Characterization of Plant Growth Promoting Rhizobacteria Isolated from the Rhizosphere of Peruvian Highlands Native Crops

Over the past few decades, research in agricultural microbiology has highlighted the efficacy of plant growth promoting rhizobacteria (PGPR) in inducing seedling emergence, promoting the increase in plant height, weight and overall crop yield. A number of microbial isolates have shown promising antagonistic activity against several soilborne pathogens. In this study, twelve bacterial strains were isolated from root rhizosphere of Peruvian native highlands crops, i.e Goldenberry (Physalis peruviana L.), Potato (Solanum tuberosum L.) and Maca (Lepidum meyenii Walp.). The Short Communication Ogata-Gutiérrez et al.; IJPSS, 11(1): 1-8, 2016; Article no.IJPSS.24573 2 strains were identified using 16S rRNA gene PCR amplification and sequencing and were characterized for their PGPR activities. Among these isolates, all strains were found to be able to produce indol-acetic acid, two strains were able to solubilize hydroxyapatite, biand tri-calcium phosphate and the remaining others were able to solubilize at least one source of inorganic phosphate. Of 12 isolates, 10 strains showed antagonistic activity against Fusarium solani, 5 showed activity against Alternaria alternata and 9 inhibited growth of Curvularia lunata. It was also found that out of 12 isolates, three were able to induce tomato seedling emergence by 75% compared to the control in in-vitro assays. 16S rRNA gene sequence based analysis of these strains confirmed that, of 12 strains, 4 were members of genus Pseudomonas, 3 belong to genus Bacillus, 2 were related to genus Rahnella and 1 each belong to the genus Stenotrophomonas, Serratia and Streptomyces.


INTRODUCTION
Peru is an important centre for a number of genetically diverse indigenous crops now cultivated across the world. A number of these native Andean plants form an important part of the routine diet of a significant proportion of population, although their true potential has not been fully realised. In the recent time, these Peruvian native Andean crops are getting recognised more and more across the world not only for their nutritional value, but also for their medicinal properties which has lead to an increase in demand of these crops in the international markets. However, international markets impose high standards for agricultural products concerning to food safety and quality and so restrict import, sales and consumption of products with high content of chemicals pesticide, fungicide and fertilizer residues. In the current scenario, the microbes inhabitating the root rhizosphere of the native crops with antagonistic activity against a variety of phytopathogens and with the ability to promote plant growth provide a better environment and health friendly alternatives to these chemical fertilizers and fungicides. A considerable number of bacterial species from the rhizosphere have been isolated and their efficiency to improve plant growth has been assessed [1]. A few sudies have also reported that the efficacy of PGPR depends on the type of crop, climate and soil composition [2]. To expand our knowledge about the useful microbial species associated to Andean crops, the present investigation was directed towards use of microbes isolated from Peruvian highlands soils exhibiting traits associated with plant growth promoting ability, assessed under in vitro conditions. Tomato plants are known to be susceptible to several phytopathogenic fungi, which in turn compel farmers to extensively use chemical products to get rid of them as well as for improving crop yield. Microbial inoculants discussed here are environmentally friendly alternative for chemical fungicides and fertilizers against some of fungal diseases of tomato plants.

