Nematicidal rhizobacteria with plant growth-promoting traits associated with tomato in root-knot infested polyhouses

In polyhouse cultivation of tomato (Solanum lycopersicum) in root-knot (Meloidogyne incognita) infested soils, several accessions remained free of nematode infection. It was hypothesized that such plants that were free of root-knot infection in the selected polyhouses were possibly harboring potent nematode antagonistic microbes. Therefore, attempts were made to isolate root-associated rhizobacteria from nematode infected and uninfected roots of 7 tomato accessions. Eighty morphologically distinct isolates were multiplied in nutrient broth for 48 h to test the presence of nematotoxic metabolites. Laboratory bioassays were conducted using their cell-free filtrates to observe mortality in M. incognita juveniles (J2s) at 24 h. The most potent ones were identified through molecular characterization (16S rDNA) and assayed for plant growth promotion traits, seed germination and seedling vigor and bioefficacy against M. incognita infecting polyhouse tomato. Rhizobacterial densities were estimated from the root samples using 3 media. In all the media, the average numbers of colony forming units (cfu) from uninfected roots were significantly higher than the infected roots. The maximum numbers of colonies were observed in soil plate count agar. The extracellular metabolites caused juvenile mortality in the range of 60.67–100% from isolates associated with nematode uninfected roots of accessions H308, H299 and H266, as compared to isolates from nematode infected roots of accessions H195, BSS99, H178/F4 and H88, which caused mortality in the range of 52.33 to 73.67%. Four isolates (B9, B16, B26 and B31) caused 100% J2 mortality, followed by 13 isolates (B7, B8, B13, B14, B17, B18, B21, B22, B25, B28, B29, B32 and B33) that caused 90–95%, 12 isolates caused 80–85% and 51 isolates caused < 85% J2 mortality, when exposed to bacterial metabolites in cell-free filtrates for 24 h. The isolates B9, B16, B26 and B31 were identified based on 16S rRNA sequence analysis as Bacillus pumilus, B. megaterium, B. subtilis and B. cereus, respectively. These 4 bacterial isolates possessed plant growth promotion traits like production of IAA, ammonia, catalase and chitinase with the ability to solubilize zinc and phosphate. These enhanced tomato seed germination and seedling vigor, and their application in soil resulted in significant increase in root and shoot length of tomato seedlings. In the present investigation, the 21% bacterial colonies (17 isolates) that caused nemato toxicity in the range of 90 to 100% and another 15% (12 isolates) that caused juvenile mortality in the range of 80 to 90% were from uninfected tomato accessions, indicating their role in protecting the plants against root-knot nematode (RKN) infection. The 4 bacterial isolates characterized from uninfected plants possessed high nematicidal potential with plant growth promotion (PGP) traits that enhanced tomato seed germination and seedling vigor index. The isolates can be utilized for root-knot nematode pest management in polyhouse cultivation.

Page 2 of 11 Devindrappa et al. Egyptian Journal of Biological Pest Control (2022) 32:51 Background In India, the National Horticulture Mission enacted the promotion of polyhouse and greenhouse; presently it occupies an area of 30,000 ha cultivating vegetables and ornamental crops (Reddy 2016). Though the cultivation of crops in polyhouses enhances the yield per unit area, the altered environment promotes the build-up of soil-borne pests and pathogens. RKN species, Meloidogyne incognita and M. javanica, are serious constraints to vegetable crops, especially tomato (Solanum lycopericum) under polyhouse cultivation (Phani et al. 2021) causing yield losses by 25-100% (Seid et al 2015). Application of chemical nematicides is the most common short-term management strategy for the nematode pest. However, several chemical nematicides are withdrawn from the market due to environmental concerns (Xiang et al. 2018). With increasing awareness on environmental protection and food safety, there is a need to exploit the microbial resources present in the rhizosphere. Variable infestation of the nematode is often observed in plants of the same genotype due to differences in the soil microbiome. The rhizosphere bacteria like Bacillus subtilis, B. licheniformis, B. megaterium, B. coagulans and Pseudomonas fluorescens directly and indirectly suppressed M. incognita due to nematicidal metabolites released by them (Xiang et al 2018). Various Bacillus spp. stimulated the plant growth promotion, enhanced acquisition of nutrients and excited plant defense mechanism against soil-borne pathogens and nematodes (Alooa et al. 2019). The composition and function of microbial communities in the rhizosphere can play a vital role in plant growth and nematode infection. Though the soil microbiome effects on root-knot infection using 16s RNA genes of bacterial communities have been reported for open field conditions (Zhou et al. 2019), the identification of culturable nematode antagonistic bacteria associated with uninfected roots in polyhouses has not been reported. In the present study, it was hypothesized that tomato plants free of root-knot infection in the selected polyhouses are harboring more nematode antagonistic bacteria. Therefore, root-associated rhizobacteria from nematode infected and uninfected tomato in polyhouse cultivation were isolated and evaluated for nematode antagonism. The most potent ones were identified through molecular characterization and assayed for plant growth promotion characters.

