Antagonism of Pseudomonas putida Against Dematophora nectarix A Major Apple Plant Pathogen and Its Potential Use as a Biostimulent

Plant growth promoting rhizobacteria prove a key to sustainable agriculture provided they effectively colonize roots, survive, proliferate in the rhizosphere and enhance plant growth by a variety of mechanisms. In the present study, several samplings were conducted in different apple growing regions of Himachal Pradesh in order to isolate a strain capable of showing multifarious plant growth promoting activities and antagonism against Dematophora nectarix a major apple plant pathogen. In this research five isolates of fluorescent Pseudomonas sp. were isolated from apple rhizosphere. Out of five isolates, one isolate DE-18 was selected on the bases of high production of PGP activities and was identified by 16S rRNA gene sequencing. The isolate DE-18 showed maximum phosphate solubilisation and siderophore production of 425 μg/ml and 42.18 %SU respectively. Among the five isolate, DE-18 showed production of both protease (40 mm) and chitinase (28 mm). The isolate DE-18 also showed antagonism against Dematophora nectarix (38.46%) and Phytopathora cactoram (36.18%) and can act as Bioprotectant. The 16S rDNA based phylogenetic analysis demonstrated that the isolate DE-18 belonged to the Pseudomonas putida and sequence was deposited in the GenBank nucleotide sequence databases under accession number KU139388.

Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria that aggressively colonize plant roots and benefit plants by providing growth promotion 1 .In the last few years, the number of PGPR that have been identified has seen a great increase, mainly because the role of the rhizosphere as an ecosystem has gained importance in the functioning of the biosphere.Various species of bacteria like Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia have been reported to enhance the plant growth [2][3][4] .There are several PGPR inoculants currently commercialized that seem to promote growth through at least one mechanism; suppression of plant disease (termed Bioprotectants), improved nutrient acquisition (Biofertilizers), or phytohormone production (Biostimulants).Inoculant development has been most successful to deliver biological control agents of plant disease i.e. organisms capable of killing other organisms pathogenic or disease causing to crops.
Pseudomonas sp. is ubiquitous bacteria in agricultural soils and has many traits that make them well suited as PGPR.The most effective strains of Pseudomonas have been Fluorescent Pseudomonas sp. and have been studied for decades for their plant growth-promoting effects through effective suppression of soil borne plant diseases.Among various biocontrol agents, Fluorescent pseudomonads, equipped with multiple mechanisms for biocontrol of phytopathogens and plant growth promotion, are being used widely as they produce a wide variety of antibiotics, chitinolytic enzymes, growth promoting hormones, siderophores, HCN and catalase, and can solubilize phosphorous [5][6][7][8] .Considerable research is underway globally to exploit the potential of fluorescent Pseudomonads.Specific strains of the Pseudomonas fluorescensputida group have recently been used as seed inoculants on crop plants to promote growth and increase yields 9 .
Biological control has gained considerable attention and appears to be promising alternative to chemical control.Biological control has significant potential in terms of both environmental and economic issues for incorporation into organic temperate fruit production.A number of studies have demonstrated benefits resulting from application of plant growth promoting and disease suppressive rhizobacteria to subsequent growth of apple in replant soil.Replant problem is caused by abiotic and biotic factors 10 .Various species of fungi like Fusarium equiseti, F. oxysporum, F. solani, Rhizoctonia sp., Cylindrocladium sp., Rosellinia necatrix, Penicillium claviforme, P. janthinellum, Phytophthora sp., Pythium sp., Cylindrocarpon sp., and of nematodes like Pratylenchus penetrans and Xiphinema sp. have been found associated with replant disease by various workers 11 .Root rot is a very serious soil-borne disease infecting temperate fruits especially apple.It is caused by Rosellinia necatrix Berl.ex Prill.(Anam.Dematophora necatrix Hartig).Agarwala 12 (1961) observed this disease on apple trees in Himachal Pradesh.The annual losses estimated due to this disease are about Rs. 1.3 million 13 which are expected to be much more as the disease is reported to occur in all apple growing regions of the country.
A diversity of bacterial species has been identified that suppress individual causal elements and enhance growth of plants in replant soil.Biological control of Phytophthora cactorum which contributes to replant disease has been reported in response to application of Enterobacter aerogenes 14 .Biological control of soil borne pathogens with antagonistic bacteria has gained considerable attention and appears to be promising alternative to chemical control.So the present research was undertaken to isolate and identify a PGPR inoculant that will promote growth and can act as bioprotectant, biofertilizer and a biostimulant.

