Isolation and functional characterization of bacterial endophytes from Carica papaya fruits

Authors


Correspondence

Puja Ravikumar, Vittal Mallya Scientific Research Foundation, 94/3 & 94/5, 23rd cross, 29th main, BTM II Stage, Bangalore 560076, India. E-mail: puja@vmsrf.org

Abstract

Aims

To isolate and characterize the endophytes from papaya fruits and to determine the fermentative potential of the strains.

Methods and Results

Endophytes provide potential sources for novel natural products for the use in agriculture and nutrition. There is very limited information on isolation and characterization of bacterial endophytes from papaya. We describe isolation and characterization of eighteen endophytes of papaya fruit from four economically important papaya varieties viz ‘Red lady’, ‘Solo’, ‘Coorg Honey’ and ‘Bangalore’. The phylogenetic analysis based on the 16S rRNA sequence revealed that isolated endophytes are genetically distinct and cluster as discrete clades in the dendrogram. The Bacillus species is a predominant bacterial endophyte across papaya varieties. The seeds and the endocarp of papaya fruits harbour Kocuria, Acinetobacter and Enterobacter species. The Staphylococcus species were detected in the fruit mesocarp of two papaya varieties used in the study. The endophytes isolated from papaya fruits were capable of producing extracellular enzymes like amylase, cellulase, pectinase and xylanase. Three isolates, Bacillus (PE-LR-1 and PE-LR-3) and Kocuria (PE-LR-2), were selected for fruit fermentation, and antioxidant potential of the fermented product was evaluated. PE-LR-3 fermented product has the free radical scavenging activity of 61·2% and a microbial cocktail of PE-LR-3 with Saccharomyces cerevisiae MTCC 2918 enhances the antioxidant potential to 75·7%.

Conclusion

These findings suggest that different parts of papaya fruits harbour an array of bacterial endophytes that could be important agents in attributing the high nutritive status to the fruit and can serve as potent microbial cocktails for developing value-added fermented products of this important fruit.

Significance and Impact of study

This study describes isolation of a bacterial endophyte from papaya fruit that is capable of improving the antioxidant potential of raw papaya after fermentation.

Introduction

Papaya (Carica papaya L.) is a fruit crop widely grown in tropical and subtropical environments. There has recently been increased interest in the study of the papaya genome because of its small genome size of 372 Mb and its short life cycle as compared to many other tropical fruit tree crops. The complete genome sequencing of Hawaiian papaya is in progress, and a draft genome sequence of a transgenic (red) papaya has already been published (Ming et al. 2008). In addition, there is sizeable information on the nutritive importance of papaya fruit, and the high carotene and lycopene content has been implicated in imparting the antioxidant potential to the fruit. However, the bacterial endophytes associated with papaya fruit are yet to be elucidated and functionally characterized. The endophytic bacteria are detected inside surface-sterilized plants or extracted from inside plants and have no visibly harmful effects on the plants (Hallmann et al. 1997). Many endophytes have bestowed on their host plants their beneficial potential like producing several secondary metabolites and protecting the plants from disease attacks. Endophytic bacteria in a single plant host are not restricted to a single species but comprise several genera and species. Endophytic bacteria are found in roots, stems, leaves, seeds, fruits, tubers, ovules and also inside legume nodules (Hallmann et al. 1997; Sturz et al. 1997; Benhizia et al. 2004). In most plants, roots have higher numbers of endophytes compared with above-ground tissues (Rosenblueth and Romero 2004). Endophytic bacteria have been studied mainly after culturing in laboratory media, but a more complete scheme is emerging, using methods that do not require the bacteria to be cultured and that make use of the analysis of sequences from bacterial genes obtained from DNA isolated from inside plant tissues (Chelius and Triplett 2000; Engelhard et al. 2000; Sessitsch et al. 2002; Reiter et al. 2003; Miyamoto et al. 2004). The functional characterization of the endophytes provides vital information about the contribution of the endophytes to the nutritional properties of the plants. Papaya is nutritionally rich in antioxidants like beta carotene, vitamins like B, folate and pantothenic acid, and minerals like magnesium and potassium. Fermentation of the fruit using selected microbes can help retain its nutrition, add value to the fruit and prevent severe postharvest losses. Papaya fruit has been used as a nutritional substrate for biomass production of Saccharomyces cerevisiae for single cell protein production (Ojokoh and Uzeh 2005) and for wine production (Maragatham and Panneerselvam 2011). Papaya, a sugar crop, possessing many saccharides like glucose, fructose and sucrose, harbours numerous endophytes, and studying the fruit endophytes can provide a few important clues about the characteristic biochemical features of the fruit. This study was initiated to isolate, characterize and correlate the presence of endophytes in different parts of papaya fruit. This study includes isolation of endophytic bacteria from four different varieties of papaya viz. ‘Red lady’, ‘Solo’, ‘Coorg Honey’ and ‘Bangalore’ followed by their characterization based on Gram staining, 16S rRNA sequence analysis, production of extracellular enzymes and their ability to ferment papaya fruit.

