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Antagonistic potential of bacterial endophytes and induction of systemic resistance against collar rot pathogen Sclerotium rolfsii in tomato
Biological Control 137 (2019) 104014
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Antagonistic potential of bacterial endophytes and induction of systemic resistance against collar rot pathogen Sclerotium rolfsii in tomato
T
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Pramod Kumar Sahua, , Shailendra Singha, Amrita Guptaa, Udai B. Singha, G.P. Brahmaprakashb, Anil K. Saxenaa a b
ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan, UP 275103, India University of Agricultural Sciences, GKVK, Bengaluru, KA 560065, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial endophyte Biocontrol Sclerotium rolfsii Antagonism Induced systemic resistance
A diverse pool of 310 bacterial endophytes isolated from tomato plants growing in the Indo-Gangetic plains of India was evaluated for their antagonistic potential against three soil-borne fungal pathogens causing root rot (Rhizoctonia solani), collar rot (Sclerotium rolfsii) and, fungal wilt (Fusarium oxysporum f. sp. lycopersici). Preliminary screening indicated that most of the isolates could inhibit R. solani and F. oxysporum f. sp. lycopersici to the greater extent but failed to reduce mycelia growth of S. rolfsii by more than 50 percent. The only exceptions were 1PR7a, 2P2, and 2PR9b which were further characterized and evaluated against S. rolfsii. Molecular identification of 15 isolates based on 16s rRNA gene sequence similarity indicated the presence of three genera viz. Bacillus, Lysinibacillus, and Stenotrophomonas. In planta trial in tomato challenged with pathogen S. rolfsii in presence and absence of endophyte inoculation revealed that Bacillus sp. 2P2 showed the highest protection against S. rolfsii. These strains elicited induced systemic resistance of plant and significantly higher activity (p ≤ 0.05) of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and ascorbate oxidase indicating the further strengthening of cell wall barrier through lipid peroxidation, cross linking of cell walls, lignifications, suberization and other cell wall strengthening processes. It was further confirmed by confocal scanning laser micrographs of upper collar region. It was evident that the inoculation of endophyte inhibited the colonization and movement of the pathogen. In addition, endophytes upregulated the expression of three pathogenesis-related genes PR1a, PR2a, and PR3, which are responsible for production of glucanases and chitinases contributing to pathogen inhibition. Further, oxidative stress alleviation was evident from decreased superoxide accumulation and enhanced dry matter content. Results of the present study indicated suppressive potential of endophyte Bacillus sp. 2P2 against S. rolfsii which could be useful in collar rot management in the nursery as well as after transplantation.
1. Introduction Tomato is an economically important crop in which a number of fungal diseases cause a huge loss in its production and quality of the produce. Sclerotium rolfsii is one of the most devastating soil-borne phytopathogens, which causes wilt, blight, basal stem rot and fruit rot in tomato (Amselem et al., 2011). In hot and humid climates, disease progression is even more severe (Punja, 1988). Disease management strategies like the use of ammonia fertilizers, deep ploughing, destruction of debris and solarization are used for management (Agrios, 2005). Fungicides are used to control the disease, but there is some concern for the environment (Saeed et al., 2016). Since biological control of soil-borne plant pathogens has been
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reported (Singh et al., 2013a), microorganism from different niches are being explored for sustainable management of S. rolfsii. Among these microorganisms, endophytes are one of the potential candidates to be explored for suppression of the pathogen. Endophytes are microorganisms living inside the plants and do not cause any negative impact to its host (Compant et al., 2010). Endophytes are being prospected for production of various bioactive compounds useful in industry, agriculture, and other sectors (Backman and Sikora, 2008; Nicoletti and Fiorentino, 2015). The tremendous potential of these endophytes are being harnessed in plant health and growth promotion (Oteino et al., 2015). Endophytes are reported to suppress plant pathogens by various mechanisms such as production of ammonia, hydrogen cyanide (HCN), inhibitory volatile organic compounds (VOCs), antibiotics,
Corresponding author. E-mail addresses: [email protected], [email protected] (P.K. Sahu).
https://doi.org/10.1016/j.biocontrol.2019.104014 Received 21 February 2019; Received in revised form 9 June 2019; Accepted 24 June 2019 Available online 25 June 2019 1049-9644/ © 2019 Elsevier Inc. All rights reserved.
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2.3. Identification and molecular characterization of endophytes
biosurfactant, oxidative enzymes, hydrolytic enzymes, antioxidants, secondary metabolites, siderophore, quorum sensing degraders, antimicrobial allelochemicals, and induction of systemic resistance (Cheong et al., 2017; Wicaksono et al., 2018). It was reported that bacterial endophytes have faster and intense defense induction in host plants as compared to rhizospheric microorganism against variety of plant pathogens (Pieterse et al., 2014; Martinez-Medina et al., 2016), which is mediated through different signaling pathways leading to production of defense related molecules (Backman and Sikora, 2008; Glaeser et al., 2016) that act against pathogens through different mechanisms. All these effects make endophytes a suitable candidate to be explored for suppressing collar rot pathogen. Looking at the physiology of the pathogen, it is important to have biocontrol agents that are able to colonized plant system internally apart from strengthening the structural defense of plants by inducing systemic resistance. Since there are some reports on control of soilborne pathogens using endophytic bacteria (Eljounaidi et al., 2016). Endophytes from different tomato cultivars are being explored with hypothesis of having cultivar specificity and variation in suppressive ability (Feng et al., 2013; Upreti and Thomas, 2015). Therefore, it was considered worthwhile to explore antagonists from tomato varieties grown in different districts of Indo-Gangetic plains and the following objective of present investigation was set up: (a) develop a pool of endophytic bacteria obtained from different varieties of tomato grown in Indo-Gangetic plain, (b) screen these endophytes for their biocontrol potential against soil-borne pathogens (S. rolfsii, F. oxysporum f. sp. lycopersici and R. solani) of tomato, (c) assess the physio-bio-chemical and molecular changes in tomato plants grown under S. rolfsii stressed conditions in response to inoculation of selected bacterial endophytes to unravel some of the possible mechanisms that may elucidate their mode of action.
A loopful of 24 h bacterial culture was taken for DNA isolation from 15 endophytes selected from in vitro screening. Genomic DNA from bacterial endophytes was extracted using ZR fungal/bacterial DNA Mini PrepTM genomic DNA isolation kit (ZYMO Research Corporation, USA) as per manufacturer’s instruction and stored at −20 °C. The 16S rRNA gene was amplified by universal primers PA forward (5′-AGAGTTTGA TCCTGGCTCAG-3′) and PH reverse (5′-AAGGAGGTGATCCAGCC GCA-3′). Further, the 16S rRNA gene was sequenced by the Sanger method and identified by matching sequence similarity in EZbiocloud database (https://www.ezbiocloud.net). Sequences were submitted to NCBI-GeneBank database. Phylogenetic relatedness of these endophytes with other close relatives was assessed using the neighbor-joining method and tree was developed using MEGA7 (version = 7.0.25). Bacterial endophytes were also screened for presence of antimicrobial genes nonribosomal protein synthase (NRPS; ituC and srfA), polyketide synthase (pks) and chitinase (chiA) (Supplementary data Table 1). 2.4. In planta assay The results of in vitro studies revealed that the ability among endophytes from the tomato was limited to inhibit S. rolfsii to a greater extent as compared to that for R. solani and F. oxysporum f. sp. lycopersici. Therefore, the further in planta studies were carried out with S. rolfsii as the pathogen and endophytes Bacillus sp. 1PR7a, Bacillus sp. 2P2 and Bacillus sp. 2PR9b as test organisms. 2.4.1. Preparation of planting material, inoculum, and diseased soil The inoculum of S. rolfsii was developed by inoculating mother culture on flask containing the autoclaved sorghum grains for mass multiplication (Singh et al., 2012). Inoculated flasks were incubated for 15 days at 28 ± 2 °C. These grains were used for developing pathogeninoculated pots. Mass multiplication of endophytes was done as per the methods described by Singh et al. (2016). The experimental soil was homogeneously mixed and moistened before autoclaving. Thereafter, the soil was autoclaved and added to pots (4 kg soil in 5.5 kg capacity pots) followed by 3 cycles of wetting and drying to bring the compaction similar to natural soil. After this, S. rolfsii (5 g inoculum/pot) was added on upper 3 cm, one set without pathogen kept as negative control). The pots were covered with aluminum foil and incubated for 2 days. After two days, the inoculum developed in pots was mixed uniformly in the upper soil layer to make the pathogen-inoculated pot. Surface sterilized tomato seeds (cv. Selection-22) were inoculated with endophytes as per different treatments (as mentioned in Section 2.4.2) and sown in a seedling tray containing the autoclaved potting mixture. Seedlings were raised for one month using standard practices of irrigation, thinning and planting.
2. Material and methods 2.1. Isolation of endophytes Bacterial endophytes were isolated from tomato plants grown at farmers’ field of four different districts of Indo-Gangetic plains. Root and hypocotyl portions were detached and washed in running tap water to remove adhering soil. Further, the surface sterilization was done as per the protocol described by Zinniel et al. (2002). Surface sterilized plant tissue was ground in 12.5 mM potassium phosphate buffer using sterile pestle and mortar. The 10−2 and 10−3 dilutions were made and inoculated on nutrient agar medium. Further, morphologically distinct colonies were taken and purified for further use.
2.2. In vitro bio-efficacy The bacterial endophytes were screened for antagonism against three soil-borne pathogens of tomato Rhizoctonia solani (NAIMCC-F02899), Sclerotium rolfsii (NAIMCC-F-03053), Fusarium oxysporum f. sp. lycopersici (NAIMCC-F-00899) obtained from National Agriculturally Important Microbial Culture Collection (NAIMCC), ICAR-NBAIM, India. Circular disc (7 mm) of the fungal pathogen was taken from fully colonized one week old mother plate and incorporated upside down at the center of PDA plate. Actively grown endophyte cultures (24 h old culture diluted to 0.2 OD at 600 nm) were streaked equidistantly on two sides of pathogen disc. S. rolfsii was incubated for 4 days, F. oxysporum f. sp. lycopersici was incubated for 10 days and R. solani was incubated for 5 days. Fungus inoculated plates without the bacterial endophyte served as control. This experiment was conducted with three replications and repeated twice for consistency in the results. The reduction in the radial mycelium growth was measured and percent inhibition was recorded.