Collection of soil samples
Soil samples were collected from rhizosphere of apple plants from Shimla district which is the main apple belt of Himachal Pradesh.The randomly selected apple plants were used for collection of soil samples along with feeder roots from different replant sites of Shimla district viz., Magawta, Shrontha, Siao and Deola.The roots and soil samples from each tree basin were drawn from 15 cm soil depth with the help of soil auger.The samples were carried to the laboratory in polythene bags and were stored in refrigerator at 4° C till further analysis.

Isolation and purification of fungal pathogen
The rhizospheric soil samples were used for isolation of fungal pathogens.The media employed for the isolation of fungus was Potato Dextrose agar (PDA).The plates were incubated at 28 ± 2ºC for 3-4 days and the well isolated fungal colonies appeared on plates were purified by subsequent sub culturing.The purified colonies were grown on PDA slants and stored thereafter at 4ºC and were subcultured periodically on the same media at 28 ± 2ºC.The predominant isolates were identified on the basis of their spore arrangement and fungal isolates were finally identified from National Centre for Fungal Taxonomy, New Delhi.

Isolation of fluorescent Pseudomonas, media and growth conditions
Isolation of fluorescent Pseudomonas sp. was made from apple rhizhosphere in Shimla district of Himachal Pradesh (India).Ten grams of each rhizospheric soil sample were mixed and shaken in 90 ml sterile distilled water blank in 250 ml Erlenmeyer flask for 20-30 minutes to obtain standard soil suspension.Isolation of fluorescent Pseudomonas sp. was made by following the serial dilutions and pour plate method using the specific King's B medium 15 .Plates were incubated at 28±2°C and enumerations of fluorescent Pseudomonas colonies were done after 48 h of incubation.
These were identified on the bases of morphological, biochemical and physiological tests viz., Gram's staining, pigment production, oxidase test, catalase test, gelatine liquification, denitrification test and growth at optimum temperature i.e 4°C and 41°C.

Evaluation of PGPR traits
All the Pseudomonas isolates from apple rhizosphere were qualitatively and quantitatively characterized by standard protocols for the presence of PGPR traits viz., phosphate solubilisation, siderophore production, HCN production, ammonia production, antifungal activity and for production of hydrolytic enzymes which are known to play an essential role in growth promotion of plants.

Siderophores detection and quantification
Siderophore production by Pseudomonas isolates was detected by observing orange halos production around the bacterial colony on CAS agar plates 20 after 72 h of growth.For quantification of siderophores, to 0.5 ml of cell free culture supernatant of each Pseudomonas isolate grown in liquid CAS medium, 0.5 ml of CAS reagent and 10µl of shuttle solution was added and absorbance was measured at 630 nm against a blank.Siderophores content was expressed as percentage siderophore units using the formula: % Siderophore units (%SU) = (Ar-As)/ Ar × 100 Where Ar = absorbance of reference at 630 nm (CAS reagent) and As = absorbance of sample at 630 nm HCN and ammonia detection Bacterial cultures were tested for the production of ammonia in peptone water.Freshly grown cultures were inoculated in 10 ml peptone water and incubated for 48-72 h at 28±2°C.After incubation, Nessler's reagent was added in each tube.Development of brown to yellow colour will be a positive test for ammonia production 21 and King's B medium amended with 0.44% of Glycine was used for detection of hydrogen cyanide following the method of Bakker and Schippers 22 (1987)

Antifungal activity
Isolated bacterial cultures were tested for growth inhibitory effect on the mycelium growth of Dematophora sp., Fusarium sp., Phytopathora sp, Pythium sp. and Rhizocotina sp, the major fungal pathogens of apple plants by well plate assay method 23 .

Chitinase and Protease activity
Chitinase activity was measured according to Chernin 24 et at.(1995) and protease activity according to Kaur 25 et at.(1988).