Materials and methods

Fruit material

Four agronomically important ruling varieties of papaya were used in this study. Three varieties – Red Lady, Solo and Coorg Honey – were obtained from Lalbagh Horticulture Department, Bangalore, and Bangalore variety maintained and cultivated by local farmers was used. The variety Red Lady has oblong moderate-sized (weighing about 1·2 kg) fruits with red pulp; Solo variety has small-sized, round-shaped fruits (weighing about 0·51 kg) with orange pulp; and Coorg Honey has oblong-shaped fruits (weighing about 0·52 kg) with yellow pulp. The endophytes were isolated from mesocarp, endocarp and seeds of ripe papaya fruits. The fruits with soft yellow pulp were considered ripe. These regions were selected for isolating the endophytes as we were specifically exploring for microbes with good fermentative potential. The fruits were transported to the laboratory immediately after the harvest.

Isolation of endophytic bacteria

The ripe fruits were surface sterilized to prevent the interference of epiphytic bacteria and other contaminants by washing them with sterile water, wiping dry and disinfected using 95% ethanol. The fruits were peeled with a sterilizing knife. The mesocarp, endocarp and seeds of the fruit were cut and scraped respectively using sterilized knife to 1 cm2 area. The tissues of the fruit were mashed with sterile water using autoclaved mortar and pestle. One millilitre of tissue suspension from different fruit parts was pour plated with Luria–Bertani Agar (Hi Media Labs, Mumbai, India) and incubated at 27°C for 2 days. The colonies obtained were subcultured and maintained for further investigation.

Morphological characterization

The morphological characterization was carried out by recording colony characters and staining. The colonies obtained were further subcultured by quadrant streaking to obtain isolated colonies to study the morphological characters based on shape, margin, colour and consistency of the colonies followed by Gram staining. The pure culture from isolated colonies was used for Gram staining. The KOH String test was also carried out using a drop of 3% potassium hydroxide and a bacterial colony on a glass slide. This method serves as a valuable adjunct to the traditional Gram staining (Graevenitz and Bucher 1983).