2.4.2. Experimental setup Thirty days old tomato seedlings were planted as per treatments- T1 Negative control (un-inoculated), T2 Positive control (inoculated with collar rot pathogen S. rolfsii), T3 inoculated with Bacillus sp. 1PR7a + S. rolfsii, T4 inoculated with Bacillus sp. 2P2 + S. rolfsii, T5 inoculated with Bacillus sp. 2PR9b + S. rolfsii and T6 inoculated with Trichoderma viride + S. rolfsii (taken as standard biocontrol agent). Tomato plants were transplanted to the pathogen-inoculated pots. Two seedlings were maintained in each pot. Soil moisture was maintained by spraying autoclaved water on alternate days. 2.4.3. Assessment of plant growth and disease incidence Disease incidence (%) was recorded from different treatments after 7 days of pathogen inoculation to plants. Root and shoot biomass was recorded by drying in a hot air oven at 60 °C ± 5 till stable weight was 2
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expressed) gene as a control for comparing the intensity of amplified product in the gel. There were two sets of five treatments- control, 1PR7a, 2P2, 2PR9b and Trichoderma, one set was ‘without S. rolfsii’ inoculation and another ‘with S. rolfsii’ inoculation (total ten treatments). The pathogen was inoculated and plants were raised as mentioned in Section 2.4.1. Total RNA was isolated from tomato plants with different treatments using PureLink RNA isolation kit (Invitrogen) and DEPC treated water following manufacturers’ instruction. The isolated RNA was subject to RT-PCR for cDNA synthesis. Reverse transcriptase reaction was performed using iScript Select cDNA synthesis kit (BioRad) at 42 °C for 30 min. The heat deactivation of enzyme was done by incubating at 85 °C for 5 min. RT-PCR amplification of constitutively expressed gene and three other genes was done by method described by Leblanc et al. (1999) with modifications. Briefly, cDNA was used for amplification using primer sequences GaPDH F 5-GAAATGCATCTTGC ACTACCAACTGTCTTGC-3, R 5-CTGTGAGTAACCCCATTCATTATCATA CCAAGC-3; PR1b F 5-CCAAGACTATCTTGCGGTTC-3, R 5-GAACCTAA GCCACGATACCA-3; PR2a F 5-TATAGCCGTTGGAAACGAAG-3, R 5TGATACTTTGGCCTCTGGTC-3; and PR3 F 5-CAATTCGTTTCCAGGTT TTG-3, R 5-ACTTTCCGCTGCAGTATTTG-3. PCR amplification of cDNA was performed using Thermal Cycler (PaqBio) with the conditions: 95 °C for 4 min, 35 cycles of 94 °C for 45 s, 49.3 °C (GaPDH) or 57.80 °C (PR1b) or 57.4 °C (PR2a) or 53.9 °C (PR3) for 45 s; 72 °C for 60 s, followed by final extension of 72 °C for 10 min. The final product obtained with RT-PCR were separated by electrophoresis in 1.2% agarose gel in TAE buffer using Bangalore GeNei, India gel electrophoresis apparatus and visualization was done in gel documentation system (BioRAD, India). The intensity of amplified product in the gel was compared with constitutively expressed gene GaPDH.
achieved. 2.4.4. Effects of endophytes on induced systemic resistance of the plant Induced systemic resistance in tomato plants by inoculation of pathogen and endophytes were tested using standard protocols (Singh et al., 2013b) after 7 days of pathogen inoculation to plants. Freshly harvested tissues were used for each assay. Phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and ascorbate oxidase (AO) were assayed. The protocol of Havir (1987) was followed for preparing enzyme extract for estimation of phenylalanine ammonia lyase (PAL). Briefly, the leaf tissue sample (0.5 g) was extracted in 4 ml 0.2 M borate buffer (pH 8.7) with 1.4 mM β-mercaptoethanol. Enzyme extract (200 µl) was taken with 500 µl borate buffer and 1 ml of 0.1 M L-phenylalanine and 1.3 ml distilled water. This mixture was incubated at 32 °C for 30 min and the reaction was terminated by 500 µl 1 M trichloroacetic acid. The activity was measured at 290 nm and expressed in µmole of trans-cinnamic acid per g fresh weight. Peroxidase (PO) activity in tomato plants was determined as per the method described by Hammerschmidt et al. (1982). Enzyme extract (200 µl) was taken with 3 ml 50 mM phosphate buffer, 0.5 ml 20 mM guaiacol and 300 μl of 12.3 mM H2O2. The absorbance was recorded at 436 nm. Polyphenol oxidase (PPO) activity was determined according to Gauillard et al. (1993). Enzyme extract was prepared by homogenizing 100 mg leaf sample in 2 ml 0.1 M phosphate buffer and centrifuged. Enzyme activity was measured by taking 1.4 ml of 0.1 M citrate–phosphate buffer in 0.5 ml TNB and 1 ml 2 mM catechol solution. The reaction was started by adding 100 µl of enzyme extract and absorbance was read at 412 nm in 30-sec intervals for 3 min. The polyphenol oxidase activity was expressed by the change in absorbance (change in optical density; ΔOD) per min per mg fresh weight. Ascorbate oxidase (AO) in plant tissue was estimated using the method described by Drumm et al. (1972). The substrate solution (3 ml 10 mM ascorbic acid) was added in 100 µl enzyme substrate to start the reaction. The change in absorbance was recorded at 265 nm and expressed as units of µmole ascorbate disappeared (min−1 mg−1 protein).
2.4.8. Statistical analysis Laboratory experiments were carried out in a complete randomized design (CRD), however, a greenhouse experiment was taken in a randomized complete block design (RCBD) with five replications. The experiments were repeated twice to evaluate the consistency in the results. The comparison has been done based on standard deviation values and means were separated by DMRT at p ≤ 0.05 (Little and Hills, 1978).
2.4.5. Reduction in superoxide radicals Nitroblue tetrazolium (NBT) reacts with O2− (superoxide radical) and a dark blue color formazan is produced as a precipitate. The intensity of the blue color is used for qualitative comparison as described in Gupta et al. (2017) after 7 days of pathogen inoculation. The leaf samples were collected and immediately immersed into tubes containing NBT solution. Tubes were covered with aluminum foil. It was incubated for 1 h and chlorophyll was bleached by bleaching solution (acetone and DMSO in 1:1 ratio). After removal of chlorophyll, leaves were directly visualized for NBT staining under the stereomicroscope.
3. Results 3.1. Bacterial endophytes and in vitro bio-efficacy A total of 310 bacterial endophytes were isolated from tomato. Preliminary screening of 310 bacterial endophytes by dual plate assay against soil-borne pathogens causing root rot (R. solani), collar rot (S. rolfsii) and fungal wilt (F. oxysporum f. sp. lycopersici) indicated that 45 endophytes had antagonistic potential (Fig. 1); out of which, 15 were antagonistic to more than one pathogen. Table 1 indicates the list of 15 antagonists used for further study. The inhibition in radial growth of three pathogens was recorded and presented in Table 2 for 15 potential isolates. Isolates 2PR9b and 2P2 were the only isolates that could inhibit more than 50% radial growth of S. rolfsii by more than 50% and were significantly different from other isolates. Isolate 1PR7a suppressed the radial growth of S. rolfsii nearly 50%. However, with regards to suppression of radial growth of R. solani, all the endophytes appeared to have the potential to reduce by more than 50%. Among them, 2PR9b was significantly superior to all other isolates (70% inhibition). The radial growth of F. oxysporum f. sp. lycopersici was also suppressed by 50% by at least six endophytes and the highest inhibition was in the presence of 2P2 (74.56%) closely followed by 3PH2a (73.5%). The results revealed that in general, the selected isolates were more effective at suppression of R. solani and F. oxysporum f. sp. lycopersici than S. rolfsii (Table 2).
2.4.6. Confocal microscopy for studying the effect of pathogen and endophytes Fine sections of tomato roots pre-inoculated with bacterial endophytes were examined under Confocal Scanning Laser Microscope (CSLM; Nikon Eclipse 90i) 7 days after pathogen inoculation. Thin sections prepared from freshly harvested tissue was treated with SYTO9, Propidium iodide (as described by Upreti and Thomas, 2015) and 4′,6-diamidino-2-phenylindole (DAPI) for 3 min and excess dye was gently removed by using phosphate buffer saline and observed under CSLM. The X, Y and Z plane images were taken using the NIS element 3.2.3 program (Nikon) and used for comparison among the treatments. 2.4.7. Expression of three PR genes in tomato In another trial, expression of three PR genes PR1b (unknown PR protein), PR2a (β-1, 3-glucanase) and PR3 (chitinase) was studied using the semi-quantitative reverse transcriptase PCR (semi-q RT-PCR) using glyceraldehyde 3-phosphate dehydrogenase (GaPDH, constitutively 3
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Fig. 1. Graph showing the distribution of antagonist endophytes among a population of 310 bacterial endophytes against three soil-borne pathogens R. solani, S. rolfsii, and F. oxysporum f. sp. lycopersici; Same alphabets above the error bars indicate non significance between two treatments. Table 1 Detailed list indicating fifteen selected bacterial endophytes, their source cultivar, and NCBI accession numbers. Sl. No.
Identity of the endophytes
Tomato cultivar isolated from
NCBI Accession No.
1. 2.