MATERIALS AND METHODS
Twelve microbial strains were used in this study. These strains were isolated from the root rhizosphere of three Andean crops, i.e Goldenberry (Physalis peruviana L.) and Maca (Lepidium meyenii Walp.) from Junin and Potato (Solanum tuberosum L.) from Puno [3], located in the highlands of Peru over 3300 meters above sea level. Soils at the collection sites had a high content of organic matter and were moderately acidic, with pH between 4.5 and 5.5. All strains were maintained in nutrient broth (NB) plus 25% (v/v) glycerol at -80°C. Strains were Gramstained [4]. Colony morphology: size, color, shape, texture, height and edge were recorded after 48 h of growth on nutrient agar plates at 28°C [modified for 5 ] Phosphate solubilisation assays were performed as described by Nautiyal et al. [6] and formation of a solubilisation halo was observed up to 20 days. Indolacetic acid (IAA) production by these isolates was determined using the method described by Glickmann and Dessaux [7] and appearance of pink colour was considered to be indicative of IAA production. All the strains were also screened for antagonistic activity against phytopathogenic fungi Fusarium solani, Alternaria alternata and Curvularia lunata. Antagonistic activity was tested through the dual culture technique and fungal growth inhibition was quantified as described by Rahman et al. [8]. The ability of the strains to promote the emergence of seedling after microbial inoculation was also tested. For this assay, seeds of Solanum lycopersicum var. Río Grande were used. Bacterial strains were grown to obtain a population of 10 8 cfu/ml. The culture was resuspended in 10 ml of saline solution 0.85%. The surface-sterilized tomato seeds were then soaked in the bacterial suspension for 15 minutes. After that, the seeds were sown in plastic containers using sterile sand as substrate. The plants were irrigated daily with hydroponic solution A and B (provided by Centro de Investigación de Hidroponía y Nutrición Mineral in UNALM, Peru). Inoculated seeds were placed in a greenhouse with controlled temperature (20-25°C) and light cycle (12 hours/day) for 7 days. The assays were performed using 4 replicates per bacterial strain and container. Results were recorded as emergence percentage, when the first two leaves appeared out of the sand. Seeds not inoculated were used as controls. For the molecular identification of the strains isolated, total DNA was extracted from overnight grown cultures broth using AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences,Union city, CA, USA) following the manufacturer's instructions. Primers fD1 and rD1 were used to PCR amplification of the 16S rRNA gene [9]. Phylogenetic neighbours of the isolates were identified using the EzTaxon web server (http: //www. ezbiocloud.net/eztaxon) [10]. The ClustalX2 software [11] was used to align both studied and selected sequences. Phylogenetic analysis was performed by the neighbor-joining (NJ) method and distances were calculated according to the Kimura-2 method using the MEGA Version 6 [12].

