-labelled Rhizobium strain R4 cells were observed at the site of emerging primary laterals especially at the lateral root junctions, and at the main root tip. Interestingly, in some regions of a protruding lateral root, curled rice root hairs containing GFP-labelled bacteria was observed with R4(GFP)-treated rice plants (Fig. 1a–c). Under light microscopy, an internal structure resembling that of an infection thread that extended towards the base of the rice root hair was also observed. Whether these structures actually protruded into the base of the hair cell was not clear. GFP fluorescence was also confined to this infection thread-like structure and GFP-labelled bacteria were observed throughout the root hair. At some sites where the GFP-labelled bacteria were observed in root hairs, a spreading of fluorescence was observed below the infected root hairs at the base of the lateral roots (Fig. 2a–d). Whenever spreading bacterial cells were observed within rice roots they were always intercellular. Curled rice root hairs with infection thread-like structures containing GFP-labelled bacteria were observed at a frequency of 10–15% during the first 24–48 h after inoculation with strain R4(GFP) (Fig. 1h). It appeared that it was the root hairs near the site of emerging lateral roots that contained GFP-labelled bacteria (Fig. 1). While at other infection sites the GFP-labelled bacteria showed signs of spreading into rice root tissues forming intercellular lines (Figs 2, 3). By day 7, these formations of intercellular long lines of GFP-labelled bacteria were quite extensive in many roots and lateral roots (Figs 2, 3). Many of the long lines showed regions where the bacteria formed regions of clumps along the intercellular lines (Fig. 2). Some of the root hairs, which initially were infected with GFP-labelled bacteria, completely lost their fluorescence. In Fig. 2, lines of intercellular bacterial growth can be seen together with a very prominent cluster of concentrated multiplying bacteria. In addition, long lines of R4(GFP) intercellular growth down to the tip of a lateral root were also observed (Fig. 2d). In contrast to the strain R4 rice interactions, at 24 h, no motility was observed with GFP-labelled Rhizobium strains ANU843 and E4 around the base of the protruding primary lateral roots.
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After 48 h, all GFP-labelled rhizobia strains R4, ANU843, and E4 were observed within the root hairs near the base of the growing lateral roots on the first main root and starting to spread as intercellular lines of cells (Fig. 3). Occasionally, GFP-labelled bacteria were observed in root hairs in regions with no protruding lateral roots in proximity. Also epidermal cells were infected by GFP-labelled rhizobia. Thus, within the 24–48 h period following inoculation, the GFP-labelled rhizobia had initiated infection of rice root tissues.
At 72 h, GFP-labelled ANU843 infected more root hairs at the base of the lateral roots and along the first main root while apparently no new root hairs were infected with the GFP-labelled strain R4. With strain R4(GFP)-treated rice plants, some root hairs which had fluorescing bacteria had begun to lose fluorescence and this was more obvious by day 7 post-inoculation. At 96 h, additional lateral roots contained intercellular long lines of GFP-labelled Rhizobium R4, while it appeared that some GFP-labelled bacteria that had originally infected rice root hairs were not fluorescent. In some cases, the intercellular long lines of GFP-labelled bacteria were attached to the tip of the lateral roots or the first main root until 7 d. Strains ANU843(GFP) and E4(GFP) had infected more epidermal cells and the root hairs by day 7, but their intercellular lines of colonization were shorter (Fig. 3) and this correlated with the general dying off of the infected seedlings.
In the Magenta jar assays, root hairs containing GFP-labelled rhizobia R4, ANU843, E4, and SmA818 after 48 h were observed at a frequency of about 5% of root hairs, which was lower than found in the glass slide assays. Collectively, however, these observations suggest that Rhizobium strain entry into rice seedling roots is via a crack entry and perhaps also by structures resembling infection threads in root hairs near the newly emerging lateral roots. By 7 d it was clear that the R4-infected plants were growing very well, had about 12–13 emerging lateral roots, and were green with lots of lateral roots forming and this entry did not require a Sym-plasmid (Fig. 2) (Perrine et al., 2005). By contrast, the strain ANU843-infected and E4-infected plants were turning yellow and the majority of the lateral roots were stunted. These plants would eventually die off which is the finding with most Rhizobium–rice inoculations under these growth conditions. As internal structures resembling infection threads inside rice roots were observed, nodulation assays were done with clover seedlings to determine if these structures were similar to infection threads within the legume berseem clover. After 14 d, under clover growth conditions, GFP-labelled ANU843 and E4 formed pink nodules on the clover roots (Fig. 4f). Under fluorescence microscopy, GFP-labelled bacteria were found within infection threads inside curled roots hairs and within the sectioned nodule structure (60 mm thickness). This process differed in that the infection process observed in rice plants treated with GFP-labelled strain R4 were not confined to the structures resembling infection threads in legumes. By using light and fluorescence microscopy at the same time, such structures appeared to contain fluorescence i.e. GFP-labelled bacteria. Interestingly, strain R4 did not form nodules on 20 berseem clovers under clover growth conditions (20–25 °C; medium light intensity, photon flux of 140–160 µmol m–2 s–1 using a 12 h 30/20 °C day/night cycle and 70% relative humidity) (Fig. 4h). However, strain R4-inoculated berseem clover plants formed white nodules when grown under rice growth conditions (plants were incubated in a growth chamber with a photon flux of 575–600 µmol m–2 s–1 using a 12 h 30/20 °C day/night cycle and 70% relative humidity.) (Fig. 4i, j). Under the fluorescence microscope, GFP-labelled bacteria were observed only outside the nodule structure (Fig. 4j) suggesting that strain R4 is a Rhizobium strain that may have lost some ability to nodulate berseem clover under clover growth conditions and evolved to become a ‘rice-Rhizobium-specific strain’. In addition, the strain R4 had also lost its rice growth inhibition ability in contrast to the clover nodulating strains ANU843 and E4.
