Screening and Characterization of Plant Growth Promoting Endophytic Microbes of Tomato ( Solanum lycopersicum L.) combats Pythium aphanidermatum

About 45 bacteria, 9 fungi, 3 yeast, and 1 actinobacterium were isolated from plant tissues of tomato and screened for their plant growth-promoting traits and antagonistic activity against damping-off fungus Pythium aphanidermatum . The bacterial isolate, Bacillus pumilus TEB10 exhibited pathogen inhibition of about 55 and 79%, while yeast isolate Candida tropicalis TEY1 showed 30 and 68% in the dual plate and inverted plate assay, respectively. In addition, the Bacillus pumilus TB10 showed siderophore, HCN, cellulase, and phosphate solubilization activities. Similarly, yeast isolate Candida tropicalis TEY1 exhibited catalase, phosphate, and zinc solubilization activities. The sterol biosynthesis pathway end product ergosterol was profiled in secondary metabolites of P. aphanidermatum , suggesting the potentiality of this pathogenesis.


INTRODUCTION
Tomato (Solanum lycopersicum L.) is one of the important vegetable crops cultivated worldwide. Pathogens infect the tomato plant at different stages and caused yield loss and sometimes plant death. Among them, one of the important nursery pathogens is Pythium spp., an oomycete that causes damping-off disease. The two types of damping-off, such as preemergence and postemergence, make a loss of seed germination by killing the seed before emergence, and after emergence it kills the seedling by the formation of a water-soaked lesion in the collar region. It is more widespread in the case of moist environments with high relative humidity. A tender stem with cotyledonary leaves is more susceptible to the pathogen. Reducing the inoculum in the seeding medium may minimize the disease infection. Several researches comprise of understanding different mechanisms involved in reducing the disease inoculum are the utilization of chemicals and microorganisms. These microorganisms act as biocontrol agents isolated *Corresponding author's e-mail: sridharanaps@gmail.com from the phyllosphere, rhizosphere, and endophytes (Strobel, 2006). The endophytes are found to be second genome of plants and, as a whole are called as phytobiome (Lopez et al., 2018). Endophytes play a significant role in alleviating the biotic stress caused by pathogens.
Endophytic microbes live entire or part of their life cycle inside the tissues of living plants and cause unapparent and asymptomatic infections in plant tissues, but cause no symptoms of disease (Wilson, 1995). Similarly, endophytes are isolated from inner plant parts or from surface-disinfected tissues, which do not visibly harm the plant (Hallmann et al., 1997). Endophytes enter plants from different entry points and live inside without harming their host (Malfanova et al., 2011). They may originate from seeds and vegetative material and enter the phyllosphere and rhizosphere. They are transmitted vertically between generations by seed and vegetative material. In seeds, they can persist for years and arise during favorable conditions (Rudgers et al., 2009). The microbiome richness outside the host alters the endophyte population within the host. The endophytic microbiome changes even within same plant species with different varieties. The rich nutrients inside the plant facilitate favorable environment for endophyte's survival.
The endophytic microorganisms comprise bacteria, actinobacteria, filamentous fungi, and yeast, forming the microbiome. Cultural and molecular methods are employed for the detection, differentiation and identification of endophytes inside the host (Rosenblueth and Martinez-Romero, 2006). Among the endophytes, the population of bacteria tends to present higher specifically in root than in the shoot region (Soad A. Algam, 2005). Several endophytes are involved in plant growth promotion activities and also suppress the disease-causing pathogens associated with the host plant (Turner et al., 2013). The plant growth-promoting attributes include fixing atmospheric nitrogen, production of enzymes to solubilize the available nutrients, synthesis of plant hormones, production of siderophore, lytic enzymes, and secondary metabolites to inhibit the plant pathogen and to induce systemic resistance (Malusa et al., 2016).