Bacillus sp. 1PR7a Stenotrophomonas sp. 1PR10 Lysinibacillus sp. 1TH16a Bacillus sp. 2PR1 Bacillus sp. 2P2 Bacillus sp. 2PR9b Stenotrophomonas sp. 3A Bacillus sp. 3PH2a Bacillus sp. 3TR2b1 Bacillus sp. 3PH7 Bacillus sp. 4R4 Bacillus sp. 4PH5a Bacillus sp. 4PR8 Lysinibacillus sp. 4PR19 Bacillus sp. 6TH4b
S-22 S-22
MG786842 MG786841
S-22 TMTH1 TMTH1 TMTH1 Dev (Nunhems) Dev (Nunhems) Dev (Nunhems) Dev (Nunhems) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Selection 120
MH194246 MH194244 MG786836 MG786837 MG786835 MG786838 MH194245 MG786844 MH194242 MG786839 MG786840 MH194243 MG786843
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Table 2 Inhibition in radial growth of three soil-borne pathogens by selected endophytes in dual plate experiment. Sl. No.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
3.2. Identification and molecular characterization
Isolates
Bacillus sp. 1PR7a Stenotrophomonas sp. 1PR10 Lysinibacillus sp. 1TH16a Bacillus sp. 2PR1 Bacillus sp. 2P2 Bacillus sp. 2PR9b Stenotrophomonas sp. 3A Bacillus sp. 3PH2a Bacillus sp. 3TR2b1 Bacillus sp. 3PH7 Bacillus sp. 4R4 Bacillus sp. 4PH5a Bacillus sp. 4PR8 Lysinibacillus sp. 4PR19 Bacillus sp. 6TH4b
Inhibition in radial growth (%) R. solani
S. rolfsii
F. oxysporum f. sp. lycopersici
70.15ab 62.59a-e
47.00b 19.36ef
20.73fg 42.03de
52.60e 64.92a-e 64.26a-e 70.81a 53.33e 56.16cde 69.03abc 52.50e 55.50de 67.00a-d 57.60b-e 67.59a-d 64.26a-e
6.45g 27.03cde 72.93a 78.02a 37.38bc 17.67ef 31.19cd 22.33de 17.67ef 22.33de 26.59cde 9.00fg 30.78cd
6.67hi 61.89ab 74.56a 18.67fgh 55.00bc 73.56a 30.56ef 64.33ab 11.67ghi 67.67ab 45.92cd 22.00fg 5.67i
Note: Data is shown as percent radial growth inhibition of pathogen by bacterial endophytes and analyzed in complete randomized design (CRD); the means were separated by DMRT at p ≤ 0.05, values with same superscript are not significantly different.
Based on 16S rRNA gene sequence homology, these fifteen isolates represented three genera Bacillus, Lysinibacillus and Stenotrophomonas (Table 1) in which eleven isolates belonged to Bacillus, two to Lysinibacillus and two to Stenotrophomonas. These sequences were submitted to NCBI GenBank and accession numbers were obtained (Supplementary data Fig. 1 and Table 2). The fifteen endophytes were further characterized for the presence of genes for ituC (NRPS), srfA (NRPS), chiA and pks (Supplementary data 1) indicating that some of these antimicrobial genes were found in the endophytes under study, however, the expression of these genes in endophytes is yet to be tested.
Table 3. The highest disease incidence (93.33%) was recorded for plants treated only with S. rolfsii (positive control) followed by plants treated with endophyte Bacillus sp. 2PR9b (56.67%), Bacillus sp. 1PR7a (46.67%), and Bacillus sp. 2P2 (26.67%). However, no disease incidence was recorded in the control plants (negative control). Results indicated that inoculation of Bacillus sp. 2P2 cause significantly lower disease incidence as compared to other treatments under pathogenic stress condition. Representative images of each treatment are shown in Fig. 2. The dry biomass (g/pot) and percent disease incidence was recorded in pots inoculated with S. rolfsii with or without selected endophytes (Table 3). Among the endophytes, 1PR7a and 2P2 significantly improved the dry mass accumulation as compared to a positive control (inoculated only with S. rolfsii). Isolate 2PR9b and T. viride could not influence the accumulation of dry mass significantly in the presence of
3.3. In planta bio-efficacy 3.3.1. Effect of endophytes on plant growth and disease incidence Disease symptoms appeared in the collar region after 7 days of pathogen inoculation to plants. The water soaked lesions formed near the collar region. These lesions were surrounded by mycelium of the fungal pathogen. The disease incidence was calculated based on occurrence in three sets of 10 plants (2 plants × 5 replications) and presented in 4
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pathogen S. rolfsii.
Table 3 Effects of different treatments on incidence of collar rot disease (in percent) and plant biomass after 7 days of pathogen inoculation under net house condition. Treatments
Disease incidence (percent)
Dry biomass (g/pot)
−ve control +ve control 1PR7a 2P2 2PR9b T
0.00f 93.33a 46.67d 26.67e 56.67c 66.67b
2.05a 0.80d 1.87ab 1.79b 0.94cd 1.11c
3.3.2. Assessment of induced systemic resistance (ISR) Phenylalanine ammonia lyase (PAL) activity was found highest in plants inoculated with Bacillus sp. 2P2 followed by Bacillus sp. 2PR9b and Bacillus sp. 1PR7a (Fig. 3A). The lowest PAL activity was recorded from the negative control. However, polyphenol oxidase activity was recorded highest in the plants inoculated with Bacillus sp. 2PR9b followed by Bacillus sp. 1PR7a and Bacillus sp. 2P2 (Fig. 3B). Effect of endophytes on polyphenol oxidase activity was significantly higher than that of the negative control. Peroxidase activity was recorded highest in the plants inoculated with Bacillus sp. 2P2 followed by Bacillus sp. 1PR7a and Bacillus sp. 2PR9b (Fig. 3C). Effect of endophytes on peroxidase activity was significantly higher than all other treatments. Similarly, ascorbate oxidase activity was recorded highest from plants treated with Bacillus sp.
Note: Treatments: −ve control = No inoculation; +ve control = Pathogen inoculated; 1PR7a = Bacillus sp. 1PR7a + Pathogen; 2P2 = Bacillus sp. 2P2 + Pathogen; 2PR9b = Bacillus sp. 2PR9b; T = Trichoderma viride + Pathogen. Data analyzed in randomized complete block design (RCBD); the means were separated by DMRT at p ≤ 0.05, values with same superscript are not significantly different.
Fig. 2. Effect of bacterial endophyte seedling dip treatment on occurrence of collar rot disease in tomato after 7 days of pathogen inoculation; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen. 5
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Fig. 3. Induction of plant defense enzymes after 7 days of pathogen inoculation by different treatments in tomato plants infected with Sclerotium rolfsii, (A) Phenylalanine ammonia lyase (PAL) in µmol of trans cinnamic acid g−1 fresh weight; (B) Polyphenol oxidase (PPO) in ΔO.D. min−1 mg−1 fresh weight; (C) Peroxidase (PO) in units min−1 g−1 fresh weight; and (D) Ascorbic acid oxidase in µmole ascorbate disappeared min-1 mg-1 protein; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen. Data with 5 replications analyzed in randomized complete block design (RCBD); the means were separated by DMRT at p ≤ 0.05, error bars shows standard deviation of mean. Same alphabets above the error bars indicate non significance between two treatments.
2PR9b followed by Bacillus sp. 2P2 and Bacillus sp. 1PR7a (Fig. 3D). Effect of 1PR7a on ascorbate oxidase activity was relatively less than other endophytes.
filaments can be seen in Fig. 5A (yellow arrows) whereas in Bacillus sp. 2P2 inoculated plants the pith region is intact and healthy (Fig. 5B, white arrows).
3.3.3. Reduction in superoxide radicals Reduction in reactive oxygen species (super oxide radical) was shown by bacterial endophytes application (Fig. 4). Treatments T3 (Bacillus sp. 1PR7a) and T4 (Bacillus sp. 2P2) were able to suppress the ROS generated from S. rolfsii infection to the level on par with the negative control. Effects of the stress alleviation could be seen from plant dry weight (Table 3). Inoculation of 1PR7a and 2P2 could lead to an increase in dry weight similar to that of negative control.
3.3.5. Expression of three PR genes in tomato Three antagonistic bacterial endophytes Bacillus sp. 1PR7a, Bacillus sp. 2P2 and Bacillus sp. 2PR9b which was effective antagonist against S. rolfsii resulted in induction of PR genes in tomato in presence and absence of S. rolfsii (Fig. 6). Plants inoculated with Bacillus sp. 2P2 had expression of PR1b and PR2a upregulated in the presence of the pathogen. Bacillus sp. 1PR7a did not cause differential expression of these three genes. Whereas, plants inoculated with only Bacillus sp. 2PR9b, had upregulation of PR1b, PR2a, and PR3 genes that was not seen in presence of the pathogen. In Trichoderma inoculated plants, PR1b, PR2a, and PR3 were not differentially expressed in plants inoculated only with the endophytes, while these genes were upregulated in Trichoderma colonized plants upon challenge with the pathogen.
3.3.4. Confocal microscopy for studying plant–microbe interactions Confocal scanning laser microscopy was done to visualize the pathogen progress in plants. Several observations could be made from imaging and indicated that the progression of S. rolfsii in the inner tissues resulted in profuse mycelium growth (Fig. 5A, yellow arrows) and the compounds secreted by the pathogen were found disintegrating the cellular arrangements (Fig. 5A, red arrows). It was also observed that cells were converted into unorganized mass. The effect of inoculation of endophyte Bacillus sp. 2P2 was evident from the imaging of the collar region of Bacillus sp. 2P2 inoculated plants. Staining with DAPI, SYTO 9 and propidium iodide has shown that the fungus started colonizing profusely above the collar region. The
4. Discussion The present study has been undertaken with an aim to identify potential endophyte antagonistic to fungal pathogens of tomato, a diverse pool of endophytes obtained from different cultivars grown in Indo-Gangetic plains. Three endophytes were selected and their effects on different plant physiological parameters related to disease 6
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Fig. 4. Effect of inoculation on reactive oxygen species generation in tomato under diseased condition after 7 days of pathogen inoculation; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen.