Molecular characterization of bacterial endophytes

The molecular characterization was carried out by amplification, sequencing and analysis of conserved 16S rRNA region. The overnight grown cultures of all the isolates were used for DNA extraction. The total genomic DNA was extracted as described by Sambrook et al. (1989), and the quality of DNA was checked by gel electrophoresis on a 1·2% agarose gel. The 1·5 kb full length 16S rRNA gene from each of the isolate was amplified using 16S universal primers (synthesized by Sigma Aldrich, Bangalore, India). The sequence of 16S forward and reverse primer was (5′-AGAGTTTGATCCTGGCTCAG-3′) and (5′-AAGGAGGTGATCCAGCCGCA-3′), respectively. The PCRs were performed in a 25-μl reaction volume that comprised 30 ng of genomic DNA, 2·5 μl of 10 × Taq A buffer, 2·5 μl of 2·5 mmol l−1 dNTPs, 0·5 μl of 16S primers and 0·3 μl of Taq polymerase (New England Biolabs, Gurgaon, India). The volume was made up with sterile Milli Q water. A negative control without DNA was included in each run. The cycling parameters were 2 min at 92°C for initial denaturation, 35 cycles each of 1 min at 92°C of denaturation, 30 s at 48°C for annealing, 2 min 10 s at 72°C for extension and a final extension at 72°C for 6 min. The 1·5 kb amplified PCR products were resolved by electrophoresis in a 1·2% agarose gel followed by staining with ethidium bromide (10 mg ml−1), and the amplicons were visualized under a UV transilluminator (UviPro, Wolflabs, UK). The gel section with desired band was carefully excised under UV light and eluted using Gel Extraction Kit (Axygen, CA, USA), as per manufacturer's instructions. The PCR products were sequenced (Eurofins Genomics India Pvt. Ltd, Bangalore, India) and analysed using BlastN. The sequences were submitted in the GenBank. Sequences obtained were aligned using Clustal W, and neighbour-joining tree (NJ tree) was constructed.

Enzyme activity

The isolates were inoculated on starch agar, casein agar, xylan agar, carboxymethyl cellulose (CMC) agar and pectin agar media for detecting the production of amylase, protease, xylanase, cellulase and pectinase, respectively, by agar well diffusion method (Vaca Ruiz et al. 2009). Substrate media plates were prepared by incorporating 1% of each of the substrates in Luria–Bertani agar. Wells of diameter 5 mm were bored using cork borer on the solidified media, and the culture suspensions (100 μl of 0·5 OD) were added into the wells along with Bacillus licheniformis MTCC 6824 as control. The plates were incubated for 48 h at 27°C and observed for zones of clearance around the wells considering a minimum zone of 15 mm as positive reaction.

Amylase and pectinase were detected by flooding the plates with iodine–potassium iodide solution, and cellulase and xylanase enzymes were detected by flooding the plates with Congo Red solution (1%) for 15 min followed by destaining with sodium chloride (1 mol l−1) for 10 min. Observation of protease production was based on formation of clear zones around the wells on casein agar.

Shake flask fermentation

‘Solo’ variety was chosen for fermentation experiment after evaluating the % free radical scavenging activity in all the papaya varieties used in study (data not shown). To check for the fermentative ability, three isolates Bacillus species (PE-LR-1 and PE-LR-3) and Kocuria species (PE-LR-2) were selected for shake flask fermentation of the ‘Solo’ fruits. S. cerevisiae MTCC 2918 was used as control for fermentation. Bacillus species (PE-LR-1 and PE-LR-3) were selected for shake flask fermentation as they produce Bacillithiol and Kocuria species (PE-LR-2) for the nitrate reductase activity that can enhance the flavour. The Staphylococcus species isolated from papaya fruit were not selected for use in fermentation experiments because of potential pathogenicity problems. The papaya fruit extract was prepared by using a raw papaya of ‘Solo’ variety. The fruit was washed with sterile Milli Q water several times followed by wiping the surface with 95% ethanol. The outer skin was peeled using a sterile knife. The seeds and placenta were removed from the sliced pulp and rinsed with sterile water. Approximately, 500 g of sliced papaya was pulverized into slurry using a mixer. The fruit extract was obtained from slurry filtered with the use of muslin cloth as described by Ojokoh and Uzeh (2005). The extract was dispensed in to sterile 250 ml conical flasks and plugged with sterile cotton wool and pasteurized for 45 min at 85°C. On cooling the medium, each flask received 150 ml of 1 : 1 ratio of papaya extract and sterile mineral salt medium (100 ml of mineral salt medium contains 0·6 g KH2PO4, 1·4 g K2HPO4, 0·04 g MgSO4.7H2O, 0·2 g (NH4)2SO4, 0·4 g NaCl, 0·3 g glycine and 1% glucose). The medium and 0·1% (v/v) trace elements solution (0·02% MnSO4.2H2O, 0·05% FeCl3, 0·001% FeSO4, 0·1% CaCl2.2H2O, 0·02% CoCl2.2H2O, 0·001% H3BO4, 0·01% ZnSO4, 0·02% CuCl2 and 0·001% NaMoO4) were sterilized by autoclaving at 15 psi at 121°C for 20 min, and sterile 0·1% (v/v) trace element solution was added to the medium just prior to inoculation of the fermenting organisms. All the flasks were inoculated with 5% of overnight grown fresh culture of the different isolates. The fermentation was carried out at 30°C for 31 h with mild shaking at 150 rev min−1. The fermentation process was terminated after 31 h as thereafter we observed a decline in antioxidant activity. The pH of the medium was maintained between 5·5 and 6·5 for the isolates (Camacho-Ruiz et al. 2003; Narendranath and Power 2005; Manikkandan et al. 2009). The antioxidant potential of the endophyte-fermented papaya product was compared with S. cerevisiae-fermented papaya product. S. cerevisiae was used as standard for comparing based on the documented literature on yeast fermentation of papaya fruit (Ojokoh and Uzeh 2005).