Isolation of root associated rhizobacteria from tomato crop Sample collection and isolation of rhizobacteria
Three polyhouses at ICAR-Centre for Protection Cultivation Technology, Indian Agriculture Research Institute (IARI), New Delhi, with root-knot infestation, were selected for sampling. Root samples were collected in December, from infected and uninfected 7 accessions of tomato. Five root samples from each row were pooled to form one composite sample, which was labeled and stored in plastic bags at 4 °C until processed. Whole plants were uprooted from the soil to examine nematode infection. Infected roots had heavy galling, as compared to uninfected roots that had very few galls or no galls. For the isolation of bacteria, 10 g of tomato roots was cut into small pieces and added to 90 ml of 0.85% sodium chloride (dilution 10 -1 ) and shaken at 200 rpm for 1 h in an orbital shaker (New Brunswick Scientific, USA). Suspensions were serially diluted with sterile distilled water to prepare 10 −4 dilutions, agitated and a 100 µl of suspension was plated on 3 nutrient media, viz. Soil Plate Count Agar (SPCA), Soil Extract Agar (SEA) and King's B procured from Hi Media, Mumbai, India. Plates were incubated for 12-48 h. in a BOD incubator at 30 °C using 5 replicates. The numbers of bacterial colonies were counted under a stereoscopic microscope at 10x. The morphologically distinct bacterial colonies were isolated based on shape, size, elevation, opacity and margin and multiplication on nutrient broth (NB) for nematicidal evaluation. The selected bacterial isolates were sub-cultured to purify and maintained in 15% glycerol vials, at − 80 °C.

In vitro screening of rhizobacteria from infected and uninfected plants for juvenile mortality
Freshly prepared NB was inoculated with the selected colonies and incubated on a shaker at 120 rpm for 48 h, followed by centrifugation at 10,000 rpm for 10 min. The supernatants were filtered using Whatman No.1 paper, followed by nitrocellulose syringe filter (0.20 µm) to obtain cell-free filtrates (CFF) with metabolites if any. The egg masses of M. incognita were hand-picked from culture pots maintained at greenhouse, Division of Nematology, ICAR-Indian Agricultural Research Institute (IARI), New Delhi. The Meloidogyne species was identified based on the perineal pattern structure of female nematode (Taylor et al. 1955).The egg masses were growth promotion (PGP) traits that enhanced tomato seed germination and seedling vigor index. The isolates can be utilized for root-knot nematode pest management in polyhouse cultivation.
Keywords: Rhizobacteria, Meloidogyne incognita, Bacillus spp., Extracellular metabolites, Plant growth-promoting traits Page 3 of 11 Devindrappa et al. Egyptian Journal of Biological Pest Control (2022) 32:51 kept for hatching at 21º C in a BOD incubator. A 1 mL of CFF was pipetted in each well of pre-sterilized multiwell plates, followed by addition of 100 J2s of freshly hatched M. incognita in 100 µl water and incubated at 25 °C, with sterile medium and sterile distilled water (SDW) as control treatments. The immobile J2s were counted at 24 h under a stereoscopic zoom binocular microscope and then transferred to distilled water for 24 h to confirm their mortality. Five replicates were maintained for each isolate.

Molecular Characterization of rhizobacterial isolates
The 24-h bacterial cultures were used for genomic DNA isolation by Zymo Research Crop quick DNA ™ Fungal/ Bacterial Miniprep kit (Cat. No. D6005) according to manufacturer's instructions and analyzed by 0.8% agarose gel. The purity and quantity of the DNA were checked in Nanodrop spectrophotometer (Thermo scientific, USA). The DNA yield was quantified in ng mL −1 , and the purity was assessed at 260/280 nm absorbance ratio. The PCR amplifications of 16S rRNA was performed using the universal primers were 27F (5-GTT TGA TCC TGG CTCAG-3) and 1494R (5-ACG GCT ACC TTG TTA CGA CTT-3) used for 16S rRNA gene amplification. The PCR was carried out using standardized protocol, and the amplicons were sent for Sanger sequencing to ABA Biotech Pvt. Ltd. The nucleotide sequences obtained were aligned with the existing nucleotide database of NCBI GenBank, and the reference sequences were retrieved. Phylogenetic tree was constructed using MEGA6 (Molecular Evolutionary Genetics Analysis version 6.0) software to understand the phylogenetic relationship of the selected 4 isolates. The nucleotide sequences were compared against nucleotide database using the NCBI BLASTn and EzTaxon SERVER 2.1 programs to identify the closest known taxa. The 16S rRNA gene along with the closest homology sequences was aligned using multiple sequence alignment program CLUSTAL W algorithm implemented in MEGA 6 SOFTWARE using default parameters. The phylogenetic tree was constructed by neighbor joining method, using MEGA 6 PROGRAM, and evolutionary distances were compared with the help of Kimura's parameter models. The 16S rRNA gene sequences obtained were submitted under accession numbers MZ675428-MZ675431.

