Disease suppression, growth promotion and colonization attributes of resident endophytic bacteria against white root rot (Dematophora necatrix Hartig) of apple

doi:10.21203/rs.3.rs-3251034/v1

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Disease suppression, growth promotion and colonization attributes of resident endophytic bacteria against white root rot (Dematophora necatrix Hartig) of apple

https://doi.org/10.21203/rs.3.rs-3251034/v1

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published 03 Jan, 2024

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The inherent potential of apple trees was investigated to explore bacterial endophytes and their role in suppressing Dematophora necatrix, the incitant of white root rot disease. Resultantly 34 endophytic bacteria secluded from healthy apple plants, and subsequently 6 most efficient isolates viz. Bacillus megaterium strain EA3, Enterobacter sp. strain EA7, Bacillus megaterium strain EK2, Stenotrophomonas maltophilia strain EK6, Acinetobacter nosocomialis strain ES2 and Pseudomonas aeruginosa strain ES8 depicting anti-pathogen interactions through preliminary screening were further assessed under in vitro, glasshouse and field conditions against white root rot disease. Maximum mycelial growth inhibition (80.37%) was obtained in S. maltophilia strain EK6 encouraging for its seed treatment and soil application thereby providing significant effective control (87.91%) of white root rot under controlled culture situations as compared to other five bacterial endophytes evaluated simultaneously, followed by field efficacy of 83.70 per cent. In addition, it has significantly enhanced the growth parameters of apple trees under both the situations. The inoculated healthy plants were assessed for endophytic colonization which revealed maximum endosphere colonialism by S. maltophilia strain EK6. Additionally, confocal microscopic explication of transverse sections of root cells colonized by bacterial endophytes as compared to untreated control implied their persistence and establishment in endosphere of apple seedlings. This investigation emphasized the first evidence of interaction between apple associated bacterial endophytes and D. necatrix, therefore these endophytes could be employed as alternative for mitigating white root rot disease in future.

Antagonism

bacterial endophytes

colonization

root rot

sequence analysis

sustainable

Apple (Malus × domestica Borkh.) is one of the most valued fruit crop cultivated in wet and dry temperate as well as in cold desert regions of the world (Spadoni et al. 2015). Few of the Indian hill states are confessed to be suitable for apple cultivation owing to altitudinal advantage and are well renowned for quality apple production. During recent years apple cultivation has become major source of livelihood for the rural families and it has also brought phenomenal economic boon amongst some growers of the Himachal Pradesh. However, biotic stresses due to fungal, bacterial and viral pathogens are posing serious threats for successful raising of desired production level. Amongst major diseases harnessing apple crops, white root rot (Dematophora necatrix Hartig [Rosellinia necatrix (Hartig) Berl.]) is asserted as one of the major limiting factors. The disease is serious in both nurseries and orchards, as a consequence render huge economic losses to orchardists of the state (Sharma et al. 2005). The infected trees manifest disease commencement on above and underground plant parts through decline in the vigour of the plant bolstered by bronzing, wilting and eventually defoliation. The level of underground infection determines the damage to fibrous root system, and subsequently encircling of primary and secondary roots with greenish grey hyphal stands, leading to formation of flocculent web of whitish ribbons during monsoon season (Sharma 1993). Initially, the total estimated annual losses due to this disease were recorded to be $1.3 million, which has jumped to about $1.6 million dollars in 2002 (Agarwala and Sharma 1966; Williamson 2003). Recently, during 2016-17, the collective annual crop losses of ₹ 265.64 million were estimated in major apple growing areas of Himachal Pradesh and may be expected much more in the coming years (Pal 2019).

Hitherto, no suitable measures are accessible across the world, which could be pumped to achieve the desirable level of disease management. Some of the fungicides have been registered to manage the disease to some extent, but are accountable for environment pollution, persuading resistance strains of deleterious pathogens and by biomagnifications jeopardizing human health (Zhang 2004). Keeping in view the menace and complexities arising from their usage have encouraged the researchers to explore alternative practices for disease management. The use of endosymbionts as a biological control agent has become an attractive, hopeful, and eco-friendly alternative since they have the ability to inhabit the same ecological niche as plant pathogens (de Lamo et al. 2018). Endophytes are microbes that reside in the interior tissues of living plants, without visibly harming the host (Hardoim et al. 2015).Various mechanisms, for instance antibiotics production, synthesis of cell wall-degrading enzymes, competition for minerals and nutrients, and systemic resistance induction, are documented in various pathogen inhibition while plant growth stimulation was elicited by endosymbionts through nitrogen fixation, phosphate solubilization, siderophore and indole-3-acetic acid (IAA) production (Rosenblueth and Martínez-Romero 2006; Mohamad et al. 2018).

Endophytic bacteria are known for their interior tissue colonization behavior, the dwelling of endophytic bacteria inside the plant parts has been documented immensely by researchers (Thomas and Reddy 2013). However, little is known about role of bacterial endosymbiots in management of white root rot pathogen in apple. Therefore, the present study was anticipated with the objectives to screen plant growth promotion (PGP), biocontrol and colonization behavior of bacterial root endophytes against white root rot of apple.