Surface sterilization of plant samples
Shoot and root samples of tomato (var. PKM 1) were collected from TNAU orchard and immediately used for isolation of endophytic microbes. The samples were washed in tap water to remove the adhering soil and debris and allowed to dry in shade. The plant parts were separated as shoot and root and its weight was measured. The samples were placed at laminar air flow chamber and cut into 2 cm pieces. It was first soaked in 1 % Sodium hypochlorite (NaOCl) for 1 min for external sterilization and then rinsed in sterile distilled water, followed by soaking at 70 % ethanol for 30 sec and thrice rinsed in sterile distilled water. It was allowed to dry on sterile filter paper. About 100 µL last wash water was plated in the medium for confirmation of sterility and to make sure that no epiphytic microbes associated with the sample (Cao et al., 2004).

Isolation of endophytes from tomato
The samples were placed in separate mortars containing 5 mL of sterile distilled water, crushed finely, and diluted serially to 10 -3 and 10 -4 . About 100 µL of each diluent was poured into Petri plate containing respective media for fungi (Potato dextrose agar, Rose Bengal agar), bacteria (Nutrient agar, Tryptic soy agar), yeast (Yeast extract and malt extract agar) and actinobacteria (Kenknight agar, actinomycetes agar). The plates were replicated thrice and incubated at 28±2 ℃ for 15 days. The colony was observed periodically, and CFU was calculated. The purified colonies were stored in glycerol stock at -80 ℃.

Plant pathogen
The damping-off pathogen, Pythium aphanidermatum was obtained from the Department of Plant Pathology, Tamil Nadu Agriculture University, Coimbatore.

Dual plate assay
The dual plate assay was conducted according to the method of Rabindran and Vidyasekaran, (1996). Mycelial plugs of 8 mm diameter of the pathogen were placed near the periphery of PDA plates, and antagonists such as bacteria, yeast, and actinobacterium were streaked respectively on the opposite end. In case of antagonistic fungi, an 8 mm mycelial plug was placed. A plate with pathogen alone served as control. These plates were inverted and maintained at 28 ± 2 ºC, for five days. Percent inhibition was calculated using the formula, where PI = per cent inhibition; C = radial growth of the pathogen in the control plate; T = radial growth of the pathogen in treatment

Inverted plate assay
An 8 mm mycelial plug of the endophytic fungi was placed at the center of a 90 mm bottom Petri plate containing PDA medium. The inoculum was spread in the bottom Petri plate for endophytic bacteria and yeast isolates. The mycelial disc of the pathogen was placed on another bottom plate containing PDA. The pathogen plate was upturned and positioned over the antagonist plate. These plates were sealed with parafilm to prevent the volatile compounds from escaping. The control consisted of a pathogen plate upturned and positioned over an uninoculated PDA plate (without antagonist). These plates were incubated

HCN production
The effective isolates of bacteria (TB10) and yeast (TEY1) were screened for the production of hydrogen cyanide. The nutrient agar media was amended with 4.4 g glycine -1 was inoculated with effective isolate. A Whatman no.1 filter paper soaked in 2 % sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with parafilm and incubated at 28±2 °C for 4 days. The development of yellow to reddish brown color indicated HCN production (Tsegaye, 2019).

Siderophore assay
Chrome Azurol Sulphonate (CAS) assay was used for the assessment of siderophore production (Schwyn and Neilands, 1987). The effective isolates of bacteria (TB10) and yeast (TEY1) were streaked in a single line on the center of the plate containing CAS medium. The uninoculated plate act as control. These plates were incubated at 28 °C for 5-7 days for the formation of an orange zone around the bacterial colonies.

Hydrolytic enzymes activity Pectinase Assays
Bacteria (TB10) and yeast (TEY1) were separately streaked on the Petri plate containing one fourth of nutrient agar medium amended with 0.5% pectin and incubated at 28± 2 °C. The appearance of the halo region after 2-4 days indicate pectinase positive (Tsegaye, 2019).