Biochemical assays for plant growth-promoting (PGP) traits Indole acetic acid (IAA) production (Gordon and Weber 1951)
The IAA was estimated using 48-h bacterial cultures grown in NB at 30 °C. The cultures were centrifuged at 10,000 rpm for 10 min, and supernatant (4 ml) was mixed with 2 drops of ortho-phosphoric acid and 4 ml of Salkowski reagent (48 mL of 35% perchloric acid and 2 mL of 0.5 M ferric chloride solution) in 1:2 ratio (bacterial supernatant/reagent) at room temperature. The appearance of pink color indicated production of IAA. The optical density was observed at 530 nm after 25 min in 96-well microtiter plates using multimode reader (Thermo scientific, USA).

Phosphate and zinc solubilization test (Pikovaskya 1948)
The estimation of phosphate solubilization was conducted by spotting the bacterial cultures on the Pikovaskya agar plates and incubated at 30 °C for 5 days. The appearance of clear zone around the colony was considered positive for phosphate solubilization. Similarly, 24-h-old bacterial culture was inoculated on Tris mineral salts agar medium amended with 0.2% soluble zinc oxide (ZnO) and incubated for 3-5 days at 30 °C. The Zn solubilization was determined by appearance of clear zone around the bacterial colony (Gaur 1990).

Hydrogen cyanide (HCN) production (Bakker and Skipper 1987)
The 24-h-old bacterial cultures were inoculated on nutrient agar amended with glycine (0.44%) by spread plate method. Whatman No. 1 filter paper pre-saturated with 0.5% picric acid in 2% sodium carbonate solution (w/v) was placed in the lid of Petri plate, and the plates were sealed with parafilm (to prevent leakage), followed by incubation at 30 °C for 3-5 days. The HCN production was observed by change in color of filter paper from yellowish brown to deep brown.

Catalase production test (Schaad 1992)
The catalase test was performed by adding drop of 3% hydrogen peroxide to 48-h bacterial colony on a clean glass slide and mixed through sterile toothpick. The appearance of effervescence indicated catalase production.

Chitinase production test (Rodriguez-Kabana et al. 1983)
The M9 Agar media amended with 1% (w/v) colloidal chitin was prepared and plates were divided into 4 equal sectors, followed by spot inoculation with 24-h-old bacterial cultures and incubation at 30 °C for 5 days. The appearance of clearance zone around the bacterial colonies indicates chitinase production.

Ammonia (NH3) production test (Cappuccino and Sherman 1992)
The overnight bacterial culture (100 μl) was mixed, inoculated into 10 ml peptone water and incubated at 30 °C for 4 days. After incubation, an 1 mL of culture broth was added with 1 ml Nessler's reagent. Formation of a yellow to deep orange color was considered positive for ammonia production.

Seed germination test
In the seed germination test, by paper towel method, the bacterial isolates B9, B16, B26 and B31 were used for seed treatment of tomato variety Pusa Ruby. Seeds were surface sterilized with 0.1% HgCl2 (5 min), washed 4-5 time with sterile distilled water and soaked in liquid broth culture (10 8 cells/mL) for 1 h. Each seed was placed on wet blotting paper using sterile forceps, rolled the paper and incubated in BOD for 16 days at 30 °C. Seeds treated with sterile nutrient broth and distilled water served as control, and 5 replications for each isolate were maintained. Germination percentage and seedling vigor index were assessed as per protocols of International Seed Testing Association (1985).