The causative fungal pathogen

Dematophora necatrix, the incitant of white root rot in apple, was isolated from diseased root portions of apple seedlings as per the methodology illustrated by Sharma et al. (2005). Pathogenicity of fungus was assessed on one-year-old apple seedlings grown in pots. The soil around the seedlings was removed delicately to divulge the fine root system, and 5g wheat grain culture (Sharma and Gupta 1997) of D. necatrix was put in the soil embracing roots, and covered with soil. A control was distinctly maintained in which all the operations were identical except the addition of pathogen culture. Treated pots were watered regularly and monitored for the symptoms emergence. Koch’s postulates were confirmed by re-isolating the pathogen from symptomatic roots, and compared with the original test fungus. The pathogen was cultured on Potato Dextrose Agar (PDA) dishes incubated at 25 ± 2°C for 7 days. The cultures were stored in PDA slants at 4°C for further use.

Collection of samples, isolation and purification of endophytic bacteria

Healthy root sections of apple seedlings (Malus domestica) were collected from major apple growing districts viz., Kullu, Shimla and Kinnaur of Himachal Pradesh (India) during 2016-17, brought to the plant pathology laboratory, and processed within 24–48 h. Isolation of bacterial root endophytes was performed employing methods suggested by Sharma and Roy (2015). Isolations executed from each location were replicated thrice and the bacterial counts were expressed in terms of CFU/g of root sample (Agarwal and Hasija 1986). The isolated bacteria were sub-cultured on Nutrient Agar (NA) and incubated at 37^οC for 24 h. Subsequently, the cultures recovered were revived and stored on NA slants and were designated with code EA, EK, and ES depending on isolation sites as Kullu, Kinnaur and Shimla, respectively.

Interaction of bacterial endophytes with Dematophora necatrix

Evaluation under in vitro conditions

All the bacterial isolates were screened for antagonism under in vitro conditions against D. necatrix employing streak plate method (Jinantana and Sariah 1997). The Petri dish containing sterilized PDA media was streaked on both sides/periphery with 48 h old colonies of respective bacterial isolate. Mycelial disc of (5mm diameter) D. necatrix (test pathogen) was placed at the center of the streaked culture. A separate replicated set of Petri dishes without bacterial streaks were kept as control. The inoculated Petri dishes were incubated at 25 ± 1^oC for 7 days until the test pathogen in the control Petri dish grew completely. The colony interactions were assessed as per cent inhibition of the radial growth by the following formula of Fokkema (1976) i.e. R₁-R₂/R₁×100, where, R₁ signify diameter of the radial growth of the pathogen towards opposite side and R₂ imply the radial growth of the pathogen towards the opponent antagonist. The zone of interaction of test pathogen and bacterium was observed further in order to endure the type of antagonistic behaviour under compound microscope (Yurnaliza et al. 2014). The alteration in the hyphal tip/ mycelial growth of D. necatrix at the interaction point were viewed at 40X magnification and compared with control. The bacterial isolates which had rendered excellent antagonistic activity were selected for further characterization and in vivo assessment.

Identification of bacterial endophytes

Morphological and biochemical characterization of isolated endophytes have been carried out employing standard methodology depicted in Bergey’s Manual of Determinative Bacteriology (Vos et al. 2009). DNA extraction was executed using Zymo Research DNA Extraction Kit (Murphy Ave., Irvine, USA) following manufacturer’s instructions. Universal 16S rDNA primers 27F (AGA GTT TGA TCM TGG CTC AG) and 1492R (CGG TTA CCT TGT TAC GAC TT) were employed for the amplification of 16S rDNA gene from selected bacterial isolates. PCR was performed in 25µl reaction volume containing 0.5µl Taq DNA polymerase, 2.5µl Taq buffer A, 1.0µl dNTPs mixture (GeNei, Bangalore, India); 1.0µl 27F primer, 1.0µl 1492R primer (John Wiley & Sons, New York, USA), 1.5µl genomic DNA and 17.5 µl molecular grade water. The PCR amplification phases were governed in a thermal cycler with a total of 45 cycles. Each cycle comprised of initial denaturation at 94^oC for 2 min 30 sec, denaturation at 94^oC for 30 sec, annealing at 58^oC for 30 sec, extension at 72^oC for 1.5 min, and final extension at 72^oC for 5 min. The electrophoresis of the amplified DNA was executed over agarose gel (2%) containing ethidium bromide (0.5 mg/ml). To each PCR sample, 4µl of 6X loading dye was added. 100bp DNA ladder marker (GeNei, Bangalore, India) was used as standard and the gel was run at 110V. The amplified DNA was scrutinized under the UV trans-illuminator and the image was regulated through gel documentation apparatus (Syngene). The sequencing of the purified and amplified DNA products of endophytic bacterial isolates was outsourced to Eurofins Genomics India Pvt. Ltd. Bangalore, India. Sequenced data so procured was computed employing BLAST (Basic Local Alignment Search Tool) (www.ncbi.nlm.nih.gov) for homology. All the sequences used for phylogenetic analysis were interpreted by MEGA 6.0 software using neighbor-joining method.