RESULTS AND DISCUSSION
Four out of the twelve isolates were Grampositive rods. One of them (Aa9) showed a filamentous morphology with a branching growth pattern characteristic of actinobacteria [13], while other isolates showed the ability to resist high temperatures (80°C for 30 min). Eight strains were Gram negative. Five of them were diazotrophic bacteria since they were capable to growth in mineral medium (MM) without nitrogen and to acidify it [14]. Other three isolates were classified into pseudomonad group, as they were capable to grow in cetrimide agar and showed fluorescence in Pseudomonas Agar F (DIFCO) [15]. The colony morphology of each group was different. Diazotrophic strains showed translucent colonies with entire edge, sizes between 0.5 to 1.5 mm and convex. As well, pseudomonads showed sizes between 0.5 and 1 mm,umbonated elevation and were cream or iridescent. Bacillus colonies were bigger than the other two bacterial groups with sizes between 1 and 3 mm. They appeared opaque, mucoid with entire edge and umbonated elevation. The actinomycete on the other hand, showed punctiform colonies with undulate edges and yellow pigment.
The majority of the isolates showed one or more PGPR attribute. All strains were able to produce auxin in the range of 8.1 -67.5 ppm in the presence of IAA precursor tryptophan. Strain Ba60 produced the maximum IAA level (67.5 ppm) followed by two diazotrophic isolates, Azo16M2 and LMTZ064-66 (Table 1). However, most of the pseudomonads produced low amounts of IAA compared to the other bacterial groups, showing values between 7 and 10 ppm. Agaras et al (2015) [16] reported similar values of this phytohormone in different species of pseudomonads isolates and their positive control P. putida GR12-22. Bacillus sp. and Streptomyces strains were documented with a production up til 76 ppm [17] and 5 ppm [18], respectively. IAA production by PGPR can vary among different species and it depends on culture conditions, growth stage and substrate availability [19]. Auxins produced by the PGPR strains play an important role in increasing the overall surface area of the plant root. The larger surface area of the roots enable the plant to absorb the water and other important nutrients more efficiently, which in turn leads to a better growth and development of the plant PGPR. All the isolates were able to solubilize at least one inorganic phosphate source tested in the assays. Two isolates (Da29 and Azo16M2) were capable of solubilizing hydroxyapatite, bi-and tri-calcium phosphate. Nine strains were capable to solubilize bicalcium phosphate with maximum halo sizes varying between one and 17.5 mm and two isolates (Ba60 and Ps42) could solubilize only hydroxyapatite (Table 1). It is well documented the fact that a number of microorganisms can make insoluble soil phosphorous available to plants by solubilizing mineral phosphates through the production of organic acids or phosphatases. The results obtained in the present study showed that most of the bacterial strains tested could solubilize bicalcium phosphate better than tricalcicum phosphate and hydroxyapatite. These results are similar to those obtained by Ben Farhat et al. [20], who report tricalcium phosphate and hydroxyapatite as bacterial inorganic phosphate sources, with a low rate of conversion than the bicalcium phosphate.
In antagonistic assays, strain Ba60 could inhibit the growth of Alternaria alternata only, while strains LMZ064-66 and Pa86 could inhibit the growth of only Fusarium solani. Azo16M2 and Ba8 were able to restrict the growth of both Fusarium solani and Alternaria alternata. The percenteges fungal growth inhibition for all the strains was greater than 20%. Azo16M2 and Ba8 isolates were also able to inhibit the growth of Curvularia lunata with percentages inhibition 0.7 and 5.2% respectively. Bacillus isolates and the diazotrophic bacteria Azo16M2 showed greater antagonistic activity against Alternaria alternata (Fig. 1). Ba8 have shown antagonistic activity against all the phytopathogenic fungi tested in our study. A number of species from the genus Bacillus are widely known to produce various antifungal and antibacterial secondary metabolites effective against a range of phytopathogenic fungi [21]. Some species like B. subtilis, B. amyloliquefaciens, B. pumilus and B. cereus are already being used as biological control agents against many soil-borne plant pathogens [8].
Based upon the previous results, five of the twelve isolates were further tested for their ability to induce tomato seedling emergence. The results obtained in the test clearly indicated a positive effect of the strains on the seedling emergence of tomato plants. Three out of 5 chosen strains (Ps42, Da29 and Aa25) significantly increased the seedling emergence of tomato (75%) compared to the non-inoculated control, that showed a level of 50% seedling emergence. Increase in the seed emergence due to the production and secretion of some phytohormones and enzymes by microbes can be used as an attribute for a primary rapid screening of PGPR strains [22]. An increase in the seedlings emergence may favor an early development of the efficient root system of the plant which is in itself an indicative of better plant health and growth.
The NJ phylogenetic tree (Fig. 2) showed that all the studied isolates were clustered in 6 groups and showed close affiliation with the genus Pseudomonas, Rhanella, Serratia, Stenotrophomonas, Bacillus and Streptomyces. Within Pseudomonas genus, the isolates Pa86 and LMTZ064-90 were clustered with P. azotoformans group, while Ps42 and Pa82 with P.syringae and P. putida groups respectively.  . Similarity values were over 99%. Further up to species level details could be inferred from the tree provided (Fig. 2) though the accurate identification for some of the strains in this study require phylogenetic analysis using a few more marker genes sequences.

CONCLUSIONS
The results obtained in this study clearly illustrate the potential of these isolates as PGPR under in vitro conditions. Though, it cannot be also ruled out that the interaction between PGPR and plants could have different results under field conditions. However, the present work is among the first few which provides primary information about the microbial strains with antagonistic and plant growth promoting activity from these Peruvian Andes region which has not been exploited yet up to its true potential. The study also creates a platform for the future field trial using the strains described in the present study as potential PGPRs.