Cellulase assay
Bacteria (TB10) and yeast (TEY1) were streaked on the sodium carboxymethyl cellulose (CMC) amended medium and incubated for 2-3 days at 28 ± 2 °C. After incubation period, the plates were then flooded with Gram's iodine to visualize the halo zone around the colony indicates positive result (Tsegaye, 2019).

Protease assay
Proteolytic activity was observed in the medium containing skim milk. The overnight grown culture was streaked onto the medium and incubated at 28 ± 2 °C for 2-3 days. The formation of clear halo zone around the colony indicates positive for protease activity (Tsegaye, 2019).

Amylase assay
The effective isolate of bacteria (TB10) and yeast (TEY1) were streaked on soluble starch agar medium and incubated at 28 ± 2 °C for 2-3 days. The plates were then flooded with iodine solution for a minute and then poured off. Iodine reacts with starch to form a blue compound, and later it became colorless around the colony, indicating the production of amylase (Tsegaye, 2019).

Catalase assay
Petri dish containing overnight grown bacteria (TB10) and yeast (TEY1) were poured with hydrogen peroxide. The formation of oxygen molecules indicates positive reaction (Amaresan, 2012).

Zinc and Phosphate solubilization
The overnight grown culture was spot inoculated on the zinc and phosphate solubilization medium and incubated at 28 ℃. The appearance of halo region shows a positive effect for solubilization.

Collection and extraction of non-volatile soluble metabolites
Pythium aphanidermatum was grown in PD broth in three replicates and incubated at 28 ± 2 ºC for 7 days. An uninoculated PD broth served as control. After passing through muslin cloth, the culture filtrate was centrifuged at 6000 rpm for 15 min, mixed with equal quantity of ethyl acetate, and incubated overnight under shaking conditions. The solvent phase was separated and concentrated in a vacuum flash evaporator. After sufficient drying, the crude metabolites were extracted using 1 mL methanol and filtered through a 0.2 µm bacteriological membrane filter for GC-MS analysis (Meena, 2017).

Non-volatile soluble metabolites analysis by GC-MS
The purified crude methanolic extract was subjected to GC-MS analysis in a Perkin Elmer GC-MS Clarus® SQ 8 equipped with DB-5MS (Agilent, USA) capillary standard non-polar column with dimensions 0.25 mm OD x 0.25 µm ID x 30 m length. The instrument was set to an initial temperature of 40˚C, and the injection port temperature was ensured at 220 ˚C, interface temperature set 250 °C, source kept at 220 °C, oven temperature-programmed as 75 °C for 2 min, 150 °C @ 10 °C/min, up to 250 °C at 10 °C per min. The GC conditions were: 1:12 split, helium carrier at 20 psi. The MS conditions were: positive ion mode, electron impact spectra at 70 eV.

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The mass spectral scan range was set at 50 to 600 Da. The MS peaks were determined by their scatter pattern. The linear regression coefficient was used to calculate the concentrations in the samples from peak areas obtained in the chromatographs. The bioactive molecules were identified by comparison of mass spectra with NIST 08 Mass Spectra Library (National Institute of Standards and Technology). The name, molecular weight, and structure were ascertained from NIST, PubChem, and HMDB databases (Leylaie and Zafari, 2018).

Statistics analysis
All the experiments were analyzed independently. The treatment means were compared by Duncan's multiple range test (DMRT) Gomez et al. (1984). All the data were analyzed statistically by AGRESS and interpreted.

Morphology of P. aphanidermatum
The microscopic observation of Pythium aphanidermatum revealed aseptate and colorless hyphae with the presence of antheridia and oogonia. This sexual reproductive structure observed was similar to the P. aphanidermatum causing damping-off in Cucumber Priyanka et al. (2019) (Figure 1a, b). The pathogenicity test on tomato showed the symptoms of shrinking of the stem near the collar region and toppling down of seedling due to rotting. (Figure 1c).