Evaluation under in vivo conditions

Six bacterial endophytes which had evinced considerable antagonism under in vitro conditions were further scrutinized for their plant growth and biocontrol potential against white root rot under glasshouse conditions as well as field conditions at the Research Farm of Regional Horticultural Research and Training Station, Sharbo, Kinnaur (H.P.) INDIA. The suspension culture of in vitro effective bacterial isolates were prepared on Nutrient Broth (NB) and incubated in shaking incubator for 5 days. Subsequently, the stratified apple seeds were dipped in respective bacterial suspension (5×10⁸ cfu/ml) for 30 minutes. The treated seeds were dried in a sterile stream of air at room temperature for 1 h before planting in the pots. The potting mixture (soil, sand, and cow dung @ 2:1:1) used was sterilized by tyndallysing the soil in polybags for 30 minutes for 3 successive days at 100^oC. Four seeds were sown at 2 cm depth in each small sized plastic pot (10 cm high and 8 cm internal diameter) containing sterilized potting mixture. The seedlings upon attaining 8–10 cm height after germination were drenched 2 times (100 ml×2) at weekly intervals with 200 ml of bacterial suspension around the root zone however, in field conditions drenching was executed after 2 months of germination when the seedlings get established in the field. After a week, the treated seedlings were challenge inoculated with the culture of D. necatrix (5g) grown on wheat grains @ 0.1% (w/w). In untreated seedlings no bacterial suspension was used (seeds were dipped in non-chlorinated water). The experiment was arranged in a completely randomized design or randomized block design with 4 replications in glasshouse and field, respectively. A total of 672 plants were used for carried out glasshouse and field experiments. In glasshouse, the temperature was maintained from 22 to 25^oC with 12 h photoperiod. The plants were watered regularly and data on seedlings representing disease symptoms were monitored and recorded at 15, 30 and 45 days of pathogen inoculation following Sharma and Gupta (1997):

Disease incidence (%) = [Total number of diseased plants/ Total number of plants assessed] × 100; Disease control (%) = [(Incidence in control - Incidence in treatment / Incidence in control] × 100

Different plant growth characters such as per cent germination (number of seeds germinated/total number of seeds sown), shoot length (cm), root length ((cm) and vigour index (VI) were also assessed. The vigour index was calculated using the formula given by Abdul Baki and Anderson (1973).

Vigour Index = (Mean root length + Mean shoot length) × Germination (%)

Colonization of bacterial endophytes in apple roots and elucidation through confocal laser scanning microscopy

The bacterial isolates depicting antagonism against D. necatrix were assayed for their proficiency to colonize the apple roots through confocal laser scanning microscopy (CLSM) [Advanced Materials Research Center (AMRC), Indian Institute of Technology, Mandi (H.P.) India]. Apple seedling possess hardy and woody nature under field conditions therefore, to examine interior colonization and for ease in preparation of samples for confocal microscopy, root specimens from seedlings treated with different bacterial isolates in glasshouse were executed for microscopic studies. The treated plants were permitted to develop for 3 weeks and watered through sterile distilled water once in 2 days. Consequently, the seedlings were uprooted after 7 and 21 days, rinsed multiple times with sterile water and preserved for examination under confocal microscope. The root samples were surface-sterilized with sodium hypochlorite (1%) for 3 min, rinsed twice in sterile distilled water, and placed on sterile tissue paper in a laminar flow, and stored in ethanol (70%). Thin transverse sections were prepared from fresh root tissue using sterilized dissecting blade. Afterward, root sections were placed on microscopic slide and immersed with 2–3 drops of 4’, 6-diamidino-2-phenylindole (DAPI) stain. Stained samples after 10–20 min were scrutinized using confocal laser scanning microscope equipped with a 488nm laser and supported by Laser scanning Microscopy (LSM) software. The samples stained with DAPI were excited at 488 and 405nm. Confocal data were analyzed using the Zeiss LSM Image Browser version 4.0 program. Two-dimensional images were processed with LSM image browser, MS-Power Point and Adobe Photoshop 7.0 (Thomas and Reddy 2013). Confocal depiction of different endophytic suspensions treated root sections were compared with control root section images. The colonized roots were further assessed for enumeration of bacterial population at dilution 10^− 2 (cfu/g of root samples) on Nutrient Agar by serial dilution and plate count technique (Aggarwal and Hasija 1986).

The data obtained from laboratory, glasshouse and field experimentation were subjected to appropriate statistical analysis employing standard procedures, and as prescribed by Gomez and Gomez (1984). Critical differences (CD) amongst the treatments in various experiments at 5% level were also calculated to deduce the least significant differences (LSD).

Pathogenicity test evinced that symptoms of disease initiated after 13 days of inoculation of D. necatrix which died completely after 45 days. The Koch’s postulates were affirmed by re-isolating the same fungus from the diseased roots, and were established to be the same fungus (D. necatrix) by correlating with original culture through microscopic observation of fungal characters i.e presence of pear shaped swellings at the septa in the hyphae of both the cultures.

The data obtained on isolation and number of bacterial endophytes divulged that samples accreted from different sites harboured variable bacterial endophytic populations. The maximum bacterial endophytic population (84.00×10²) was recorded from Shimla followed by district Kullu (76.67×10²). However, least endophytic bacterial population was noted in Kinnaur district (70.33×10²). Overall, a total of 34 bacterial endophytes were assessed from roots of apple trees. Of these, 14 isolates were recovered from Shimla, 11 isolates from Kullu however, least isolates were evinced in samples collected from district Kinnaur (9 No.). In brief, endophytic bacterial isolates were registered supremely in Shimla as compared to other districts.

Evaluation under in vitro conditions

Among 34 bacterial endophytes, the 6 best performance bacterial isolates which proved efficient in the course of preliminary screening were analyzed further for antifungal activity by dual culture technique. The data dispensed in Table (1) implied that all the bacterial endophytic cultures were competent of impeding the mycelial growth of D. necatrix, however the level of suppression varied with endophytes. Amongst them, S. maltophilia strain EK6 was most effective, and resulted in maximum growth inhibition of 80.37 % followed by P. aeruginosa strain ES8 (76.67%). However, A. nosocomialis strain ES2 was noticed to be least efficient in restricting the mycelial growth (52.59 %) of the pathogen. The antagonistic isolates were examined further under compound microscope to explicate their mechanism of action at interaction zone (Fig.1). The distortions and deformity of hyphal tips of D. necatrix as a result of interactivity with promising bacterial endophytes have been summarized in Table (1).