compared to rhizospheric microorganisms, they could contribute in strengthening the structural defense of the plants effectively (Pieterse et al., 2014; Martinez-Medina et al., 2016). Bacillus sp. 2P2 induced the highest level of PAL and PO production as compared to other treatments. The effect could be seen from tissue disintegration at the collar region in Fig. 1 where the structural defense of host could be compared with control and endophyte treated plants. Ascorbate oxidase (AO) controls glutathione and NADPH levels during stress (Jacob et al., 2018). The level of PPO and AO was found highest in Bacillus sp. 2PR9b and was comparable to Trichoderma. The combined strengthening of plant tissue might be protected from the hydrolytic enzymes produced by the pathogen. The strengthening of plant tissue by activation of ISR is also endorsed by other workers (Singh et al., 2013b; Chung et al., 2018). There are several reports indicating that the accumulation of lignin was suppressive against F. oxysporum, Botrytis cinerea and Phoma exigua in Linum usitatissimum (Hano et al., 2006). Similarly, Streptomyces sp. RP1A-12 was reported to have a suppressive effect on S. rolfsii causing peanut stem rot (Jacob et al., 2018). The induction of hydrolytic enzymes like β 1, 3-glucanase and chitinases also contribute to defense against fungal pathogens. In the present study, indirect evidence for the induction of these enzymes was provided through expression of pathogenesis-related genes PR1b, PR2a and PR3 producing glucanase and chitinase (Fig. 6). The three endophytes showed variation among themselves with regard to the
development such as activation of defense enzymes, expression of pathogenesis-related proteins, reduction in superoxide radicals, and extent of tissue disintegration was studied. S. rolfsii is a necrotrophic pathogen (Agrios, 2005) which during the infection process, attacks on healthy tissue directly and produces oxalic acid, pectinolytic, cellulolytic and other enzymes to disintegrate and kill the host tissues. Here, strengthening of plant cell walls could provide a protective barrier against collar rot pathogen. As seen in the present study, the inoculation of endophytes provided protection to tomato plants challenged with S. rolfsii. The plants treated with endophytes showed enhanced production of defense-related enzymes like phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and ascorbate oxidase (AO). Induction of these enzymes contribute to the strengthening of the cell wall through lignifications and suberization which in turn acts as a physical barrier to the entry of pathogen at the point of first interaction (Nejad and Johnson, 2000; Singh et al., 2013b; Jacob et al., 2018). Many studies indicated that enhanced activity of PAL, a key enzyme of the phenylpropanoid pathway in higher plants, is chiefly linked with resistance to the pathogen (Tonnessen et al., 2015). The PO play an important role in biosynthesis of lignin while PPO catalyzes the oxidation of phenolics to quinines and hydrogen peroxide that are toxic to fungi and microbes in general (Jia et al., 2016). As the earlier findings suggest, endophytes have faster and intense induction of plant systemic resistance as 7
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Fig. 5. Confocal scanning laser micrograph indicating TS of upper collar region after 7 days of pathogen inoculation (A) pathogen challenged plant, and (B) inoculated with Bacillus sp. 2P2 and S. rolfsii; samples were stained with SYTO9, Propidium Iodide, and DAPI; yellow arrows indicating heavy colonization of S. rolfsii mycelia inside the tissue, red arrows indicating the disintegrated plant tissue, white arrows indicating healthy internal tissue and reduced internal colonization of S. rolfsii mycelia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
implicated in suppression of different pathogens through production of antimicrobials (Bahroun et al., 2017; Rafi and Cheah, 2018). On the other hand, these endophytes found to harbor antimicrobial genes (Supplementary data Table 1) indicating their potential of producing compounds that could inhibit the pathogen; however, expression of these genes has not been tested. This result suggests only the presence of the antimicrobial genes in the endophytes, but its expression during plant-endophyte-pathogen interaction need to be tested. The combined effect of induction of defense-related enzymes and production of ammonia, lipopeptide surfactants, hydrogen cyanide and iron quenching activity by endophytes led to the strengthening of the cell wall barrier and reduction in colonization and migration of S. rolfsii. This was evident from the intact root and shoot portions (Fig. 2) and confocal scanning laser micrographs of both the endophytes inoculated and uninoculated treatments challenged with S. rolfsii (Fig. 5). CSLM imaging has shown that the upper collar region of endophyte inoculated plants could prevent the upward movement of S. rolfsii in the stem (Fig. 5B), whereas, in endophyte uninoculated plants, the pathogen progressed upward and covered the stem (Fig. 5A). Similarly, the downward movement of pathogen and the destruction of the root system is visible in Fig. 2, in which the Bacillus sp. 2P2 inoculation indicated apparently healthy roots. This indirectly indicates that there are suppressive effects of Bacillus sp. 2P2 on S. rolfsii which is further confirmed by a reduction in disease incidence (Table 3).
expression of these genes in the presence and absence of this pathogen. Inoculation of Bacillus sp. 2P2, and Trichoderma (used as positive control) induced expression of all three PR genes in plants challenged with S. rolfsii. In contrast, Bacillus sp. 2PR9b upregulated expression of three genes only in the absence of pathogen. Kavroulakis et al. (2006) also reported expression of PR genes in tomato plants, even in the absence of any pathogen. The result indicated that 2PR9b might elicit defense through activation of PR genes. Enhanced expression of PR genes against S. rolfsii has been found to have co-linear relations with resistance (Jogi et al., 2016). Plant protection by inducing resistance against pathogens like Alternaria solani, S. rolfsii, Xanthomonas campestris, Stemphilium solani, Corynespora cassiicola and Oidium lycopersici by beneficial microbes has also been reported (Silva et al., 2004; Jogi et al., 2016; Volpiano et al., 2018). Besides induction of defense-related and hydrolytic enzymes, Bacillus sp. 2P2 and Bacillus sp. 2PR9b found to produce ammonia, lipopeptide surfactants and iron quenching activity as shown in our previous studies (Sahu and Brahmaprakash, 2018). Bacillus sp. 2P2 also produced hydrogen cyanide. Ammonia and hydrogen cyanide could also be one of the weapons of endophytes in destroying the fungal mycelia in the plant. These effects altogether could be one of the reasons to suppress the invasion and colonization of pathogen in the root and stem as the suppressive ability on pathogen growth was seen in vitro (Table 2). There are several reports where endophytes have been
Fig. 6. Semi-quantitative RT-PCR estimation of PR genes expression in tomato plants treated with bioagents with and without challenge of test pathogen S. rolfsii after 7 days of pathogen inoculation under net house conditions. Gel sequence: (1) Control- Glyceraldehyde 3- phosphate dehydrogenase (GaPDH), (2) PR1b Basic, (3) PR2a- glucanase acidic, and (4) PR3- chitinase (acidic). Sample sequence: M = 100 bp ladder, C = Control, 1PR7a = Bacillus sp. 1PR7a, 2P2 = Bacillus sp. 2P2, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride (this five sets of treatment each for plants inoculated without S. rolfsii and with S. rolfsii).
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Apart from disease incidence, these endophytes were also found to have a role in reducing the secondary effects of pathogen attack i.e. oxidative stress. Reactive oxygen species (ROS) are generated in plants in response to biotic and abiotic stress and trigger hypersensitive response causing cell death (Apel and Hirt, 2004). However, plants harbor antioxidant enzyme system which protects plants from the deleterious effect of very high ROS generation due to stress. Such protective activity is enhanced by endophytes which can be detected by nitro blue tetrazolium (NBT) reduction (Potocky et al., 2007). The accumulation of superoxides in the leaves was determined and found that inoculation of selected endophytes has reduced the accumulation of superoxides in leaves. It represents the ability of these endophytes in suppressing secondary effects of biotic and abiotic stresses. In a similar way, alleviation of oxidative stress in chickpea challenged with S. rolfsii by application of rhizospheric microbes was reported by Singh et al. (2013b). The multiple mechanisms exhibited by endophytes and in particular by Bacillus sp. 2P2 for suppression of S. rolfsii could be further translated to field to minimize collar rot incidence in tomato. Further work has been initiated to inoculate Bacillus sp. 2P2 in microplots for raising tomato nursery. It will be worthwhile to study the translocation of endophytes along with the seedlings after transplanting.
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Author contributions PKS, AKS, GPB, and UBS conceived and designed the experiments. PKS, SS, and AG performed the experiments. UBS and PKS analyzed the data. PKS, UBS, and AKS contributed reagents, materials, and analysis tools. PKS, AKS, and UBS wrote the main manuscript text and other parts of the manuscript. All authors reviewed the manuscript. Declaration of Competing Interest The authors have declared no competing interest. Acknowledgment The authors gratefully acknowledge ICAR-NBAIM, Mau and UAS, GKVK, Bengaluru for conducting experiments and financial support extended during these experiments. We acknowledge Dr. Harsh Vardhan Singh for careful reading of the manuscript. We also acknowledge Confocal Scanning Laser Microscopy Facility, ICAR-NBAIM for microscopy; In-charge NAIMCC, and the technical persons associated, Mr. Manish Roy and Mr. Alok Upadhyay for supply and preservation of microbial cultures. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2019.104014. References Agrios, G.N., 2005. Plant Pathology, fifth ed. Academic Press, New York, USA. Amselem, J., Cuomo, C.A., Van Kan, J.A., Viaud, M., Benito, E.P., Couloux, A., Coutinho, P.M., De Vries, R.P., Dyer, P.S., Fillinger, S., Fournier, E., et al., 2011. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 7 (8), e1002230. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. https://doi.org/10.1146/annurev.arplant.55. 031903.141701. Backman, P.A., Sikora, R.A., 2008. Endophytes: an emerging tool for biological control. Biol. Control. https://doi.org/10.1016/j.biocontrol.2008.03.009. Bahroun, A., Jousset, A., Mhamdi, R., Mrabet, M., Mhadhbi, H., 2017. Anti-fungal activity of bacterial endophytes associated with legumes against Fusarium solani: assessment of fungi soil suppressiveness and plant protection induction. Appl. Soil Ecol. 124, 131–140. Cheong, S.L., Cheow, Y.L., Ting, A.S.Y., 2017. Characterizing antagonistic activities and host compatibility (via simple endophyte-calli test) of endophytes as biocontrol
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gene OsPAL4 is associated with broad spectrum disease resistance. Plant Mol. Biol. 87 (3), 273–286. Upreti, R., Thomas, P., 2015. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects. Front. Microbiol.. https://doi.org/10.3389/fmicb.2015.00255. Volpiano, C.G., Lisboa, B.B., São José, J.F.B., de Oliveira, A.M.R., Beneduzi, A., Passaglia, L.M.P., Vargas, L.K., 2018. Rhizobium strains in the biological control of the phytopathogenic fungi Sclerotium (Athelia) rolfsii on the common bean. Plant Soil 432 (1–2), 229–243. Wicaksono, W.A., Jones, E.E., Casonato, S., Monk, J., Ridgway, H.J., 2018. Biological control of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, using endophytic bacteria recovered from a medicinal plant. Biol. Control. https://doi.org/10.1016/j.biocontrol.2017.03.003. Zinniel, D.K., Lambrecht, P., Harris, N.B., Feng, Z., Kuczmarski, D., Higley, P., et al., 2002. Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. Appl. Environ. Microbiol.. https://doi.org/10.1128/AEM. 68.5.2198-2208.2002.