Microbial cocktail fermentation

PE-LR-3 was used in a microbial cocktail fermentation along with S. cerevisiae in two different ratios of the microbial cultures viz. PE-LR-3/S. cerevisiae (1 : 1) and PE-LR-3/S. cerevisiae (2 : 1). The cocktail fermentation was taken up as it has been reported that synergism between microbes leads to improved process potential. (Takagi et al. 2010; Zhou et al. 2011). PE-LR-3 was chosen for use in microbial cocktail fermentation as PE-LR-3 fermentation improved the antioxidant potential of the product, and the optimal pH for PE-LR-3 and S. cerevisiae is identical. The ratios of the inocula were taken based on the OD of the cultures at 600 nm. All the flasks were incubated for 31 h at 30°C with mild shaking at 150 rev min−1. The antioxidant activity was evaluated at intervals of 19, 23, 27 and 31 h by DPPH (2,2-diphenyl-1, 1-picrylhydrazyl) method. These time points were chosen for sampling as the increase in the antioxidant activity was observed after 20 h of fermentation.

Measurement of DPPH radical scavenging activity

The antioxidant activity of the fermented product was assessed by quenching of a stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and is represented as % radical scavenging activity (Aoshima et al. 2004). The DPPH (Sigma Aldrich) reagent was prepared at a concentration of 1 mg ml−1 in methanol (Merck, Mumbai, India). To 2 ml of methanol as solvent, 100 μl of DPPH reagent solution and 50 μl aliquot of fermented filtrate were added, mixed and incubated at room temperature for 20 min. The remnant DPPH was quantified by measuring absorbance at 517 nm. Methanol was used as blank and methanol with DPPH reagent as control. The degree of DPPH quenching that directly correlates with the antioxidant activity was calculated using the following formula; Radical scavenging activity (%) = 100(A‒B)/A, where A and B are 517 nm absorption of the control and test, respectively.

Statistical analysis

The mean values of % free radical scavenging activities exhibited by endophytic bacterial isolates Bacillus species (PE-LR-1 and PE-LR-3) and Kocuria species (PE-LR-2) individually and in microbial cocktail fermentation using S. cerevisiae and Bacillus species (PE-LR-3) were obtained from triplicate experiments and are presented as mean ± SE.