Table 1 In vitro antagonistic activity of bacterial endophytes against Dematophora necatrix

Isolate	Mycelial growth (mm)	Mycelial growth inhibition (%)	Changes in mycelial growth at interaction zone
*Bacillus megaterium* strain EA3	24.00	73.33 (58.89)	Tight encircling over pathogen hyphae
*Enterobacter* sp. strain EA7	23.33	74.07 (59.37)	Tight encircling over hyphae
*Bacillus megaterium* strain EK2	33.00	63.33 (52.71)	Shrinkage and malformation of hyphae
*Stenotrophomonas maltophilia* strain EK6	17.67	80.37 (63.69)	Shrinkage and buldzing of hyphae
*Acinetobacter nosocomialis* strain ES2	42.67	52.59 (46.47)	Distortion and thickening on pear shaped swelling of hyphae
*Pseudomonas aeruginosa* strain ES8	21.00	76.67 (61.09)	Pear shaped hyphae which is likely to become near bursting
Control	90.00	0.00 (0.00)
CD _0.05		1.43

Figures in parentheses are arc sine transformed values

Identification of bacterial endophytes

On the basis of morphological and biochemical tests

Six bacterial isolates which had evinced maximum antagonism against D. necatrix under in vitro conditions, were subjected further for morphological, and biochemical assay. All the isolates tested conferred variation in terms of their morphological, and biochemical characteristics. The colonies of these isolates were small to large, creamish-white to light brownish, moist or smooth, entire to undulate, slightly raised or flat, and circular to irregular edged on Nutrient Agar plates. Based on Gram’s reaction, 57 % of the total isolates were Gram positive while 43 % were Gram negative. On the basis of biochemical characterization, 86 % isolates were positive for oxidase, 24 % for indole production and 67 % for nitrate reduction, and for catalase production.

On the basis of morphological, and biochemical reactions which was executed using Bergey’s Manual of Determinative Bacteriology, the isolates EA3 and EK2 were tentatively recognized as Bacillus sp., ES2 as Acinetobacter sp., EA7 as Enterobacter sp., EK6 as Stenotrophomonas sp., whereas, ES8 was identified as Pseudomonas sp.

On the basis of molecular analysis

The identity of 6 bacterial endophytes which displayed significant biocontrol activity; PGP and colonization under in vitro, glasshouse and field assay were re-confirmed employing 16S rRNA gene sequencing. Phylogenetic evaluation studies corroborated the affiliation of the 6 representative bacterial root endophytes segregated from apple. The 16S ribosomal RNA gene amplicons obtained from the tested isolates exhibited homology to the library sequences accessible in NCBI databases. These sequences were proffered to GeneBank (NCBI) and were assigned with accession numbers as given in the Table (2).

Table 2 Identification of the selected bacterial root endophytes isolated from apple roots

Isolate	Identified as	Closest Neighbour	GenBank accession no.	Location	Maximum identity (%)
EA3	Bacillus megaterium strain EA3	Bacillus megaterium strain B2P2	MH712295	Kullu	99
EA7	Enterobacter sp. strain EA7	Acidovorax wautersii strain NF 1598	MH707254	Kullu	98
EK2	Bacillus megaterium strain EK2	Bacillus megaterium strain HS1J	MH712296	Kinnaur	99
EK6	Stenotrophomonas maltophilia strain EK6	Stenotrophomonas maltophilia strain NVTC10498	MH712279	Kinnaur	99
ES2	Acinetobacter nosocomialis strain ES2	Acinetobacter nosocomialis strain STO2	MH714743	Shimla	99
ES8	Pseudomonas aeruginosa strain ES8	Pseudomonas aeruginosa strain NVJT	MH712282	Shimla	99

The phylogenetic analysis of bacterial endophytes depicted two major clads viz., (i) clad I consisted of two sub groups: I(a) S. maltophilia whereas, I(b) contained Enterobacter sp. On the other hand, clad II divided further in to two sub groups- clad II(b) contained Acinetobacter sp. however, clad II(a) encompassed all the Gram +ve Bacilli dominated by endophytes genus Bacillus (Fig. 2). The efficient endophytic bacterial genera from the current study highlighted with bold letters in the phylogenetic tree illustrated resemblance with similar other sequences deposited in the NCBI GenBank with 50 % bootstrap value.

Evaluation under in vivo conditions

The 6 most efficient bacterial endophytes, which had evinced the best antagonism against D. necatrix, under in vitro conditions, were scrutinized in pot experiment under glasshouse conditions for their potential to stimulate growth of apple seedlings, and biocontrol competency against white root rot pathogen. The data furnished in the Table (3) revealed the substantial increase in vigour index of seeds perusing application of culture filtrate of endophytes as they exhibited noticeably increased shoot, and root length compared with the untreated control. Overall, the germination of seeds varied between 41.67 to 91.67 %. Upon bacterization with different treatments, maximum germination (91.67%), shoot length (12.33cm), root length (12.67cm), and vigour index (1129.33) were recorded with Enterobacter sp. strain EA7 which was comparatively higher than that of untreated control in all respect. The S. maltophilia strain EK6 exhibited 83.33 % germination, 13.33cm shoot length, 12.33cm root length, and 1120.67 vigour index.