Singh, U.B., Malviya, D., Singh, S., Pradhan, J.K., Singh, B.P., Roy, M., Imram, M., Pathak, N., Baisyal, B.M., Rai, J.P., Sarma, B.K., 2016. Bio-protective microbial agents from rhizosphere eco-systems trigger plant defense responses provide protection against sheath blight disease in rice (Oryza sativa L.). Microbiol. Res. 192, 300–312. Singh, U.B., Sahu, A., Singh, R.K., Singh, D.P., Meena, K.K., Srivastava, J.S., Manna, M.C., 2012. Evaluation of biocontrol potential of Arthrobotrys oligospora against Meloidogyne graminicola and Rhizoctonia solani in Rice (Oryza sativa L.). Biol. Control 60 (3), 262–270. Singh, S.P., Singh, H.B., Singh, D.K., 2013a. Trichoderma harzianum and Pseudomonas sp. Mediated management of Sclerotium rolfsii rot in tomato (Lycopersicon esculentum Mill.). Life Sci. 8, 801–804. Singh, A., Sarma, B.K., Upadhyay, R.S., Singh, H.B., 2013b. Compatible rhizosphere microbes mediated alleviation of biotic stress in chickpea through enhanced antioxidant and phenylpropanoid activities. Microbiol. Res.. https://doi.org/10.1016/j.micres. 2012.07.001. Tonnessen, B.W., Manosalva, P., Lang, J.M., Baraoidan, M., Bordeos, A., Mauleon, R., Oard, J., Hulbert, S., Leung, H., Leach, J.E., 2015. Rice phenylalanine ammonia-lyase
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Antagonistic potential of bacterial endophytes and induction of systemic resistance against collar rot pathogen Sclerotium rolfsii in tomato
T
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Pramod Kumar Sahua, , Shailendra Singha, Amrita Guptaa, Udai B. Singha, G.P. Brahmaprakashb, Anil K. Saxenaa a b
ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan, UP 275103, India University of Agricultural Sciences, GKVK, Bengaluru, KA 560065, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial endophyte Biocontrol Sclerotium rolfsii Antagonism Induced systemic resistance
A diverse pool of 310 bacterial endophytes isolated from tomato plants growing in the Indo-Gangetic plains of India was evaluated for their antagonistic potential against three soil-borne fungal pathogens causing root rot (Rhizoctonia solani), collar rot (Sclerotium rolfsii) and, fungal wilt (Fusarium oxysporum f. sp. lycopersici). Preliminary screening indicated that most of the isolates could inhibit R. solani and F. oxysporum f. sp. lycopersici to the greater extent but failed to reduce mycelia growth of S. rolfsii by more than 50 percent. The only exceptions were 1PR7a, 2P2, and 2PR9b which were further characterized and evaluated against S. rolfsii. Molecular identification of 15 isolates based on 16s rRNA gene sequence similarity indicated the presence of three genera viz. Bacillus, Lysinibacillus, and Stenotrophomonas. In planta trial in tomato challenged with pathogen S. rolfsii in presence and absence of endophyte inoculation revealed that Bacillus sp. 2P2 showed the highest protection against S. rolfsii. These strains elicited induced systemic resistance of plant and significantly higher activity (p ≤ 0.05) of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and ascorbate oxidase indicating the further strengthening of cell wall barrier through lipid peroxidation, cross linking of cell walls, lignifications, suberization and other cell wall strengthening processes. It was further confirmed by confocal scanning laser micrographs of upper collar region. It was evident that the inoculation of endophyte inhibited the colonization and movement of the pathogen. In addition, endophytes upregulated the expression of three pathogenesis-related genes PR1a, PR2a, and PR3, which are responsible for production of glucanases and chitinases contributing to pathogen inhibition. Further, oxidative stress alleviation was evident from decreased superoxide accumulation and enhanced dry matter content. Results of the present study indicated suppressive potential of endophyte Bacillus sp. 2P2 against S. rolfsii which could be useful in collar rot management in the nursery as well as after transplantation.
1. Introduction Tomato is an economically important crop in which a number of fungal diseases cause a huge loss in its production and quality of the produce. Sclerotium rolfsii is one of the most devastating soil-borne phytopathogens, which causes wilt, blight, basal stem rot and fruit rot in tomato (Amselem et al., 2011). In hot and humid climates, disease progression is even more severe (Punja, 1988). Disease management strategies like the use of ammonia fertilizers, deep ploughing, destruction of debris and solarization are used for management (Agrios, 2005). Fungicides are used to control the disease, but there is some concern for the environment (Saeed et al., 2016). Since biological control of soil-borne plant pathogens has been
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reported (Singh et al., 2013a), microorganism from different niches are being explored for sustainable management of S. rolfsii. Among these microorganisms, endophytes are one of the potential candidates to be explored for suppression of the pathogen. Endophytes are microorganisms living inside the plants and do not cause any negative impact to its host (Compant et al., 2010). Endophytes are being prospected for production of various bioactive compounds useful in industry, agriculture, and other sectors (Backman and Sikora, 2008; Nicoletti and Fiorentino, 2015). The tremendous potential of these endophytes are being harnessed in plant health and growth promotion (Oteino et al., 2015). Endophytes are reported to suppress plant pathogens by various mechanisms such as production of ammonia, hydrogen cyanide (HCN), inhibitory volatile organic compounds (VOCs), antibiotics,
Corresponding author. E-mail addresses: [email protected], [email protected] (P.K. Sahu).
https://doi.org/10.1016/j.biocontrol.2019.104014 Received 21 February 2019; Received in revised form 9 June 2019; Accepted 24 June 2019 Available online 25 June 2019 1049-9644/ © 2019 Elsevier Inc. All rights reserved.
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2.3. Identification and molecular characterization of endophytes
biosurfactant, oxidative enzymes, hydrolytic enzymes, antioxidants, secondary metabolites, siderophore, quorum sensing degraders, antimicrobial allelochemicals, and induction of systemic resistance (Cheong et al., 2017; Wicaksono et al., 2018). It was reported that bacterial endophytes have faster and intense defense induction in host plants as compared to rhizospheric microorganism against variety of plant pathogens (Pieterse et al., 2014; Martinez-Medina et al., 2016), which is mediated through different signaling pathways leading to production of defense related molecules (Backman and Sikora, 2008; Glaeser et al., 2016) that act against pathogens through different mechanisms. All these effects make endophytes a suitable candidate to be explored for suppressing collar rot pathogen. Looking at the physiology of the pathogen, it is important to have biocontrol agents that are able to colonized plant system internally apart from strengthening the structural defense of plants by inducing systemic resistance. Since there are some reports on control of soilborne pathogens using endophytic bacteria (Eljounaidi et al., 2016). Endophytes from different tomato cultivars are being explored with hypothesis of having cultivar specificity and variation in suppressive ability (Feng et al., 2013; Upreti and Thomas, 2015). Therefore, it was considered worthwhile to explore antagonists from tomato varieties grown in different districts of Indo-Gangetic plains and the following objective of present investigation was set up: (a) develop a pool of endophytic bacteria obtained from different varieties of tomato grown in Indo-Gangetic plain, (b) screen these endophytes for their biocontrol potential against soil-borne pathogens (S. rolfsii, F. oxysporum f. sp. lycopersici and R. solani) of tomato, (c) assess the physio-bio-chemical and molecular changes in tomato plants grown under S. rolfsii stressed conditions in response to inoculation of selected bacterial endophytes to unravel some of the possible mechanisms that may elucidate their mode of action.
A loopful of 24 h bacterial culture was taken for DNA isolation from 15 endophytes selected from in vitro screening. Genomic DNA from bacterial endophytes was extracted using ZR fungal/bacterial DNA Mini PrepTM genomic DNA isolation kit (ZYMO Research Corporation, USA) as per manufacturer’s instruction and stored at −20 °C. The 16S rRNA gene was amplified by universal primers PA forward (5′-AGAGTTTGA TCCTGGCTCAG-3′) and PH reverse (5′-AAGGAGGTGATCCAGCC GCA-3′). Further, the 16S rRNA gene was sequenced by the Sanger method and identified by matching sequence similarity in EZbiocloud database (https://www.ezbiocloud.net). Sequences were submitted to NCBI-GeneBank database. Phylogenetic relatedness of these endophytes with other close relatives was assessed using the neighbor-joining method and tree was developed using MEGA7 (version = 7.0.25). Bacterial endophytes were also screened for presence of antimicrobial genes nonribosomal protein synthase (NRPS; ituC and srfA), polyketide synthase (pks) and chitinase (chiA) (Supplementary data Table 1). 2.4. In planta assay The results of in vitro studies revealed that the ability among endophytes from the tomato was limited to inhibit S. rolfsii to a greater extent as compared to that for R. solani and F. oxysporum f. sp. lycopersici. Therefore, the further in planta studies were carried out with S. rolfsii as the pathogen and endophytes Bacillus sp. 1PR7a, Bacillus sp. 2P2 and Bacillus sp. 2PR9b as test organisms. 2.4.1. Preparation of planting material, inoculum, and diseased soil The inoculum of S. rolfsii was developed by inoculating mother culture on flask containing the autoclaved sorghum grains for mass multiplication (Singh et al., 2012). Inoculated flasks were incubated for 15 days at 28 ± 2 °C. These grains were used for developing pathogeninoculated pots. Mass multiplication of endophytes was done as per the methods described by Singh et al. (2016). The experimental soil was homogeneously mixed and moistened before autoclaving. Thereafter, the soil was autoclaved and added to pots (4 kg soil in 5.5 kg capacity pots) followed by 3 cycles of wetting and drying to bring the compaction similar to natural soil. After this, S. rolfsii (5 g inoculum/pot) was added on upper 3 cm, one set without pathogen kept as negative control). The pots were covered with aluminum foil and incubated for 2 days. After two days, the inoculum developed in pots was mixed uniformly in the upper soil layer to make the pathogen-inoculated pot. Surface sterilized tomato seeds (cv. Selection-22) were inoculated with endophytes as per different treatments (as mentioned in Section 2.4.2) and sown in a seedling tray containing the autoclaved potting mixture. Seedlings were raised for one month using standard practices of irrigation, thinning and planting.
2. Material and methods 2.1. Isolation of endophytes Bacterial endophytes were isolated from tomato plants grown at farmers’ field of four different districts of Indo-Gangetic plains. Root and hypocotyl portions were detached and washed in running tap water to remove adhering soil. Further, the surface sterilization was done as per the protocol described by Zinniel et al. (2002). Surface sterilized plant tissue was ground in 12.5 mM potassium phosphate buffer using sterile pestle and mortar. The 10−2 and 10−3 dilutions were made and inoculated on nutrient agar medium. Further, morphologically distinct colonies were taken and purified for further use.