DNA Extraction and amplification of 16S rRNA gene fragments
The fluorescent Pseudomonas isolate DE-18 was selected on the basis of overall plant growth promoting and disease suppressing activities for their genotypic characterization by 16S rRNA gene sequencing.Genomic DNA was extracted using conventional method (Phenol: Chloroform method) and the DNA was quantified via agarose gel electrophoresis (using 1.0% agarose).16S rRNA gene analysis was done using Pseudomonas specific oligonucleotide primer sequences viz., FP-1 (20-mer): GGTCTGAGAGGATGATCAGT and RP-1 (18-mer): TTAGCTCCACCTCGCGGC in MJ Mini BIO-RAD personal thermal cycler-100 (PTC-100) with a total of 35 cycles.These primers were designed and were used to find their taxonomic affiliation to fluorescent Pseudomonas species of group I which mainly comprise Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas fluorescence.The PCR amplification was carried out in 0.2 ml PCR tubes with 50 µl reaction volume consisting of following components: 5.0 µl Taq buffer A, 3.0 µl dNTP Mix, 1.0 µl FP-1, 1.0 µl RP-1, 0.40 µl Taq DNA polymerase, 2.0 µl genomic DNA, 37.6 µl double distilled water.A PCR program was implemented as follows: denaturation at 94ºC for 1min, two minutes annealing at 55ºC and extension for 2 min at 72ºC.The final extension was for 10 min at 72ºC.For DNA sequencing, eluted amplified DNA product was first purified followed by sequencing in B. Genei, India pvt.Ltd. (Bangalore)

Phylogenetic analysis
The sequencing results were aligned to 16S rRNA gene sequences from GenBank database at NCBI (National Centre of Biotechnological information) through web site http:// www.ncbi.nlm.nih.gov using BLAST program for screening of sequence similarity.Phylogenetic reconstruction was accomplished with the