Table 3 Effect of bacterial endophytes on seed germination, shoot and root length of apple seedlings in glasshouse conditions

Isolate	Germination (%)	Shoot length (cm)	Root length (cm)	Vigour Index (VI)
*Bacillus megaterium* strain EA3	83.33 (69.98)	11.67	11.00	994.33
*Enterobacter* sp. strain EA7	91.67 (79.99)	12.33	12.67	1129.33
*Bacillus megaterium* strain EK2	75.00 (59.98)	11.33	9.67	859.67
*Stenotrophomonas maltophilia* strain EK6	83.33 (69.98)	13.33	12.33	1120.67
*Acinetobacter nosocomialis* strain ES2	75.00 (59.98)	10.67	11.67	811.67
*Pseudomonas aeruginosa* strain ES8	83.33 (69.98)	12.67	10.67	1082.00
Control	41.67 (39.98)	6.67	6.33	289.67
CD _0.05	21.01	1.95	1.85	-

The promising endophytic treatments were screened further for their effectiveness in controlling white root rot disease. Notably, all the bacterial isolates screened were recorded very efficient in reducing disease incidence in comparison to control. Data furnished in Table (4) revealed that, seed treatment with S. maltophilia strain EK6 was highly effective which exhibited minimum disease incidence of 10.18 % followed by P. aeruginosa strain ES8 (13.89%), and Enterobacter sp. strain EA7 (15.74%). Additionally, it was noted that up to 15 days of pathogen inoculation there was no disease in treatments EA3, EA7, EK2, EK6, and ES8. The overall maximum disease control after 45 day of inoculation was registered in S. maltophilia strain EK6 (87.91%) pursued by P. aeruginosa strain ES8 (83.52%), and B. megaterium strain EA3 (81.32%). However, B. megaterium strain EK2 was least effective accounting for 75.83 per cent disease control.

Table 4 Effect of apple seed treatment with bacterial endophytes on white root rot in glasshouse conditions

Treatment	Disease incidence (%) after pathogen inoculation			Mean (%)	Per cent Disease Control
Treatment	15	30	45	Mean (%)	Per cent Disease Control
*Bacillus megaterium* strain EA3	0.00 (0.00)	16.66 (24.08)	38.88 (38.54)	18.51 (20.87)	78.03
*Enterobacter* sp. strain EA7	0.00 (0.00)	16.66 (24.08)	30.55 (33.49)	15.74 (19.19)	81.32
*Bacillus megaterium* strain EK2	0.00 (0.00)	19.44 (26.04)	41.66 (40.18)	20.37 (22.08)	75.83
*Stenotrophomonas maltophilia* strain EK6	0.00 (0.00)	8.33 (16.77)	22.22 (28.02)	10.18 (14.93)	87.91
*Acinetobacter nosocomialis* strain ES2	0.00 (0.00)	16.66 (24.08)	41.66 (40.18)	19.44 (21.42)	76.93
*Pseudomonas aeruginosa* strain ES8	0.00 (0.00)	8.33 (16.77)	33.33 (35.25)	13.89 (17.34)	83.52
Control	52.78 (46.58)	100.00 (89.39)	100.00 (89.39)	84.26 (75.12)
Mean	9.44 (11.15)	26.94 (31.64)	46.11 (44.16)
CD _0.05	Treatments (T)=1.29 Interval (I)=0.71 Treatments (T)×Interval (I)=2.24

Six bacterial endophytes which had expressed considerable plant growth and biocontrol potential against white root rot under glasshouse conditions were screened further in root rot infested field conditions at the Research Farm of Regional Horticultural Research and Training Station, Sharbo, Kinnaur (H.P.) for evaluating their efficacy in field conditions. The data illustrated in the Table (5) divulged substantial increase in vigour index of seeds treated with suspension culture of bacterial endophytes. Conspicuously, treated seeds depicted significant increase in shoot, and root length compared to the untreated control. The germination percentage of treated seeds aligned between 33.33 to 83.33 %. Upon bacterization, maximum germination (83.33%) was noted in P. aeruginosa strain ES8, while shoot length (54.67cm), root length (26.67cm), and vigour index (4582.22) were recorded in S. maltophilia strain EK6 which were remarkably greater than untreated control in every respect.

Table 5 Effect of bacterial endophytes on seed germination, shoot and root length of apple seedlings under field conditions

Isolate	Germination (%)	Shoot length (cm)	Root length (cm)	Vigour Index (VI)
*Bacillus megaterium* strain EA3	75.00 (59.98)	48.33	21.33	3646.33
*Enterobacter* sp. strain EA7	75.00 (59.98)	51.00	24.67	3849.67
*Bacillus megaterium* strain EK2	66.67 (54.98)	45.33	25.00	3047.20
*Stenotrophomonas maltophilia* strain EK6	83.33 (69.98)	54.67	26.67	4582.22
*Acinetobacter nosocomialis* strain ES2	66.67 (54.98)	43.67	23.33	2934.44
*Pseudomonas aeruginosa* strain ES8	83.33 (69.98)	47.00	19.67	3936.33
Control	33.33 (34.99)	27.33	16.33	927.44
CD _0.05	16.17	2.42	2.18	--

Data illustrated on efficacy of bacterial endophytes (Table 6) revealed that all the treatments greatly reduced the incidence of white root rot in comparison to control. Seed treatment with S. maltophilia strain EK6 suspension was highly effective, and resulted in minimum disease incidence of 13.89 % up to 45 days of pathogen inoculation. It was pursued by Enterobacter sp. strain EA7 (17.59%), and P. aeruginosa strain ES8 (19.44%). Additionally, no pertinent disease symptoms were detected in these treatments up to 15 days of pathogen inoculation. The overall maximum disease control was achieved with S. maltophilia strain EK6 (83.70%) followed by Enterobacter sp. strain EA7 (79.35%) and P. aeruginosa strain ES8 (77.18%). However, B. megaterium strain EK2 was observed as least efficient treatment rendered only 69.57 per cent disease control.