Results

Phenotypic characterization of endophytic bacteria

A total of 18 endophytic bacteria (ranging from 1 × 103 to 2·5 × 103 CFU g−1 of tissue) were isolated from mesocarp, endocarp and seeds of different varieties of papaya fruit. The edible mesocarp of papaya fruit comprised of 72%, seeds 21% and endocarp 7% of total endophytes isolated. Five bacterial endophytes from Red Lady variety; five isolates from Bangalore variety, seven isolates from Solo and one from Coorg Honey variety were isolated. The bacterial isolates were identified morphologically and by molecular methods. Pigment production was not observed in the isolates. The details of the colony characteristics are summarized in Table 1. Majority of the isolates were Gram-positive cocci (PE-2, PE-3, PE-LR-2, SV-1, SV-2, SV-3 and SV-4). The isolates PE-1, PE-4, PE-Lg-1, PE-LR-1, PE-LR-4, CH1, SS1 and SS3 were Gram-positive rods, while PE-5 and SP1 were Gram-negative coccobacilli.

Table 1. Morphological characterization of endophytic bacterial isolates isolated from different regions of papaya fruits of four cultivated varieties
VarietyIsolateTissue sourceColony characteristics
ShapeMarginColourConsistencyGram character
Red LadyPE-1MesocarpIrregularWrinkledOff whiteSlimyGram-positive rods
PE-2MesocarpCircularEntireYellowSlimyGram-positive cocci
PE-3MesocarpCircularEntireYellowSlimyGram-positive cocci
PE-4MesocarpCircularEntireOff whiteSlimyGram-positive rods (endospores)
PE-5EndocarpCircularEntireWhiteButyrousGram-negative coccobacilli
BangalorePE-Lg-1MesocarpIrregularIrregularWhiteRoughGram-positive rods (endospores)
PE-LR-1SeedIrregularIrregularWhiteRoughGram-positive rods (endospores)
PE-LR-2SeedCircularEntireYellowSlimyGram-positive cocci clusters and tetrads
PE-LR-3SeedCircularEntireOff whiteSlimyGram variable Spindle rods
PE-LR-4MesocarpIrregularIrregularOff whiteSlimyGram-positive rods (endospores)
SoloSV-1MesocarpIrregularUndulateBeigeButyrousGram-positive cocci (clusters)
SV-2MesocarpCircularEntireOff whiteSlimyGram-positive cocci (clusters)
SV-3MesocarpIrregularUndulateWhiteSlimyGram-positive cocci (clusters)
SV-4MesocarpCircularEntireWhiteSlimyGram-positive cocci (clusters)
SP1MesocarpCircularEntireYellowSlimyGram-negative coccobacilli
SS1SeedIrregularIrregularWhiteButyrousGram-positive rods (endospores)
SS3SeedCircularEntireOff whiteSlimyGram-negative rods
Coorg HoneyCH1MesocarpIrregularIrregularWhiteButyrousGram-positive rods

Enzyme activity

Amylase activity was detected in Gram negative and variable PE-5 and SP-1 as well as Gram-positive rods like PE-LR-3 and PE-LR-4. Pectinase activity was observed in six isolates, and xylanase and cellulase activity was observed in four isolates (Table 2). PE-1, PE-5, PE-LR-2, PE-LR-3, PE-LR-4 and SP-1 produced pectinase (Fig. 1). Xylanase activity was detected only in Gram-positive/variable rods isolated from the Bangalore variety and CH-1 from Coorg Honey variety. Cellulase production was observed in PE-1, PE-LR-1, PE-LR-4 and CH-1, whereas the PE-2, PE-3, PE-4, SV-1, SV-2, SV-3, SV-4, SS-1 and SS-3 did not show any enzymatic activity. Protease production was absent in all the isolates.

Table 2. Production of enzymes by papaya isolates by agar well diffusion method
IsolatesProteaseAmylasePectinaseCellulaseXylanase
PE-1++
PE-2
PE-3
PE-4
PE-5++
PE-Lg-1
PE-LR-1++
PE-LR-2+
PE-LR-3+++
PE-LR-4++++
SV-1
SV-2
SV-3
SV-4
SP-1++
SS-1
SS-3
CH-1++
Figure 1.