2.2. In vitro bio-efficacy The bacterial endophytes were screened for antagonism against three soil-borne pathogens of tomato Rhizoctonia solani (NAIMCC-F02899), Sclerotium rolfsii (NAIMCC-F-03053), Fusarium oxysporum f. sp. lycopersici (NAIMCC-F-00899) obtained from National Agriculturally Important Microbial Culture Collection (NAIMCC), ICAR-NBAIM, India. Circular disc (7 mm) of the fungal pathogen was taken from fully colonized one week old mother plate and incorporated upside down at the center of PDA plate. Actively grown endophyte cultures (24 h old culture diluted to 0.2 OD at 600 nm) were streaked equidistantly on two sides of pathogen disc. S. rolfsii was incubated for 4 days, F. oxysporum f. sp. lycopersici was incubated for 10 days and R. solani was incubated for 5 days. Fungus inoculated plates without the bacterial endophyte served as control. This experiment was conducted with three replications and repeated twice for consistency in the results. The reduction in the radial mycelium growth was measured and percent inhibition was recorded.
2.4.2. Experimental setup Thirty days old tomato seedlings were planted as per treatments- T1 Negative control (un-inoculated), T2 Positive control (inoculated with collar rot pathogen S. rolfsii), T3 inoculated with Bacillus sp. 1PR7a + S. rolfsii, T4 inoculated with Bacillus sp. 2P2 + S. rolfsii, T5 inoculated with Bacillus sp. 2PR9b + S. rolfsii and T6 inoculated with Trichoderma viride + S. rolfsii (taken as standard biocontrol agent). Tomato plants were transplanted to the pathogen-inoculated pots. Two seedlings were maintained in each pot. Soil moisture was maintained by spraying autoclaved water on alternate days. 2.4.3. Assessment of plant growth and disease incidence Disease incidence (%) was recorded from different treatments after 7 days of pathogen inoculation to plants. Root and shoot biomass was recorded by drying in a hot air oven at 60 °C ± 5 till stable weight was 2
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expressed) gene as a control for comparing the intensity of amplified product in the gel. There were two sets of five treatments- control, 1PR7a, 2P2, 2PR9b and Trichoderma, one set was ‘without S. rolfsii’ inoculation and another ‘with S. rolfsii’ inoculation (total ten treatments). The pathogen was inoculated and plants were raised as mentioned in Section 2.4.1. Total RNA was isolated from tomato plants with different treatments using PureLink RNA isolation kit (Invitrogen) and DEPC treated water following manufacturers’ instruction. The isolated RNA was subject to RT-PCR for cDNA synthesis. Reverse transcriptase reaction was performed using iScript Select cDNA synthesis kit (BioRad) at 42 °C for 30 min. The heat deactivation of enzyme was done by incubating at 85 °C for 5 min. RT-PCR amplification of constitutively expressed gene and three other genes was done by method described by Leblanc et al. (1999) with modifications. Briefly, cDNA was used for amplification using primer sequences GaPDH F 5-GAAATGCATCTTGC ACTACCAACTGTCTTGC-3, R 5-CTGTGAGTAACCCCATTCATTATCATA CCAAGC-3; PR1b F 5-CCAAGACTATCTTGCGGTTC-3, R 5-GAACCTAA GCCACGATACCA-3; PR2a F 5-TATAGCCGTTGGAAACGAAG-3, R 5TGATACTTTGGCCTCTGGTC-3; and PR3 F 5-CAATTCGTTTCCAGGTT TTG-3, R 5-ACTTTCCGCTGCAGTATTTG-3. PCR amplification of cDNA was performed using Thermal Cycler (PaqBio) with the conditions: 95 °C for 4 min, 35 cycles of 94 °C for 45 s, 49.3 °C (GaPDH) or 57.80 °C (PR1b) or 57.4 °C (PR2a) or 53.9 °C (PR3) for 45 s; 72 °C for 60 s, followed by final extension of 72 °C for 10 min. The final product obtained with RT-PCR were separated by electrophoresis in 1.2% agarose gel in TAE buffer using Bangalore GeNei, India gel electrophoresis apparatus and visualization was done in gel documentation system (BioRAD, India). The intensity of amplified product in the gel was compared with constitutively expressed gene GaPDH.
achieved. 2.4.4. Effects of endophytes on induced systemic resistance of the plant Induced systemic resistance in tomato plants by inoculation of pathogen and endophytes were tested using standard protocols (Singh et al., 2013b) after 7 days of pathogen inoculation to plants. Freshly harvested tissues were used for each assay. Phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and ascorbate oxidase (AO) were assayed. The protocol of Havir (1987) was followed for preparing enzyme extract for estimation of phenylalanine ammonia lyase (PAL). Briefly, the leaf tissue sample (0.5 g) was extracted in 4 ml 0.2 M borate buffer (pH 8.7) with 1.4 mM β-mercaptoethanol. Enzyme extract (200 µl) was taken with 500 µl borate buffer and 1 ml of 0.1 M L-phenylalanine and 1.3 ml distilled water. This mixture was incubated at 32 °C for 30 min and the reaction was terminated by 500 µl 1 M trichloroacetic acid. The activity was measured at 290 nm and expressed in µmole of trans-cinnamic acid per g fresh weight. Peroxidase (PO) activity in tomato plants was determined as per the method described by Hammerschmidt et al. (1982). Enzyme extract (200 µl) was taken with 3 ml 50 mM phosphate buffer, 0.5 ml 20 mM guaiacol and 300 μl of 12.3 mM H2O2. The absorbance was recorded at 436 nm. Polyphenol oxidase (PPO) activity was determined according to Gauillard et al. (1993). Enzyme extract was prepared by homogenizing 100 mg leaf sample in 2 ml 0.1 M phosphate buffer and centrifuged. Enzyme activity was measured by taking 1.4 ml of 0.1 M citrate–phosphate buffer in 0.5 ml TNB and 1 ml 2 mM catechol solution. The reaction was started by adding 100 µl of enzyme extract and absorbance was read at 412 nm in 30-sec intervals for 3 min. The polyphenol oxidase activity was expressed by the change in absorbance (change in optical density; ΔOD) per min per mg fresh weight. Ascorbate oxidase (AO) in plant tissue was estimated using the method described by Drumm et al. (1972). The substrate solution (3 ml 10 mM ascorbic acid) was added in 100 µl enzyme substrate to start the reaction. The change in absorbance was recorded at 265 nm and expressed as units of µmole ascorbate disappeared (min−1 mg−1 protein).
2.4.8. Statistical analysis Laboratory experiments were carried out in a complete randomized design (CRD), however, a greenhouse experiment was taken in a randomized complete block design (RCBD) with five replications. The experiments were repeated twice to evaluate the consistency in the results. The comparison has been done based on standard deviation values and means were separated by DMRT at p ≤ 0.05 (Little and Hills, 1978).
2.4.5. Reduction in superoxide radicals Nitroblue tetrazolium (NBT) reacts with O2− (superoxide radical) and a dark blue color formazan is produced as a precipitate. The intensity of the blue color is used for qualitative comparison as described in Gupta et al. (2017) after 7 days of pathogen inoculation. The leaf samples were collected and immediately immersed into tubes containing NBT solution. Tubes were covered with aluminum foil. It was incubated for 1 h and chlorophyll was bleached by bleaching solution (acetone and DMSO in 1:1 ratio). After removal of chlorophyll, leaves were directly visualized for NBT staining under the stereomicroscope.
3. Results 3.1. Bacterial endophytes and in vitro bio-efficacy A total of 310 bacterial endophytes were isolated from tomato. Preliminary screening of 310 bacterial endophytes by dual plate assay against soil-borne pathogens causing root rot (R. solani), collar rot (S. rolfsii) and fungal wilt (F. oxysporum f. sp. lycopersici) indicated that 45 endophytes had antagonistic potential (Fig. 1); out of which, 15 were antagonistic to more than one pathogen. Table 1 indicates the list of 15 antagonists used for further study. The inhibition in radial growth of three pathogens was recorded and presented in Table 2 for 15 potential isolates. Isolates 2PR9b and 2P2 were the only isolates that could inhibit more than 50% radial growth of S. rolfsii by more than 50% and were significantly different from other isolates. Isolate 1PR7a suppressed the radial growth of S. rolfsii nearly 50%. However, with regards to suppression of radial growth of R. solani, all the endophytes appeared to have the potential to reduce by more than 50%. Among them, 2PR9b was significantly superior to all other isolates (70% inhibition). The radial growth of F. oxysporum f. sp. lycopersici was also suppressed by 50% by at least six endophytes and the highest inhibition was in the presence of 2P2 (74.56%) closely followed by 3PH2a (73.5%). The results revealed that in general, the selected isolates were more effective at suppression of R. solani and F. oxysporum f. sp. lycopersici than S. rolfsii (Table 2).
2.4.6. Confocal microscopy for studying the effect of pathogen and endophytes Fine sections of tomato roots pre-inoculated with bacterial endophytes were examined under Confocal Scanning Laser Microscope (CSLM; Nikon Eclipse 90i) 7 days after pathogen inoculation. Thin sections prepared from freshly harvested tissue was treated with SYTO9, Propidium iodide (as described by Upreti and Thomas, 2015) and 4′,6-diamidino-2-phenylindole (DAPI) for 3 min and excess dye was gently removed by using phosphate buffer saline and observed under CSLM. The X, Y and Z plane images were taken using the NIS element 3.2.3 program (Nikon) and used for comparison among the treatments. 2.4.7. Expression of three PR genes in tomato In another trial, expression of three PR genes PR1b (unknown PR protein), PR2a (β-1, 3-glucanase) and PR3 (chitinase) was studied using the semi-quantitative reverse transcriptase PCR (semi-q RT-PCR) using glyceraldehyde 3-phosphate dehydrogenase (GaPDH, constitutively 3
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Fig. 1. Graph showing the distribution of antagonist endophytes among a population of 310 bacterial endophytes against three soil-borne pathogens R. solani, S. rolfsii, and F. oxysporum f. sp. lycopersici; Same alphabets above the error bars indicate non significance between two treatments. Table 1 Detailed list indicating fifteen selected bacterial endophytes, their source cultivar, and NCBI accession numbers. Sl. No.
Identity of the endophytes
Tomato cultivar isolated from
NCBI Accession No.
1. 2.