Table 6 Effect of apple seed treatment with bacterial endophytes on white root rot under field conditions

Treatment	Disease incidence (%) after pathogen inoculation			Mean (%)	Per cent Disease Control
Treatment	15	30	45	Mean (%)	Per cent Disease Control
*Bacillus megaterium* strain EA3	8.33 (16.77)	16.66 (24.08)	38.88 (38.54)	21.29 (26.46)	75.01
*Enterobacter* sp. strain EA7	0.00 (0.00)	16.66 (24.08)	36.11 (36.89)	17.59 (20.32)	79.35
*Bacillus megaterium* strain EK2	8.33 (16.77)	27.78 (31.74)	41.66 (40.18)	25.92 (29.56)	69.57
*Stenotrophomonas maltophilia* strain EK6	0.00 (0.00)	13.88 (21.64)	27.78 (31.74)	13.89 (17.79)	83.70
*Acinetobacter nosocomialis* strain ES2	8.33 (16.77)	25.00 (29.99)	36.11 (36.89)	23.15 (27.88)	72.83
*Pseudomonas aeruginosa* strain ES8	0.00 (0.00)	16.66 (24.08)	41.66 (40.18)	19.44 (21.42)	77.18
Control	55.55 (48.18)	100.00 (89.39)	100.00 (89.39)	85.18 (75.65)	0.00
Mean	13.89 (17.66)	33.05 (35.89)	49.16 (45.99)
CD _0.05	Treatments (T)=1.60 Interval (I)=0.88 Treatments (T)×Interval (I)=2.77

Colonization of bacterial endophytes in apple roots and elucidation through confocal laser scanning microscopy

The efficient bacterial endophytes were screened further for their colonization in roots of apple, and their population was inspected at 1^st, and 3^rd week following treatment with respective endophytic suspensions. The data presented in Table (7) on colonization behavior of endophytic bacteria illustrated noticeably higher endophytic count in case of endophytic suspension treated seedlings as comparison to uninoculated control. The population of endophytic bacteria increased tremendously as culturable population count of endophytic bacteria was noticed more on 3^rd week in comparison to 1^st week of inoculation. The maximum endophytic bacterial count (78.00×10¹cfu/g root) on NA medium was observed from roots which were mended with suspension of S. maltophilia strain EK6. However, the least bacterial population (41.67×10¹ cfu/g root) was registered with root samples of untreated seedlings. After completion of experimentation, isolation was carried further from each treated samples and interestingly population of inoculated samples were found to be dominant over other bacterial taxa.

Table 7 Endophytic bacterial populations after seedling dip treatment of apple in glasshouse conditions

Isolate	Endophytic bacterial population (cfu/g of roots) after days
Isolate	After 7 days	After 21 days
*Bacillus megaterium* strain EA3	43.67	61.33
*Enterobacter* sp. strain EA7	51.33	69.33
*Bacillus megaterium* strain EK2	38.67	53.67
*Stenotrophomonas maltophilia* strain EK6	58.67	78.00
*Acinetobacter nosocomialis* strain ES2	41.00	57.33
*Pseudomonas aeruginosa* strain ES8	53.33	72.67
Control	32.67	41.67
CD _0.05	Treatments (T)=1.52 Interval (I)=0.81 Treatments (T)×Interval (I)=2.15

To scrutinize and authenticate the endowment of efficient root endophytic bacteria to colonize, interact, and established in the endosphere of apple, treated root samples were procured from glasshouse experiment, processed with DAPI staining pursued by visualization under CLSM. Confocal microscopic pictures of transverse sections of root cells colonized by bacterial endophytes varied considerably, and are depicted in Fig. (3). The confocal microscopic results emphasized that each of 6 bacterial endophytes tested were capable of colonizing intercellular regions as well asouter cortex of apple roots, whereas root sections from untreated plants resulted negligible bacterial colonization.

With the advancement of science number of tedious plant diseases have been successfully managed by developing and adopting site/region specific strategies, however successful and full proof management of pernicious soil borne diseases like white root rot is still a great challenge throughout the world. All possible management practices and principles have been scrutinized by the scientific community of the world but the farmers are still longing for the steak solution of the problem. Unfortunately, there is no resistance source against the pathogen as it has very wide host range across the families and genera and about 185 plant species have been reported to be susceptible to this ubiquitous fungus. Till date, this disease is considered as major limiting constrains not only in India but throughout the globe wherever apple is grown because it is capable of decimating entire crop with high economic losses. Since last few decades, many apple orchards have been wiped out by the disease, leading to tree decline and not only ravaging whole orchard but making the soil unsuitable for raising new plantations due to heavy load of soil borne inoculums. So the continuous efforts are being made by the experts working on this pathogen to find out new ways and means to tackle the disease. In this context, utilizing the working experience for the last two decades, the authors have thought of investigating inherent abilities of the plant to fight against the pathogen. To our knowledge, this study provides the first solid authentication on use of bacterial endophytes for scrutinizing plant growth parameters, antagonistic and colonization behavior against the pathogen.