(a) Starch agar plate flooded with Iodine – Potassium iodide solution (Isolate PE-5 with highest zone of clearance), (b) Xylan agar plate flooded with Congo Red solution followed by destaining with sodium chloride (1 mol l−1) (Isolates PE-LR-3 and PE-LR-4 showed greater zones of clearance as compared to control), (c) carboxymethyl cellulose agar plate flooded with Congo Red solution followed by destaining with sodium chloride (1 mol l−1) (Isolate PE-LR-4 showed zone of clearance greater than control), and (d) Pectin agar plate flooded with iodine–potassium iodide solution (Isolate PE-5 showed higher zone of clearance than control).

16S rRNA sequence analysis

The diagnostic 16S rRNA PCR followed by BlastN of the sequence (Table 3) was used for molecular identification. Based on the BlastN results of the 16S rRNA sequences, the endophytic bacterial isolates were identified as Bacillus, Staphylococcus, Kocuria and Acinetobacter species (Table 3). The edible mesocarp and the seeds of papaya fruits show the presence of important Bacillus endophytes. An endophyte isolated from endocarp displayed 94% sequence similarity to Acinetobacter. The sequences of each of the isolates were subjected to multiple sequence alignment by Clustal W, and NJ tree was constructed (Fig. 2). The bacterial endophytes from papaya were grouped into four clusters. The two main clusters were Bacillus and Staphylococcus species. Kocuria and Acinetobacter species were distinctly grouped in the tree and did not exhibit genetic relatedness to the other endophytes. The Staphylococcus species was not detected in the Bangalore variety, whereas the Bacillus species were detected in all the papaya varieties used in the study.

Table 3. Molecular characterization of bacterial endophytes describing the GenBank ID of submitted sequences
VarietyIsolateRegionGenBank accession numberIdentification
Red LadyPE-1MesocarpFR687205Bacillus
PE-2MesocarpFR687206Staphylococcus
PE-3MesocarpFR687207Staphylococcus
PE-4MesocarpFR687208Bacillus
PE-5EndocarpFR687209Acinetobacter
BangalorePE-Lg-1MesocarpFR687210Bacillus
PE-LR-1SeedFR687211Bacillus
PE-LR-2SeedFR687213Kocuria
PE-LR-3SeedFR687212Bacillus
PE-LR-4MesocarpFR687214Bacillus
SoloSV-1MesocarpFR687215Staphylococcus
SV-2MesocarpFR687216Staphylococcus
SV-3MesocarpFR687217Staphylococcus
SV-4MesocarpFR687218Staphylococcus
SP1MesocarpFR773879Acinetobacter
SS1SeedFR773880Bacillus
SS3SeedFR773881Enterobacter
Coorg HoneyCH1MesocarpFR773878Bacillus
Figure 2.

Neighbour-Joining tree based on the 16S rRNA sequences of the bacterial endophytes.

Shake flask fermentation

The shake flask fermentation was carried out with three isolates viz. PE-LR-1, PE-LR-2 and PE-LR-3 for 31 h. The fermented product was analysed for antioxidant potential in the medium at an interval of about 4 h during the process, and the activity was compared with S. cerevisiae-fermented product. The fermentation product using PE-LR-1 and PE-LR-2 has a compromised antioxidant activity in comparison with PE-LR-3-fermented product (Fig. 3). The free radical scavenging activity is highest (61·2%) in the fermented product developed using PE-LR-3 as the fermenting organism at 23 h of fermentation after which it begins to decline (58·03%). The fermented product obtained using Kocuria species as the fermenting organism has a compromised free radical scavenging activity of 38·50% at 23 h and peaks to 48·33% at 27 h followed by a decline at 31 h. The antioxidant activity of the fermented product developed using isolate PE-LR-1 is 34·53 and 31·15% at 23 and 27 h, respectively.

Figure 3.

Antioxidant activity of the fermented papaya product (FPP) developed by using bacterial endophytes as the fermenting micro-organisms. The uninoculated fermented broth is used as control. (image) 20 h; (image) 23 h; (image) 27 h and (image) 31 h.