Isolation of endophytic microorganisms
Endophytic microorganisms were isolated from the root and shoot region of the tomato to a total of 45 bacteria, 9 fungi, 3 yeast, and 1 actinobacterium registering maximum endophytes from the root region (Table 1). All the isolated endophytes were differentiated based on morphology (Table 2). Bacteria were considered as the major group of endophytes in the tomato plants, with five major phyla considering Proteobacteria was the most represented (90 %), followed by Actinobacteria (1.5 %), Planctomycetes (1.4 %), Verrucomicrobia (1.1 %), and Acidobacteria (0.5 %) (Romero et al., 2014). Sen et al, (2019) isolated yeast endophyte Rhodotorula mucilaginosa JGTA-S1 from Typha angustifolia root. El-Tarabily et al. (2008) isolated 4.56 log10 CFU g -1 of actinobacteria from the fresh root of Cucumber and observed that the colonization was more in the root than in the shoot region.

Screening of endophytes against damping-off pathogen
The isolated endophytes were screened for their antagonistic property against P. aphanidermatum by dual plate and inverted plate assay. Among the 45 bacteria, nearly 17 were effective against the damping-off pathogen with 50 to 70 % inhibition. The bacterial isolate TEB11 exhibited a maximum inhibition of 61.66 % in the dual plate assay, and the isolate TEB10 inhibited the pathogen by 79.28 % in the inverted plate assay. The dual plate assay is the direct interaction of both organisms, whereas inverted plate assay is the indirect effect of volatile mediated inhibition. The inverted plate assay registered a maximum efficiency for inhibiting pathogen growth (Table 3). Priyanka (2019) recorded an inhibition of P. aphanidermatum at about 40 %, and 36 % by endophytic bacteria such as Pseudomonas aeurginosa (MPP EB 4 and MPP EB 3) and Achromobacter denitrificans (MPP EB 8), respectively. Similarly, Lopez et al. (2018) observed that among 11 seed endophytic bacteria, only six (4 Bacillus sp., Micrococcus sp., Paenibacillus polymyxa) bacteria efficiently inhibited the growth of pathogens viz., Alternaria sp., Stemphylium sp. and Corynespora sp. Among the 6 species, only three strains of Bacillus sp. inhibited the mycelial growth by both direct and volatile mediated interaction. Muthukumar et al. (2010) observed that the endophyte of chilli, Pseudomonas fluorescens EBS 20 inhibited the damping-off pathogen by 76 % with wide inhibition zone in a dual plate assay.
Among the 9 endophytic fungi screened for its efficiency, it was observed that the growth of the pathogen P. aphanidermatum was faster than that of the endophytes. Hence, the inhibition was between 10 to 25 % in the dual plate assay and less than 15 % in the inverted assay. The endophytic fungi, Aspergillus terreus inhibited the P. aphanidermatum with an inhibition zone of 8.7mm and reduced the spore production (Halo et al., 2018). Vinayarani and Prakash, (2018) found that endophytic T. harzianum TharDOB-31 inhibited the damping-off fungi, P. aphanidermatum registering 70 % inhibition.
In dual plate assay, the endophytic yeast inhibited the pathogen growth of up to 30 % inhibition. Whereas, the inverted plate assay demonstrated up to 68% inhibition exhibiting changes in the morphology of the mycelia. While other yeast isolates such as TEY2 and TEY3 didn't inhibit pathogen growth effectively. In inverted plate assay, when the exposure of volatile was removed,

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the pathogen gets back to its normal growth, suggesting that the volatile act as a fungistatic rather than fungicidal. A similar result was obtained for the rhizospheric yeast Torulaspora globosa DMKU-RP31 which produce significant volatile compounds that inhibited the plant pathogens Fusarium moniliforme, Helminthosporium oryzae and Rhizoctonia solani (Nutaratat et al., 2014). The endophytic yeasts such as Saccharomyces, Torulaspora, and Debaryomyces isolated from Tagetes erecta, Azadirachta indica and Carica papaya inhibited Fusarium solani, F. oxysporum and M. phaseolina (Fareed et al., 2019).
The effective isolate among the bacteria against P. aphanidermatum was Bacillus pumilus TEB10, which showed maximum inhibition in volatile mediated interaction of about 79 % and also recorded significant inhibition in a dual plate assay. Among the yeast, the isolate Candida tropicalis TEY1 was effective in inhibiting the pathogen growth in volatile-based interaction than in dual plate (Figure 2). Since indirect interaction is marked effective than direct interaction, the volatile based biocontrol mechanisms may attribute efficient management strategy. Amaresan et al. (2012) observed that, among the 82 endophytic bacteria, the isolates BECS3, BECS6, and BECS7 showed multiple attributes and demonstrated plant growth promotion properties through a tomato and chili-based bioassay under greenhouse conditions.