RESULTS AND DISCUSSION
In the present research work, five isolates of fluorescent Pseudomonas sp. were isolated from Apple rhizosphere of Magawta, Shrontha, Siao and Deola (Shimla distt., Himachal Pradesh) Table 1.These isolates were further subjected to morphological, physiological and biochemical characterization.All the isolates of fluorescent Pseudomonas were studied in detail for colony, colour, growth type, fluorescence, and cell shape.It was evident from the observations that all the five isolates viz., M1, M2, SH1, SO1 and DE-18 produced round shaped colonies with an entire margin and rod shaped cells Table 2. Through morphological and biochemical identification, all the isolates were found to be gram-negative, aerobic, oxidase positive, catalase positive with a fluorescent pigmentation.However, negative responses were also identified for some Pseudomonas isolates such as for gelatin hydrolysis and their ability to grow at 4°C and 41°C.The isolate DE-18 could not hydrolyse gelatine and isolates M1, SH1 and DE-18 did not showed growth at 41°C Table 3. Reynolds 26 (2004) also characterized isolates on the bases of biochemical tests including oxidase, catalase, gelatin hydrolysis, nitrate reduction and performing growth at 4 and 41°C and identified them as P. fluorescens.
The fungal cultures from rhizospheric soil were purified by single spore and hyphal tip isolation technique.The identification of the isolated fungus was done by studying the morphological characters and by microscopic examination of each isolate.The cultures were sent to National Centre for Fungal Taxonomy, Inderpuri, New Delhi for identification to species level and the fungi were identified as Fusarium solani and Nigrospora oryzae Table 4 Figure 1.
All the fluorescent Pseudomonas isolates were identified as potential phosphate solubilizers based on their capacity to solubilize tricalcium phosphate [Ca 3 (PO 4 ) 2 ] by the formation of halo Where, C: Growth of mycelia in control; T: Growth of mycelia in test **Protease and chitinase activities were expressed in terms of mm diameter of clear zone produced around the well by 100µl of 72 h old supernatant at 28+2 o C on skim milk agar after 48 h and minimal agar amended with 1% chitin after 4 days flooded with iodine solution respectively.zone on Pikovskaya's agar medium.The maximum P solubilisation was recorded by DE-18 (425 µgml -1 ) compared to other isolates M1, M2, SH1 and SO1 (362.5, 350, 162.5 and 225 respectively) Table 5. Verma 27 et al. (2014) also recorded phosphate solubilizing activity of Pseudomonas isolates in the range of 155µg/ml to 410 µg/ml.Most of phosphorus in soil is present in the form of insoluble phosphates and cannot be utilized by the plants.PGPR have been shown to solubilize precipitated phosphates and enhance phosphate availability to plant that represent a possible mechanism of plant growth promotion under field conditions 28 .
All the tested isolates of fluorescent Pseudomonads were positive for the production of siderophores, HCN, ammonia, lytic enzymes and plant growth-promoting hormones viz., auxins, gibberellins and cytokinins.The maximum siderophore production was showed by isolate DE-18 (42.18 %SU) and minimum by SO1 (4.68 %SU) (Table 5).In the development of the antagonism the siderophore production of the bacteria has an important role and such bacteria may function as stress factors including local and systematic host resistance 29 .Iron competition in Pseudomonads has been intensively studied and the role of the pyoverdine siderophore produced by many Pseudomonas sp. has been clearly demonstrated in the control of Pythium and Fusarium species 30 .Different types of siderophores in Pseudomonas species have antagonistic effect against F. graminearum 31 .Microbial production of HCN has been suggested as an important antifungal feature to control root fungi pathogen.Cyanide acts as a general metabolic inhibitor to avoid predation or competition.The host plants are generally not harmfully affected by inoculation with HCN Fig. 3. Phylogenetic tree of Pseudomonas sp.strain DE-18 and related species constructed on the basis of 16S rDNA gene sequence using the neighbor-joining method producing bacteria and rhizobacteria can operate as biological control agents 32 .
The production of IAA by PGPR generally affects the root system, increasing the size and number of adventitious roots and also the root subdivision, enabling a bigger soil amount to be exploited by the roots, thus providing large amounts of nutrients accessible to the plant 33 .The isolates DE-18 and M2 produced 14.0 and 10.0 µgml -1 of auxins respectively, whereas all the other isolates produced auxins between 2.0 and 8.0 µgml -1 (Table 5).Similar results were also observed by Sharma 34  The production of indole acetic acid by the strains of Pseudomonas sp.responsible for increasing root elongation was also reported by O' Sullivan and O' Gara 35 , (1992).However, IAA production by PGPR can vary among different species and strains, and it is also influenced by culture condition, growth stage and substrate availability 36 .All the isolates also produced gibberellins and cytokinins in the range of 100-250 µgml -1 and 30-600 µgml -1 respectively (Table 5).Thakur 37  The results of inhibition of mycelium growth of the assayed phytopathogenic fungi showed inhibition against only two fungal pathogens among the Fusarium oxysporium, Pythium ultimum, Dematophora nectarix, Phytopathora cactoram and Rhizocotina sp.From the assayed five bacterial cultures, the maximum inhibition of mycelium growth of 38.46 % and 36.18 % was found by isolate DE-18 against Dematophora nectarix and Phytopathora cactoram Table 6 and Figure 2. Pseudomonas putida and Pseudomonas fluorescens, are frequently isolated from soil and plant tissue and reported as potential biocontrol agents of phytopathogens [38][39][40] .Based on the antagonistic potential and other multifarious plant growth promoting characteristics isolate DE-18 was genotypically characterized by 16S rDNA gene sequencing.
The potential isolate DE-18 was identified based on their 16S rRNA gene using the universal primer set and BLAST analysis.The 16S rDNA based phylogenetic analysis demonstrated that the isolate DE-18 belonged to the genus Pseudomonas sp. Figure 3 described the relationship between the isolated strain and the nearest phylogenetic relatives.Similarity calculations after neighborjoining analysis indicated that the closest relative of isolate DE-18 was Pseudomonas putida (90 %).The determined 16S rDNA sequence of strain Pseudomonas putida DE-18 was deposited in the GenBank nucleotide sequence databases under accession number KU139388.
This study is assumed to be important as the agriculturally beneficial antibiotic-producing P. putida could be one of the potential candidates in the development of microbial pesticides for sustained crop productivity.Hence this P. putida DE-18 strain have a potential of being developed as a bio-inoculant for application in a agriculture and horticulture crops.VERMA
et al. (2013) also observed the production of gibberellins and cytokinins by fluorescent Pseudomonas isolates in the range 15.20 to 179.48 µg/ml and 51.20 to 179.48 µg/ml respectively.

Table 1 .
Details of soil samples and location used for isolation of fluorescent Pseudomonas sp

Table 2 .
Morphological characterization of fluorescent Pseudomonas sp.isolated from apple rhizosphere

Table 3 .
Physiological and biochemical characterization of fluorescent Pseudomonas sp.isolated from apple rhizosphere (-) Indicates negativity of test (+) indicates positivity of test

Table 4 .
Identification of fungal pathogens isolated from apple rhizosphere **The siderophore unit (% SU) expressed as percent reduction in blue color of chrome azurol-S as compared to reference i.e. % SU= (Ar-As)/Ar×100 where, Ar = Absorbance of reference solution at 630 nm; As = Absorbance of test solution at 630 nm