The shake flask fermentation using microbial cocktail of PE-LR-3 and S. cerevisiae at two different cell ratios shows higher antioxidant activity as compared to the fermented product developed by using the microbial strains singularly (Fig. 4). The free radical scavenging activity of the yeast-fermented product was 62·5% at 27 h followed by a decline at 31 h. The microbial cocktail fermentation using S. cerevisiae and PE-LR-3 with reference to yeast alone fermentation exhibits a 0·07 and 0·21% increase at the 1 : 1 and 1 : 2 ratios, respectively. The peak activity in cocktail fermentation was obtained at 27 h as compared to 23 h in single microbe fermentation. The microbial cocktail in ratio of 1 : 1 shows 66·16% free radical scavenging activity at 27 h and a decline to 46·56% at 31 h. The microbial cocktail PE-LR-3/S. cerevisiae (2 : 1) has highest antioxidant activity of 75·7% at 27 h followed by a decline to 60·60% at 31 h.

Figure 4.

Antioxidant activity of the fermented papaya product (FPP) developed by using a microbial cocktail of Saccharomyces cerevisiae and PE-LR-3 in different ratios. The antioxidant activity is compared with S. cerevisiae, and the control is uninoculated fermented broth. (image) 20 h; (image) 23 h; (image) 27 h and (image) 30 h.

Discussion

Endophytic bacteria known as bioprospecting microbes (Strobel and Daisy 2003) have been isolated from ovules, seeds and tubers from a variety of plants (Sturz and Nowak 2000). Population densities of endophytes are low and rarely exceed 106 CFU g−1 of fresh plant tissue (Hallmann et al. 2002). Bacterial endophytes have been isolated from the tissues of healthy tomato (Solanum lycopersicum L.) (Nejad and Johnson 2000), potato (Solanum tuberosum L.) (Sturz et al. 1998, 1999; Garbeva et al. 2001; Sessitsch et al. 2001, 2004), wheat (Triticum aestivum L.) (Germida et al. 1998; Coombs and Franco 2003), sweet corn (Zea mays L.), cotton (Gossypium hirsutum L.) (McInroy and Kloepper 1995), citrus plants (Araujo et al. 2001, 2002) and carrot plants (Surette et al. 2003). Exploring, isolating and identifying the endophytes from different plant organs helps in explaining some important physiological phenomena like production of antibiotics, root exudates and few plant growth–promoting molecules. To the best of our knowledge, this is the first report on molecular diversity and characterization of endophytes from mesocarp and seeds of papaya fruit. In this study, we report the isolation, identification and functional characterization of bacterial endophytes inhabiting fruits and seeds of four agronomically important papaya varieties viz ‘Red lady’, ‘Solo’, ‘Coorg Honey’ and ‘Bangalore’ variety. The rationale for taking up this study was to identify and explore diversity of papaya fruit endophytes as it has been reported that endophytic population varies among plant species and cultivars at different field sites and in different environmental stimuli (Siciliano et al. 1998; Dunfield and Germida 2001; Siciliano et al. 2001). The plant genotype also affects the type of microbial associations as observed in case of transgenic canola that possess a different rhizosphere and endophytic population compared to the nonengineered plants (Siciliano et al. 1998). The ‘Red lady’ variety possesses Bacillus, Staphylococcus and Acinetobacter species, the ‘Bangalore’ variety has Bacillus and Kocuria species and the ‘Solo’ variety has exclusively Staphylococcus species, Acinetobacter species, Bacillus species and Enterobacter species. Similar variations in the indigenous populations of endophytes in a variety of plants have been reported (Germida et al. 1998; Araujo et al. 2001; Garbeva et al. 2001; Sessitsch et al. 2001; Adams and Kloepper 2002; Araujo et al. 2002; Zinniel et al. 2002). These variations were attributed to plant cultivar, plant age, tissue type, time of sampling and environment (Zinniel et al. 2002). In our study, the colony morphology and Gram staining gave the first indication about the diversity of the endophytes in the papaya fruits. The varietal and tissue-specific diversity indicates specific plant–endophyte interactions and this is evident from the endophyte collections from ‘Red lady’, ‘Solo’, ‘Coorg Honey’ and‘Bangalore’ fruits. The presence of Staphylococcus, Acinetobacter and Enterobacter sp. emphasizes the inhabitation of diverse endophytic population in ‘Solo’ variety that has a long history of domestication. Molecular identification of all the eighteen isolates using 16S rRNA regions followed by BlastN analysis resulted in 99–100% similarity with the sequences present in the Genbank. We could not identify any novel endophyte; however, seed- and endocarp-specific Kocuria sp., Acinetobacter sp. and Enterobacter species were detected.