Identification of effective isolate
The PCR analysis of endophytic yeast TEY1 resulted the amplicon size of around 600bp and showed 99.8 % similarity with Candida tropicalis whereas the effective bacterial isolate TEB10 resulted the amplicon size of 1500bp and showed 99.8 % similarity with Bacillus pumilus when compared with isolates deposited in NCBI database.

Plant growth-promoting traits
The effective bacterial isolate B. pumilus TEB10, showed positive for siderophore activity by changing the blue background to an orange halo around the bacterial colony whereas C. tropicalis TEY1 did not show siderophore activity. Lopez et al. (2018) observed that seed endophytic bacteria Bacillus sp. and Acenitobacter sp. produce siderophore. Protease activity was observed for B. pumilus TEB10 by the formation of the halo zone around the colony but not in C. tropicalis TEY1.
Catalase activity was detected for the yeast C. tropicalis TEY1 by the formation of oxygen when H2O2 was added to the culture. The bacterium (B. pumilus TEB10) showed positive for HCN and cellulase activity whereas the yeast C. tropicalis TEY1 was devoid of HCN and cellulase. B. pumilus TEB10 showed positive for phosphate solubilization, while C. tropicalis TEY1 did not solubilize phosphate (Table 4 and Figure 3). Amaresan et al. (2012) isolated 82 bacterial endophytes in which many produced siderophore, solubilized phosphate, and not all isolate exhibited amylase, protease, cellulase, and lipase activities. Nutaratat et al. (2014), observed that the endophytic yeast from rice and sugarcane solubilize phosphate, zinc, and showed catalase positive. Rodotorula mucilaginosa isolated from T. angustifolia solubilize calcium phosphate and improved rice growth when inoculated in low phosphate media (Sen et al., 2019). Whereas in the case of zinc solubilization, the bacteria reacted negative, and yeast reacted positive. Abdallah (2018) reported that the isolated endophytic bacteria solubilize phosphate, while some strains, such as Pseudomonas geniculata CT19 (KR818061) and Bacillus subtilis SV5 (KR818065) did not solubilize phosphate.

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In this study, ergosterol was profiled in the metabolites of P. aphanidermatum at same retention time and mass value compared to true fungi. Trigos et al. (2005) reported for the first time that the Peronosporales genus Phytophthora drechsleri produced ergosterol.

Conclusion
The present research stated that different endophytic microorganisms were residing inside the plant with plant growth-promoting and antagonistic traits.
Among the isolated endophytic microorganisms, a bacterium (B. pumilus TEB10) and a yeast (C. tropicalis TEY1) exhibited effective antagonistic activity and PGP traits. These effective endophytes may be used as bioinoculant in combination with alleviating biotic stress and plant growth promotion.

Funding and Acknowledgment
This study was supported by the Ministry of Human Resource Development, Government of India through MHRD-FAST-CoE (F.No.5-6/2013-TSVII) sanctioned to SU.

Ethics statement
No specific permits were required for the described field studies because no human or animal subjects were involved in this research.

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Consent for publication
All the authors agreed to publish the content.

Competing interests
There were no conflict of interest in the publication of this content Data availability All the data of this manuscript are included in the MS. No separate external data source is required. If anything is required from the MS, certainly, this will be extended by communicating with the corresponding author through corresponding official mail;