The pathogen has been taken as fresh isolated culture from the diseased apple roots and its pathogenicity had been proved as a routine exercise and observed similar to previous reports available in the literature, however the established culture was utilized in conducting various experiments in the present investigation.

On geographical basis, India falls under tropical and subtropical zone although, due to high altitude of western Himalayas, the temperate climate like situations are created which have been utilized by the scientific community to cultivate temperate fruits. The western Himalaya constitutes different ranges developing into various agroclimatic zones such as wet temperate and high hills, dry temperate and cold desert etc. The plants growing in the cold desert are mostly xerophytes which inhabit lot of endophytes to survive under severe freezing conditions. Due to human efforts the apple cultivation has been introduced successfully in this region. Therefore, an idea has been generated to explore the possible role of endophytes in apple and further exploiting them for disease management. The data divulged maximum bacterial endophytic population (84.00×10²) from Shimla district. The reason for high endophytic population in Shimla may be due to congenial environment and longer adaptability period, since in Himachal Pradesh; initially the apples are grown commercially in Shimla over the last 7–8 decades. However endophytic population varied according to the sampling sites owing to positive impact exerted by root exudates in proliferation of selective microbial colonization. Though it is first attempt to explore the endophytic bacterial population in apple at varied altitudes however, some of earlier workers have elucidated that bacterial endophytes population varies with sampling site and altitude (Kloepper et al. 1980; Gangta 2008). The continuous and consistent surveys for sampling of endophytic population from varied locations at different altitudes had yielded good number of (34Nos.) bacterial endophytes.

The isolated endophytes were analysed for antagonism against D. necatrix, only few of them (6 Nos.) were inhibitory however, the level of suppression differed with endophytes. S. maltophilia strain EK6 followed by P. aeruginosa strain ES8 were most effective, and resulted in maximum growth inhibition of 80.37% and 76.67% respectively. The efficient bacterial strains exhibited distortions and deformity in hyphae of D. necatrix at interaction zone and have been interpreted distinctly. Similarly, repressing consequence of F. oxysporum was illustrated by Nandhini et al. (2012) employing P. aeruginosa HR7, Bacillus sp. TEB6, Pseudomonas sp. TEP3, Klebsiella sp. and Citrobacter sp. They emphasized that Bacillus and Pseudomonas spp. trigger extensive group of antibiotics viz., 2,4-diacetylphloroglucinol, phenazine, pyoluteorin, oligomycin, pyrolnitrin, pyocyanin, bacillomycin, zwittermycin A, iturin, and surfactin which are accountable for their disease suppressing attribute. Bivi et al. (2010) examined several abnormalities in hyphae of Ganoderma boninense in presence of P. aeruginosa, and EB4. They also noticed that the distortion, shrinkage and contraction of hyphae were more apparent in P. aeruginosa than EB4 treated mycelium. Overall, the alterations in hyphae were more with endophytes as compared to hyphae in the control (Fig. 1).

Based on morphological, and biochemical features, the isolates EA3 and EK2, were identified as Bacillus sp., ES2 as Acinetobacter sp., EA7 as Enterobacter sp., EK6 as Stenotrophomonas sp., whereas, ES8 was recognized as Pseudomonas sp. (Vos et al. 2009). Phylogenetic studies rendered executing 16S rRNA gene sequencing affirmed the identity of the 6 most efficacious bacterial endophytic isolates. The 16S ribosomal RNA gene amplicons procured from these isolates promulgated relatedness to the sequences accessible in NCBI databases. These sequences were proffered to GeneBank (NCBI) and were assigned with accession numbers as furnished in the Table (1). The dominance of Bacillus genera in the endosphere of apple trees with high PGP traits assured their potential as endophytes. The dominance of genus Bacillus corroborates with previous studies which illustrated that the bacterial isolates dominate the rhizosphere of apple trees (Guleria et al. 2014; Mehta et al. 2014).

Endophytes scrutinized under glasshouse conditions (Table 3) for their potential to stimulate growth of apple seedlings divulged considerable accretion in vigour index in comparison with the untreated control. Rajendran et al. (2007) emphasized that bacterial endophyte P. fluorescens strain Pf1 markedly increased the vigour index of rice seedlings when compared to untreated control. This may be due to the growth promoting substances viz., gibberellin, cytokinin, and indole-acetic acid produced by P. fluorescens. Similarly, Allu et al. (2014) demonstrated that inoculation of chilli seeds with P. aeruginosa (CEFR-3) led to a substantial increment in growth parameters viz., root length, shoot length, fresh weight, and vigour of chilli seedlings.

Each of 6 bacterial endophytes tested were inscribed efficient in curtailing incidence of white root rot disease while scrutinizing their effectiveness under glasshouse assay (Table 4). High biocontrol potency of endophytic isolates may be imputed to elevated levels of extracellular metabolites (siderophore, salicylic acid, HCN etc.) triggered by these microbes which have been estimated separately in another study. Additionally, antifungal compounds such as pseudobactin, HCN, salicylic acid, and 2-hydroxy phenazine produced by fluorescent pseudomonads suppressed phytopathogenic fungi proficiently (Reddy et al. 2008; Li et al. 2012).