Bioefficacy of rhizobacteria against M.incognita infecting tomato cv Pusa Ruby
The bioefficacy trial was conducted in 10 inch pre-sterilized plastic pots using 2-week-old seedlings of tomato

Statistical analysis
Data of colony forming units of bacterial isolates from infected and uninfected plants were analyzed for significant mean differences via one-way ANOVA using statistical software Statistical Tool for Agricultural Research (STAR). The multiple mean comparisons were performed using Tukey's honestly significant difference test (Tukey's HSD).

Isolation, selection and evaluation of bacterial isolates for nematicidal activities
The bacterial colonies from root-knot infected and uninfected tomato roots in 7 accessions produced distinct colonies in the range of 40.4 to 91.6 × 10 4 /g root in the 3 media (Table 1). Among the 3 media, SPCA gave significantly (P < 0.05) higher number of colonies, followed by Kings B and SEA for both nematode uninfected and infected roots. The bacterial colonies were significantly higher in nematode uninfected tomato roots than the nematode infected roots in all the 7 accessions of tomato. On an average, 78-91 cfu × 10 4 bacterial/g root was found in nematode uninfected roots, compared to 40-54 cfu × 10 4 /g root in nematode J2 mortality. The mortality bioassays revealed that the most isolates associated with uninfected roots, caused mortality in the range of 80-90%, as compared to isolates from infected roots, where the most cultivable isolates caused juvenile mortality in the range of 61-70% (Fig. 1).

Phylogenetic tree
The nucleotide sequences obtained were aligned with the existing nucleotide database of NCBI GenBank and reference sequences were retrieved. Phylogenetic tree was constructed by using MEGA6 (Molecular Evolutionary Genetics Analysis version 6.0) software. The phylogenetic tree was found to possess substantial genetic homogeneity among the 4 Bacillus isolates (Fig. 2). The isolates may be divided into 4 clades of the phylogenetic tree. Isolates B. pumilus and B. subtilis came under the same clade, whereas B. megaterium and B. cereus were in other clade of the phylogenetic tree.

Enhancement of tomato seed germination and seedling vigor index (SVI)
All the selected bacterial isolates enhanced the percentage seed germination and seedling vigor of tomato variety Pusa Ruby than in the untreated control on all 4 days of observation (Table 4). However, the maximum seed germination percentage (97%) and seedling vigor index (1789)

Effect of rhizobacteria on plant growth parameters of tomato cv Pusa Ruby
On soil application the 4 rhizobacteria resulted an enhancement in shoot length and weight and fresh biomass. The percent increase in shoot length varied from 13.96 to 28.64, root length from 11.20 to 25.68; fresh shoot weight from 17.88 to 38.05; and fresh root weight from 19.54 to 56.90, in bacteria alone treatments as compared to uninoculated control (Table 5). The maximum enhancement in all the above parameters was observed in B. subtilis treatments. On an average, the plant growth parameters showed the enhanced effect in presence of the nematode, except in root length where a decrease was observed due to twisting of roots due to nematode galling. However, a significant Pusa (P < 0.05) enhancement in root volume was observed with all the rhizobacteria, though the effect was most enhanced with Bacillus subtilis, and that of other rhizobacterial species was at par (Fig. 3). An increase of 88.31 in root volume was observed even in presence of the nematode. Despite some negative effect on root length and weight in the presence of the nematode, the total biomass increased significantly (P < 0.05) an application of rhizobacteria; the effect was the maximum in B. subtilis application, where 43.8% increase was observed even in presence of the nematode. The chemical nematicide velum prime did not show an increase in shoot length and weight and caused reduction in root length and weight in presence of nematode, though a 2.58% increase in biomass was observed. subtilis was found to result in significantly (P < 0.05) higher reduction in the abovementioned parameters of nematode infection (Table 6).

Discussion
In the past decades, the plant growth-promoting rhizobacteria (PGPR) have gained extensive attention due to their beneficial effects in protecting the host plants against biotic and abiotic stresses (AbdelRazek and Yaseen 2020). They offer sustainable alternative substitutes to agrochemicals in plant protection as well as crop production. In the present research work, bacteria associated with the tomato roots were isolated using 3 media. In all the media, the bacterial densities were found to be higher in uninfected roots, compared to the infected roots and harbored more nematode antagonistic rhizobacteria.  soil-borne pathogens and plant parasitic nematodes with the ability to produce several lytic enzymes and antimicrobial secondary metabolites (Akinrinlola et al. 2018).
In the present study, the selected isolates of B. pumilus, B. megaterium, B. subtilis and B. cereus with highly nematicidal and multiple plant growth promotion traits that play a major role in plant root architecture and nutrients acquisition. These microorganisms were reported to alter the auxin pathways in the host plant, leading to faster completion of the pre-germination metabolic