Enzyme activity assays for important enzymes like protease, amylase, pectinase and xylanase were taken up to identify the strains effective in biological control in papaya. Shi et al. (2010) identified Pseudomonas putida from papaya pericarp that could effectively control Colletotrichum gloeosporioides infection when applied at florescence stage. Bacterial endophytes from papaya fruits do not exhibit any lipolytic activity or protease activity, indicating these isolates may not be potential biocontrol microbes. Earlier studies on the postharvest variations of the cell wall degrading enzymes like polygalacturonase and xylanase show that the quantities of these enzymes in the fruit are linked to ripening, ethylene production and respiration (Paull and Chen 1983). The production of these enzymes by the fruit endophytes during various developmental stages can be directly linked to the early or late ripening characters of the variety, an important postharvest trait. PE-LR-4 isolated from the mesocarp of ‘Bangalore’ variety identified as Bacillus species produces all the three cell wall degrading enzymes viz. cellulase, pectinase and xylanase. This is in consonance with the postharvest qualities of this variety as it has a short shelf life, and higher amounts of these enzymes lead to quick ripening.

In our studies, we have exclusively screened the papaya fruits for the presence of endophytes as it is endowed with high nutritional properties and high antioxidant potential that could be contributed by the endophytes residing in the fruit pulp. The role of endophytes in contributing to the nutritional value of plant products has already been documented as in case of Pestalotiopsis microspora, an endophyte from Terminalia morobensis plant-produced compounds with antioxidant and antibacterial properties (Harper et al. 2003). The fruit endophytes detected in our study could ferment the fruit that resulted in a product with high antioxidant potential. We hypothesize that these endophytes are possibly contributing to the naturally high antioxidant content of the papaya fruit. PE-LR-3 mediated fermentation gives a product with antioxidant potential (61·14%) comparable to the S. cerevisiae-fermented (62·5%) product. The cocktail of S. cerevisiae and PE-LR-3 enhanced the antioxidant potential of the fermented product to 75·7%. The most plausible explanation of the improved fermentation process when a microbial cocktail is used is that the papaya endophytes are acting synergistically with S. cerevisiae. PE-LR-3 produces extracellular enzymes like pectinase, xylanase and amylase responsible for breaking down plant pectins and starches. Similar phenomenon has been observed in L-sorbose fermentation for the production of 2-Keto-L-Gulonic Acid where a helper strain ‘Xanthomonas maltophilia’ improved the productivity significantly described by Takagi et al. (2010). The peak antioxidant activity in cocktail fermentation was observed after 27 h as compared to 23 h in single strain fermentation. In the cocktail fermentation, the most convincing explanation for this is the extracellular enzymes produced by PE-LR-3 carry on the enzymatic digestion of the complex starches and pectins in the papaya extract and it leads to a delayed peak in the antioxidant activity. Further detailed metabolic and biochemical study is in progress and will help us in explaining this synergism. However, our initial shake flask fermentation studies suggest that these endophytes can be used in combination with yeast for developing fermented papaya product.

This study describes the morphological, molecular, physiological analysis and diversity of the endophytes harbouring papaya fruit. The isolates possess important biochemical traits, and these findings can help in designing improved microbial cocktails for developing fermented papaya product.

Acknowledgement

Authors are thankful to the Department of Biotechnology, Government of India for financial support. Authors declare no conflict of interest.

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