The data depicted (Table 5) on multifarious growth parameters in field trial conferred extensive increase in vigour index of seeds over untreated control. Muthukumar et al. (2010) observed maximum root length, shoot length, and the vigour index when the chilli seeds were treated with the 48h old culture filtrate of the P. fluorescens isolate EBS 20. Some other researchers also investigated various plant growth promoting effects displayed by endophytes in varied plant species (Kulinseky et al. 2004; Vijayaraghavan 2007). The positive influence of rhizobacterial application on germination rate of apple seeds might be due to increased synthesis of hormones like gibberellins, and auxins which may have triggered the activity of enzymes imperative for the growth amelioration (Li et al. 2012). Mehta et al. (2014) also detected remarkable augmentation in seed germination (27.07%), shoot length (42.33%), root length (52.6%), shoot dry weight (62.01%), and root dry weight (45.7%) of tomato under glass house by treatment of tomato seeds with B. methylotrophicus CKAM isolate.

Each of bacterial endophytes tested were also recorded to lower the incidence of white root rot efficiently in field trial. Sapak et al. (2008) evinced that Burkholderia cepacia, and P. aeruginosa isolated from symptomless oil palm roots exhibited potential to inhibit the spread of G. boninense (incitant of Basal stem rot) and subsequently played a role in keeping the pathogen population below threshold level. Majority of endophytic microorganisms are known to induce phytohormones, exhibit antifungal and phosphate solubilizing properties (Etesami et al. 2015).

The data inscribed (Table 7) on colonization competency of endophytic bacteria revealed higher endophytic count in case of endophytic suspension treated roots of apple trees in comparison to uninoculated control. The population count of endophytic bacteria increased exceedingly and was scrutinized high after 21 days in comparison to seventh day of pathogen inoculation. Subsequently, isolation was carried further from roots of each treatment and conspicuously numbers of inoculated bacterial strains were observed remarkably higher than other bacterial taxa henceforth, emphasizing the persistence of inoculated strains in roots of apple seedlings.

Confocal microscopic illustrations of transverse sections of root cells tenanted by bacterial endophytes varied immensely and clearly corroborated the endowment and establishment of endophytic bacteria in the interior tissues of apple plants (Fig. 3). The explication on transverse sections of roots evinced that each of 6 bacterial endophytes screened were able to inhabit intercellular spaces and the cortex regions of apple roots however, root sections from untreated plants rendered minimal colonization. Thomas and Reddy (2013) provided firm microscopic evidence on the substantial endophytic bacterial inhabitation in the cell wall and plasma membrane of banana. Similarly, Prieto et al. (2011) also emphasized successful colonization of olive roots by Pseudomonas spp. employing confocal microscopy. They explicated the persistence of PICF7, and PICP2 strains of Pseudomonas in the root cortex regions. All these findings approve endophytic bacteria as natural colonizers of plants. The conceivable colonization of apple roots via application of bacterial endophytes implies a credible collaboration of endophytes in the biology of the host besides interpreting cell wall and plasma membrane as substantial niche for endospheric bacteria. In present research, interaction between bacterial endophytes and white root rot pathogen of apple has been validated for the first time depending on the altitude level of the terrain and presumably renders the detailed evidence of apple associated endophytic bacteria in plant growth enhancing, biocontrol and colonization of roots against white root rot pathogen.

The results suggested that bacterial endophytic isolates particularly, S. maltophilia strain EK6 pursued by P. aeruginosa strain ES8 and Enterobacter sp. strain EA7 conferred ameliorated plant growth traits and biocontrol potential against white root rot pathogen/disease of apple. In addition, the colonization dynamics and confocal microscopic scrutiny of apple roots colonized by these endophytic treatments reveal their interior dwelling and symbiotic persistence. Conclusively, our findings strongly authenticated the hidden potential of indigenous bacteria and presumably the commercial formulation of these microbes could be used as safe and environment friendly option for effective management of this dreadful disease. Though this research interpret a basis for engagement of bacterial endophytes in tackling white root rot in apple however, further studies are needed that could elucidate the exact mechanisms divulged by endophytes at cellular and molecular level.

PGP: Plant growth promoting; NA: Nutrient agar; BLAST: Basic Local alignment search tool; PDA: Potato dextrose agar; NCBI: National Centre for Biotechnology Information; NB: Nutrient broth; PDC: Per cent disease control; DAPI: 4’, 6-diamidino-2-phenylindole; CLSM: Confocal laser scanning microscopy

Acknowledgements

The first author gratefully acknowledges the help granted by Directorate of Research, Professor & Head, Dept. of Plant Pathology, Associate Director, Regional Horticultural Research and Training Station, Sharbo, Kinnaur, Dr. Yashwant Singh Parmar University of Horticulture & Forestry Nauni, Solan 173230 (H.P.) India for permitting the field and laboratory facilities and other assistance. Authors also gratefully acknowledge the Indian Institute of Technology, Mandi, Himachal Pradesh (India) for providing Confocal Microscopy facility to carry out the root colonization assay.

Statements and Declarations

Funding: Not applicable

Conflicts of interest: The authors declare that they have no competing interests.

Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Disease suppression, growth promotion and colonization attributes of resident endophytic bacteria against white root rot (Dematophora necatrix Hartig) of apple

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Abstract

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Introduction

Materials and Methods

The causative fungal pathogen

Collection of samples, isolation and purification of endophytic bacteria

Identification of bacterial endophytes

Colonization of bacterial endophytes in apple roots and elucidation through confocal laser scanning microscopy

Results

Discussion

Abbreviations

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References

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