Bacillus sp. 1PR7a Stenotrophomonas sp. 1PR10 Lysinibacillus sp. 1TH16a Bacillus sp. 2PR1 Bacillus sp. 2P2 Bacillus sp. 2PR9b Stenotrophomonas sp. 3A Bacillus sp. 3PH2a Bacillus sp. 3TR2b1 Bacillus sp. 3PH7 Bacillus sp. 4R4 Bacillus sp. 4PH5a Bacillus sp. 4PR8 Lysinibacillus sp. 4PR19 Bacillus sp. 6TH4b
S-22 S-22
MG786842 MG786841
S-22 TMTH1 TMTH1 TMTH1 Dev (Nunhems) Dev (Nunhems) Dev (Nunhems) Dev (Nunhems) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Hybrid H1 (Chamki) Selection 120
MH194246 MH194244 MG786836 MG786837 MG786835 MG786838 MH194245 MG786844 MH194242 MG786839 MG786840 MH194243 MG786843
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Table 2 Inhibition in radial growth of three soil-borne pathogens by selected endophytes in dual plate experiment. Sl. No.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
3.2. Identification and molecular characterization
Isolates
Bacillus sp. 1PR7a Stenotrophomonas sp. 1PR10 Lysinibacillus sp. 1TH16a Bacillus sp. 2PR1 Bacillus sp. 2P2 Bacillus sp. 2PR9b Stenotrophomonas sp. 3A Bacillus sp. 3PH2a Bacillus sp. 3TR2b1 Bacillus sp. 3PH7 Bacillus sp. 4R4 Bacillus sp. 4PH5a Bacillus sp. 4PR8 Lysinibacillus sp. 4PR19 Bacillus sp. 6TH4b
Inhibition in radial growth (%) R. solani
S. rolfsii
F. oxysporum f. sp. lycopersici
70.15ab 62.59a-e
47.00b 19.36ef
20.73fg 42.03de
52.60e 64.92a-e 64.26a-e 70.81a 53.33e 56.16cde 69.03abc 52.50e 55.50de 67.00a-d 57.60b-e 67.59a-d 64.26a-e
6.45g 27.03cde 72.93a 78.02a 37.38bc 17.67ef 31.19cd 22.33de 17.67ef 22.33de 26.59cde 9.00fg 30.78cd
6.67hi 61.89ab 74.56a 18.67fgh 55.00bc 73.56a 30.56ef 64.33ab 11.67ghi 67.67ab 45.92cd 22.00fg 5.67i
Note: Data is shown as percent radial growth inhibition of pathogen by bacterial endophytes and analyzed in complete randomized design (CRD); the means were separated by DMRT at p ≤ 0.05, values with same superscript are not significantly different.
Based on 16S rRNA gene sequence homology, these fifteen isolates represented three genera Bacillus, Lysinibacillus and Stenotrophomonas (Table 1) in which eleven isolates belonged to Bacillus, two to Lysinibacillus and two to Stenotrophomonas. These sequences were submitted to NCBI GenBank and accession numbers were obtained (Supplementary data Fig. 1 and Table 2). The fifteen endophytes were further characterized for the presence of genes for ituC (NRPS), srfA (NRPS), chiA and pks (Supplementary data 1) indicating that some of these antimicrobial genes were found in the endophytes under study, however, the expression of these genes in endophytes is yet to be tested.
Table 3. The highest disease incidence (93.33%) was recorded for plants treated only with S. rolfsii (positive control) followed by plants treated with endophyte Bacillus sp. 2PR9b (56.67%), Bacillus sp. 1PR7a (46.67%), and Bacillus sp. 2P2 (26.67%). However, no disease incidence was recorded in the control plants (negative control). Results indicated that inoculation of Bacillus sp. 2P2 cause significantly lower disease incidence as compared to other treatments under pathogenic stress condition. Representative images of each treatment are shown in Fig. 2. The dry biomass (g/pot) and percent disease incidence was recorded in pots inoculated with S. rolfsii with or without selected endophytes (Table 3). Among the endophytes, 1PR7a and 2P2 significantly improved the dry mass accumulation as compared to a positive control (inoculated only with S. rolfsii). Isolate 2PR9b and T. viride could not influence the accumulation of dry mass significantly in the presence of
3.3. In planta bio-efficacy 3.3.1. Effect of endophytes on plant growth and disease incidence Disease symptoms appeared in the collar region after 7 days of pathogen inoculation to plants. The water soaked lesions formed near the collar region. These lesions were surrounded by mycelium of the fungal pathogen. The disease incidence was calculated based on occurrence in three sets of 10 plants (2 plants × 5 replications) and presented in 4
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pathogen S. rolfsii.
Table 3 Effects of different treatments on incidence of collar rot disease (in percent) and plant biomass after 7 days of pathogen inoculation under net house condition. Treatments
Disease incidence (percent)
Dry biomass (g/pot)
−ve control +ve control 1PR7a 2P2 2PR9b T
0.00f 93.33a 46.67d 26.67e 56.67c 66.67b
2.05a 0.80d 1.87ab 1.79b 0.94cd 1.11c
3.3.2. Assessment of induced systemic resistance (ISR) Phenylalanine ammonia lyase (PAL) activity was found highest in plants inoculated with Bacillus sp. 2P2 followed by Bacillus sp. 2PR9b and Bacillus sp. 1PR7a (Fig. 3A). The lowest PAL activity was recorded from the negative control. However, polyphenol oxidase activity was recorded highest in the plants inoculated with Bacillus sp. 2PR9b followed by Bacillus sp. 1PR7a and Bacillus sp. 2P2 (Fig. 3B). Effect of endophytes on polyphenol oxidase activity was significantly higher than that of the negative control. Peroxidase activity was recorded highest in the plants inoculated with Bacillus sp. 2P2 followed by Bacillus sp. 1PR7a and Bacillus sp. 2PR9b (Fig. 3C). Effect of endophytes on peroxidase activity was significantly higher than all other treatments. Similarly, ascorbate oxidase activity was recorded highest from plants treated with Bacillus sp.
Note: Treatments: −ve control = No inoculation; +ve control = Pathogen inoculated; 1PR7a = Bacillus sp. 1PR7a + Pathogen; 2P2 = Bacillus sp. 2P2 + Pathogen; 2PR9b = Bacillus sp. 2PR9b; T = Trichoderma viride + Pathogen. Data analyzed in randomized complete block design (RCBD); the means were separated by DMRT at p ≤ 0.05, values with same superscript are not significantly different.
Fig. 2. Effect of bacterial endophyte seedling dip treatment on occurrence of collar rot disease in tomato after 7 days of pathogen inoculation; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen. 5
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Fig. 3. Induction of plant defense enzymes after 7 days of pathogen inoculation by different treatments in tomato plants infected with Sclerotium rolfsii, (A) Phenylalanine ammonia lyase (PAL) in µmol of trans cinnamic acid g−1 fresh weight; (B) Polyphenol oxidase (PPO) in ΔO.D. min−1 mg−1 fresh weight; (C) Peroxidase (PO) in units min−1 g−1 fresh weight; and (D) Ascorbic acid oxidase in µmole ascorbate disappeared min-1 mg-1 protein; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen. Data with 5 replications analyzed in randomized complete block design (RCBD); the means were separated by DMRT at p ≤ 0.05, error bars shows standard deviation of mean. Same alphabets above the error bars indicate non significance between two treatments.
2PR9b followed by Bacillus sp. 2P2 and Bacillus sp. 1PR7a (Fig. 3D). Effect of 1PR7a on ascorbate oxidase activity was relatively less than other endophytes.
filaments can be seen in Fig. 5A (yellow arrows) whereas in Bacillus sp. 2P2 inoculated plants the pith region is intact and healthy (Fig. 5B, white arrows).
3.3.3. Reduction in superoxide radicals Reduction in reactive oxygen species (super oxide radical) was shown by bacterial endophytes application (Fig. 4). Treatments T3 (Bacillus sp. 1PR7a) and T4 (Bacillus sp. 2P2) were able to suppress the ROS generated from S. rolfsii infection to the level on par with the negative control. Effects of the stress alleviation could be seen from plant dry weight (Table 3). Inoculation of 1PR7a and 2P2 could lead to an increase in dry weight similar to that of negative control.
3.3.5. Expression of three PR genes in tomato Three antagonistic bacterial endophytes Bacillus sp. 1PR7a, Bacillus sp. 2P2 and Bacillus sp. 2PR9b which was effective antagonist against S. rolfsii resulted in induction of PR genes in tomato in presence and absence of S. rolfsii (Fig. 6). Plants inoculated with Bacillus sp. 2P2 had expression of PR1b and PR2a upregulated in the presence of the pathogen. Bacillus sp. 1PR7a did not cause differential expression of these three genes. Whereas, plants inoculated with only Bacillus sp. 2PR9b, had upregulation of PR1b, PR2a, and PR3 genes that was not seen in presence of the pathogen. In Trichoderma inoculated plants, PR1b, PR2a, and PR3 were not differentially expressed in plants inoculated only with the endophytes, while these genes were upregulated in Trichoderma colonized plants upon challenge with the pathogen.
