Seed bacterial endophytes: Common genera, seed-to-seed variability and their possible role in plants

Seeds contain endophytic bacteria that may be transmitted from generation to generation. Some of these bacteria can benefit plant growth and defense against abiotic and biotic stresses. Little is known however about the mechanisms of bacterial colonization of seeds and their transmission from generation to generation in host plants. In this study we have demonstrated that individual seeds of maize (Zea mays L.) taken from the same cob and bean seeds (Phaseolus vulgaris L.) from different pods and within individual pods differ in their bacterial content and population diversity. We suggest that this bacterial variability within seed population of individual plants may contribute to the species adaptation to diverse environments and be harnessed in the production of crop plants. INTRODUCTION Bacteria and fungi can be vertically transmitted in plants by seeds (Mundt and Hinkle, 1976; Baker and Smith, 1966). Thus one would expect natural selection to favor host plants that tightly control the kind and number of microbes that migrate into the developing seeds. Although diverse bacterial species have been found as endophytes colonizing different plant tissues, some genera are more frequently recovered as plant or seed endophytes than others (Cankar et al., 2005; Rosenblueth and Martinez-Romero, 2006; Compant et al., 2008; Hardoim et al., 2008; Mano and Morisaki, 2008; Ulrich et al., 2008; Mastretta et al., 2009; Puente et al., 2009). With a novel strategy of stable-isotope-probing of 13C-DNA containing endophytes that were recovered from plants fed with 13CO2 Rasche et al. (2009) detected Acinetobacter. A new species of Cohnella was also obtained from Phaseolus coccineus as a nodule endophyte (Garcia-Fraile et al., 2008), and a new species of Paenibacillus was isolated from surface-sterilized seeds of peas Pisum sativum (Smerda et al., 2005). Similarly, we identified several novel lineages among bacteria in bean seeds corresponding to a number of genera (Bacillus, Enterococcus, Nocardioides, Knoellia, Acinetobacter, Rhizobium, Phyllobacterium, Paracoccus and Sphingomonas) within phyla Firmicutes, Actinobacteria, and alpha, gamma and beta Proteobacteria (LópezLópez et al., 2010). A novel Rhizobium species, R. endophyticum, was also identified and found not to be symbiotic (López-López et al., 2010). The non-symbiotic rhizobia were also found in the seed interior and symbiotic R. etli on the seed coats (Perez-Ramírez et al., 1998). Symbiotic rhizobia were also found as natural maize endophytes (GutiérrezZamora and Martínez-Romero, 2001). In this study we report endophytic bacteria of individual seeds extracted from maize (Zea mays) and bean (Phaseolus vulgaris L.) plants. Both bean and maize are suitable for studies on seed endophytes as their seeds are large and can easily be extracted from surface disinfected pods or cobs. MATERIALS AND METHODS Seeds from Phaseolus vulgaris cultivars DOR 364 and BAT 477 were surface disinfected, placed in 5 lt pots containing sterilized vermiculite, irrigated with free N Proc. XXVIIIth IHC – IS on Envtl. , Edaphic & Gen. Factors Affecting Plants, Seeds and Turfgrass Eds.: G.E. Welbaum et al. Acta Hort. 938, ISHS 2012 40 Fahraeus solution (Fahraeus, 1957) or water and maintained in the greenhouse for 75 days. Bean pods that were 4 to 7 cm long (average 6 cm) were surface disinfected and immature seeds (6-8 mm) were detached, individually macerated, and seed extract were diluted and plated as described in López-López et al. (2010). Physiological mature corn-cobs of maize varieties Criollo (creole) San Gregorio and Criollo de Amatlán were supplied by INIFAP (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias) Campo Experimental Uruapan and by Verónica Pérez Escalante respectively. Cultivar San Gregorio was grown in the central highlands of Michoacán, Mexico (2400 masl and 800-1000mm precipitation) in April 2008 during the rainy season in an Andisol soil fertilized with 40 kg P2O5 ha-1 as calcium super phosphate and 60 kgNha-1 as urea at seeding stage, and 60 kgN ha-1 at silking stage. Cultivar Criollo de Amatlán was grown in Amatlán, Morelos (1750 masl and 1200-1400mm precipitation) in June 2008 in an Andisol soil fertilized with 60 kg P2O5 ha-1 as calcium super phosphate and 60 kgNha-1 as urea at seeding stage, and 80 kgN ha-1 at silking stage. Maize kernels were detached from corn-cobs and surface disinfected as described (Pereira et al. 2011), germinated in plates with agar 0.7% during two days at 28°C in darkness (five kernels per plate) and subsequently placed in slants in tubes (one kernel per tube) with agar 0.7% with Fahraeus medium (Fahraeus, 1957). They were kept under gnotobiotic conditions (sterile and hermetically closed). Tubes with plantlets were incubated for with 12 h photoperiod and at 28 ± 2°C until sampling. The lower part of the tubes was covered with aluminum foil to protect roots from light. Maize seedlings were individually macerated with MgSO4 10mM at two days after germination, as well as roots from seven and fifteen days-old seedlings. Extract dilutions were plated as for beans. Colonies were grown and bacteria identified by 16S rRNA gene sequences as described in López-López et al. (2010); some isolates were only distinguished by their different morphology (morphotypes, Table 2). Additionally, seed-borne maize endophytes were identified using a culture-independent approach. Root macerates from five-day-old plantlets of Criollo de Amatlán were fractionated on a Percoll gradient as described in Pereira et al. (2011). DNA was extracted from the bacterial fractions followed by PCR of 16S rRNA gene. The primers for these gene were design specifically to amplify DNA from bacteria and not from maize chloroplasts or mitochondria (F522 5’CGTGCCAGCAGCCGCGGTAATA and R1231 5’-CATTGTAGCACGTGTGTAGCCC). Cloning and sequencing of PCR products was performed as described (López-López et al., 2010). A potential inhibition of the growth of bacterial colonies by Bacillus spp. on culture plates was seemingly avoided by plating high dilutions (up to 10-4) of seed extracts to obtain few and wellseparated colonies. RESULTS AND DISCUSSION Seed-to-Seed Variability By a culture-dependent approach we detected seed-to-seed variability of bacterial endophytes obtained from surface-disinfected bean pods and maze cobs (Table 1). There was not a single bean or maize seed without bacteria, albeit at low numbers. One to 55 CFU were detected in each bean seed. Total endophytes from maize kernels were around 101-2 CFU/g at day two, to 105-8 CFU/g at day seven. Each seed contained either multiple or single species. Even seeds from a single pod or cob harbored different bacteria (Tables 1 and 2). In maize we found almost as high a variability of bacterial endophytes between kernels from the same cob as between kernels from different cobs (Table 2). This was also true for seeds in a single bean pod (Table 1). It is not known how seed endophytic diversity is determined; is the bacterial transfer process stochastic or selective? Do abundant bacteria compete with others and find their way to seeds more frequently? Bacteria from the genera Bacillus and Paenibacillus were abundant in maize kernels two days after germination. Seven days after germination, the variability both in each kernel and between kernels was higher. At this stage we also found Methylo-