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It has been long observed that there are many similarities between lateral root and nodule development (Nutman, 1948) such as the induction of cell division from already differentiated cells resulting in a new meristem formation, organ differentiation, regulation by external factors (nitrate, phosphate), and induction by changing plant hormone balance. These observations have led to two obvious questions: is the nodule evolutionarily derived from the lateral root (Sprent, 1989; Hirsch and LaRue, 1997) and if the pathways for nodules and laterals are so similar why don't non-legumes nodulate? It was always thought that rhizobia did not enter non-legume root tissues in any substantial manner.
Infection of rice roots via root hairs
Evidence from these studies suggest that the first point of infection by GFP-labelled rhizobia strains R4, ANU843, and E4 is via root hairs and/or crack entry located near the newly emerging lateral roots after 48 h inoculation. No epidermal cells or root hairs of rice roots were found to contain any GFP-labelled ANU843 and E4 bacteria prior to 48 h. A feature of rice root hairs containing GFP-labelled bacteria is the presence of a structure similar to infection threads observed with ANU843(GFP)-inoculated Berseem clover. The presence of a curled rice root hair in rice seedlings inoculated with R4(GFP) suggests that some of the signalling pathway involved in nodule formation in legumes may still be present in rice. Reddy et al. (1997, 1998) and Zhu et al. (2006) have shown that ENOD genes, which are involved in the nodulation signalling pathway, are present in rice. In fact, Plazinski et al. (1985) were able to induce root hair curling in maize and rice plants by the transfer of the R. trifolii root hair curling (HAC) genes in pSym plasmid-cured derivatives of the R. leguminosarum bv. trifolii strain ANU843. This is quite interesting, as strain R4 is a Rhizobium strain that was observed to cause root hair curling and formed white nodules only under rice growing conditions. The motility of Rhizobium strain R4(GFP) at the emerging lateral roots and root tips of rice seedlings suggests the release of chemotactic compounds such as flavonoids being released from the rice plants. Flavonoids and plant exudates have been known to stimulate genes involved in chemotaxis in Rhizobium (Caetano-Anollés et al., 1988). Strain R4 induces the rice seedling roots to release flavonoid compounds around their roots after R4 inoculation (J Stefaniak, CH Hocart, BG Rolfe, personal communication). Nodules on the non-legume, Parasponia, and actinorhizal plants (e.g. Alnus spp.), originate from lateral roots (Hirsch and LaRue, 1997). Furthermore, in legumes, nodules have been shown to associate with lateral roots in the mature root zone (Nutman, 1948, 1953). Mathesius et al. (2000) demonstrated that rhizobia can induce nodules by invading mature cortical cells activated during lateral root development. This could possibly explain the rhizobia at the emerging lateral roots in non-legumes and in rice.
In root hairs of berseem clover, GFP-labelled bacteria were confined to the infection threads until they reached the primordium (Fig. 4). In rice roots, the whole root hairs were infected with GFP-labelled R4 or ANU843. One possibility in these cases is that the bacteria may attempt to form infection threads inside rice roots, but such structures are incomplete, allowing the GFP-labelled bacteria to escape and to invade the whole root hair and, subsequently, the intercellular spaces of the root. Root hairs in the mature root zone of clover can be responsive to rhizobia and could support infection thread formation (Mathesius et al., 2000). It has been speculated that nodulation fails in mature roots because of a deficiency in signal transduction (Mathesius et al., 2000). Gage (2004) also suggested that the plant cytoskeleton is likely to have an important role in growth of infection threads in legume root hairs as cytoplasmic streaming and nucleus positioning are closely connected with infection thread growth. The spreading of GFP-labelled bacteria from some of the infected root hairs into intercellular spaces of rice root tissues leading to the formation of short or long intercellular lines of GFP-labelled rhizobia were observed (Figs 2, 3). Genetic mutants and recent discoveries have revealed a number of the genes and proteins underlying the symbiotic signalling process in the model legumes M. truncatula and Lotus japonicus (Oldroyd and Downie, 2006). Further work using confocal microscopy and GFP labels will advance our understanding of rice infection by rhizobia.
Agricultural scientists have long wanted to understand the legume symbiotic process, so as to transfer the system to the cereals and other non-leguminous plants. However, this study suggests that some strains may have the ability to infect rice root tissues via root hairs located at the emerging lateral roots and to spread extensively throughout the rice root. Thus, the presence of non-inhibitory rhizobia in rice roots provides an opportunity to build an inoculum for the cereals delivering a biological nitrogen fixation system and other genetically modified products (Perrine-Walker et al., 2007). Strain R4 readily infects rice roots and is a candidate inoculant organism to deliver genetic material that may interact favourably with rice and perhaps other cereals. It is much easier to genetically engineer strain R4 to achieve a desired outcome than the host plant.
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Acknowledgements |
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FMP was an ANU Postgraduate Research Scholar. We also thank John Maindonald, Dr Jeff Wood, and Christine Donnelly (Statistical Consulting Unit, ANU) for their comments and discussions about the data presented in this paper. This work was partly supported by the Australian Research Council (ARC) through the award of an ARC Centre of Excellence for Integrative Legume Research (No. CEO348212).
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Abbreviations |
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BMM, Bergersen's modified medium; F10, Fåhraeus medium containing 10 mM KNO3–; GFP, green fluorescent protein; IAA, indole acetic acid; LB, Luria–Bertani medium; LSD, least significant difference; TY, tryptone yeast medium.
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References |
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