3.3.4. Confocal microscopy for studying plant–microbe interactions Confocal scanning laser microscopy was done to visualize the pathogen progress in plants. Several observations could be made from imaging and indicated that the progression of S. rolfsii in the inner tissues resulted in profuse mycelium growth (Fig. 5A, yellow arrows) and the compounds secreted by the pathogen were found disintegrating the cellular arrangements (Fig. 5A, red arrows). It was also observed that cells were converted into unorganized mass. The effect of inoculation of endophyte Bacillus sp. 2P2 was evident from the imaging of the collar region of Bacillus sp. 2P2 inoculated plants. Staining with DAPI, SYTO 9 and propidium iodide has shown that the fungus started colonizing profusely above the collar region. The
4. Discussion The present study has been undertaken with an aim to identify potential endophyte antagonistic to fungal pathogens of tomato, a diverse pool of endophytes obtained from different cultivars grown in Indo-Gangetic plains. Three endophytes were selected and their effects on different plant physiological parameters related to disease 6
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Fig. 4. Effect of inoculation on reactive oxygen species generation in tomato under diseased condition after 7 days of pathogen inoculation; Treatments: −ve control = No inoculation, +ve control = Pathogen inoculated, 1PR7a = Bacillus sp. 1PR7a + Pathogen, 2P2 = Bacillus sp. 2P2 + Pathogen, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride + Pathogen.
compared to rhizospheric microorganisms, they could contribute in strengthening the structural defense of the plants effectively (Pieterse et al., 2014; Martinez-Medina et al., 2016). Bacillus sp. 2P2 induced the highest level of PAL and PO production as compared to other treatments. The effect could be seen from tissue disintegration at the collar region in Fig. 1 where the structural defense of host could be compared with control and endophyte treated plants. Ascorbate oxidase (AO) controls glutathione and NADPH levels during stress (Jacob et al., 2018). The level of PPO and AO was found highest in Bacillus sp. 2PR9b and was comparable to Trichoderma. The combined strengthening of plant tissue might be protected from the hydrolytic enzymes produced by the pathogen. The strengthening of plant tissue by activation of ISR is also endorsed by other workers (Singh et al., 2013b; Chung et al., 2018). There are several reports indicating that the accumulation of lignin was suppressive against F. oxysporum, Botrytis cinerea and Phoma exigua in Linum usitatissimum (Hano et al., 2006). Similarly, Streptomyces sp. RP1A-12 was reported to have a suppressive effect on S. rolfsii causing peanut stem rot (Jacob et al., 2018). The induction of hydrolytic enzymes like β 1, 3-glucanase and chitinases also contribute to defense against fungal pathogens. In the present study, indirect evidence for the induction of these enzymes was provided through expression of pathogenesis-related genes PR1b, PR2a and PR3 producing glucanase and chitinase (Fig. 6). The three endophytes showed variation among themselves with regard to the
development such as activation of defense enzymes, expression of pathogenesis-related proteins, reduction in superoxide radicals, and extent of tissue disintegration was studied. S. rolfsii is a necrotrophic pathogen (Agrios, 2005) which during the infection process, attacks on healthy tissue directly and produces oxalic acid, pectinolytic, cellulolytic and other enzymes to disintegrate and kill the host tissues. Here, strengthening of plant cell walls could provide a protective barrier against collar rot pathogen. As seen in the present study, the inoculation of endophytes provided protection to tomato plants challenged with S. rolfsii. The plants treated with endophytes showed enhanced production of defense-related enzymes like phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO) and ascorbate oxidase (AO). Induction of these enzymes contribute to the strengthening of the cell wall through lignifications and suberization which in turn acts as a physical barrier to the entry of pathogen at the point of first interaction (Nejad and Johnson, 2000; Singh et al., 2013b; Jacob et al., 2018). Many studies indicated that enhanced activity of PAL, a key enzyme of the phenylpropanoid pathway in higher plants, is chiefly linked with resistance to the pathogen (Tonnessen et al., 2015). The PO play an important role in biosynthesis of lignin while PPO catalyzes the oxidation of phenolics to quinines and hydrogen peroxide that are toxic to fungi and microbes in general (Jia et al., 2016). As the earlier findings suggest, endophytes have faster and intense induction of plant systemic resistance as 7
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Fig. 5. Confocal scanning laser micrograph indicating TS of upper collar region after 7 days of pathogen inoculation (A) pathogen challenged plant, and (B) inoculated with Bacillus sp. 2P2 and S. rolfsii; samples were stained with SYTO9, Propidium Iodide, and DAPI; yellow arrows indicating heavy colonization of S. rolfsii mycelia inside the tissue, red arrows indicating the disintegrated plant tissue, white arrows indicating healthy internal tissue and reduced internal colonization of S. rolfsii mycelia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
implicated in suppression of different pathogens through production of antimicrobials (Bahroun et al., 2017; Rafi and Cheah, 2018). On the other hand, these endophytes found to harbor antimicrobial genes (Supplementary data Table 1) indicating their potential of producing compounds that could inhibit the pathogen; however, expression of these genes has not been tested. This result suggests only the presence of the antimicrobial genes in the endophytes, but its expression during plant-endophyte-pathogen interaction need to be tested. The combined effect of induction of defense-related enzymes and production of ammonia, lipopeptide surfactants, hydrogen cyanide and iron quenching activity by endophytes led to the strengthening of the cell wall barrier and reduction in colonization and migration of S. rolfsii. This was evident from the intact root and shoot portions (Fig. 2) and confocal scanning laser micrographs of both the endophytes inoculated and uninoculated treatments challenged with S. rolfsii (Fig. 5). CSLM imaging has shown that the upper collar region of endophyte inoculated plants could prevent the upward movement of S. rolfsii in the stem (Fig. 5B), whereas, in endophyte uninoculated plants, the pathogen progressed upward and covered the stem (Fig. 5A). Similarly, the downward movement of pathogen and the destruction of the root system is visible in Fig. 2, in which the Bacillus sp. 2P2 inoculation indicated apparently healthy roots. This indirectly indicates that there are suppressive effects of Bacillus sp. 2P2 on S. rolfsii which is further confirmed by a reduction in disease incidence (Table 3).
expression of these genes in the presence and absence of this pathogen. Inoculation of Bacillus sp. 2P2, and Trichoderma (used as positive control) induced expression of all three PR genes in plants challenged with S. rolfsii. In contrast, Bacillus sp. 2PR9b upregulated expression of three genes only in the absence of pathogen. Kavroulakis et al. (2006) also reported expression of PR genes in tomato plants, even in the absence of any pathogen. The result indicated that 2PR9b might elicit defense through activation of PR genes. Enhanced expression of PR genes against S. rolfsii has been found to have co-linear relations with resistance (Jogi et al., 2016). Plant protection by inducing resistance against pathogens like Alternaria solani, S. rolfsii, Xanthomonas campestris, Stemphilium solani, Corynespora cassiicola and Oidium lycopersici by beneficial microbes has also been reported (Silva et al., 2004; Jogi et al., 2016; Volpiano et al., 2018). Besides induction of defense-related and hydrolytic enzymes, Bacillus sp. 2P2 and Bacillus sp. 2PR9b found to produce ammonia, lipopeptide surfactants and iron quenching activity as shown in our previous studies (Sahu and Brahmaprakash, 2018). Bacillus sp. 2P2 also produced hydrogen cyanide. Ammonia and hydrogen cyanide could also be one of the weapons of endophytes in destroying the fungal mycelia in the plant. These effects altogether could be one of the reasons to suppress the invasion and colonization of pathogen in the root and stem as the suppressive ability on pathogen growth was seen in vitro (Table 2). There are several reports where endophytes have been
Fig. 6. Semi-quantitative RT-PCR estimation of PR genes expression in tomato plants treated with bioagents with and without challenge of test pathogen S. rolfsii after 7 days of pathogen inoculation under net house conditions. Gel sequence: (1) Control- Glyceraldehyde 3- phosphate dehydrogenase (GaPDH), (2) PR1b Basic, (3) PR2a- glucanase acidic, and (4) PR3- chitinase (acidic). Sample sequence: M = 100 bp ladder, C = Control, 1PR7a = Bacillus sp. 1PR7a, 2P2 = Bacillus sp. 2P2, 2PR9b = Bacillus sp. 2PR9b, T = Trichoderma viride (this five sets of treatment each for plants inoculated without S. rolfsii and with S. rolfsii).
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Apart from disease incidence, these endophytes were also found to have a role in reducing the secondary effects of pathogen attack i.e. oxidative stress. Reactive oxygen species (ROS) are generated in plants in response to biotic and abiotic stress and trigger hypersensitive response causing cell death (Apel and Hirt, 2004). However, plants harbor antioxidant enzyme system which protects plants from the deleterious effect of very high ROS generation due to stress. Such protective activity is enhanced by endophytes which can be detected by nitro blue tetrazolium (NBT) reduction (Potocky et al., 2007). The accumulation of superoxides in the leaves was determined and found that inoculation of selected endophytes has reduced the accumulation of superoxides in leaves. It represents the ability of these endophytes in suppressing secondary effects of biotic and abiotic stresses. In a similar way, alleviation of oxidative stress in chickpea challenged with S. rolfsii by application of rhizospheric microbes was reported by Singh et al. (2013b). The multiple mechanisms exhibited by endophytes and in particular by Bacillus sp. 2P2 for suppression of S. rolfsii could be further translated to field to minimize collar rot incidence in tomato. Further work has been initiated to inoculate Bacillus sp. 2P2 in microplots for raising tomato nursery. It will be worthwhile to study the translocation of endophytes along with the seedlings after transplanting.
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Author contributions PKS, AKS, GPB, and UBS conceived and designed the experiments. PKS, SS, and AG performed the experiments. UBS and PKS analyzed the data. PKS, UBS, and AKS contributed reagents, materials, and analysis tools. PKS, AKS, and UBS wrote the main manuscript text and other parts of the manuscript. All authors reviewed the manuscript. Declaration of Competing Interest The authors have declared no competing interest. Acknowledgment The authors gratefully acknowledge ICAR-NBAIM, Mau and UAS, GKVK, Bengaluru for conducting experiments and financial support extended during these experiments. We acknowledge Dr. Harsh Vardhan Singh for careful reading of the manuscript. We also acknowledge Confocal Scanning Laser Microscopy Facility, ICAR-NBAIM for microscopy; In-charge NAIMCC, and the technical persons associated, Mr. Manish Roy and Mr. Alok Upadhyay for supply and preservation of microbial cultures. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2019.104014. References Agrios, G.N., 2005. Plant Pathology, fifth ed. Academic Press, New York, USA. Amselem, J., Cuomo, C.A., Van Kan, J.A., Viaud, M., Benito, E.P., Couloux, A., Coutinho, P.M., De Vries, R.P., Dyer, P.S., Fillinger, S., Fournier, E., et al., 2011. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet. 7 (8), e1002230. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. https://doi.org/10.1146/annurev.arplant.55. 031903.141701. Backman, P.A., Sikora, R.A., 2008. Endophytes: an emerging tool for biological control. Biol. Control. https://doi.org/10.1016/j.biocontrol.2008.03.009. Bahroun, A., Jousset, A., Mhamdi, R., Mrabet, M., Mhadhbi, H., 2017. Anti-fungal activity of bacterial endophytes associated with legumes against Fusarium solani: assessment of fungi soil suppressiveness and plant protection induction. Appl. Soil Ecol. 124, 131–140. Cheong, S.L., Cheow, Y.L., Ting, A.S.Y., 2017. Characterizing antagonistic activities and host compatibility (via simple endophyte-calli test) of endophytes as biocontrol
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