¹Department of Mycology and Plant Ppathology, Institute of Agricultural
Science, Banaras Hindu University, Varanasi, Uttar Pradesh, India.

²Department of Botany, Institute of Science, Banaras Hindu University,
Varanasi, Uttar Pradesh, India

³Department of Genetic and Plant Breeding, Institute of Agricultural Science,
Banaras Hindu University, Varanasi, Uttar Pradesh, India.

Corresponding author email: rakeshsingh@bhu.ac.in

Article Publishing History

INTRODUCTION

Rhizosphere is one of the most crucial zones where plant and microbes interact symbiotically. In this aspect, plants mainly benefit from microbial consortium for acquiring nutrients as a vital mineral and in return providing habitat to micro-organism. Root-knot nematodes (RKNs) are mainly involved in causing damage to vegetable crop and estimated loss upto 21.3% to agricultural products annually. M. incognita is highly destructive plant parasitic nematode with rapid multiplication in seasonal vegetable crop likes tomato, brinjal, okra, spinach etc (Razavi et al. 2017; Kumar et al. 2020).

incognita penetrate epidermis of root and produced several hydrolytic enzymes from stylet that degrade cell wall of plant root in order to enter through plant and not only caused diseases but also restricted water and minerals uptake of plants, due to hindering biochemical, physiological and morphological status of plants. Farmers recognized insect pests and other constraints as production problems but overlooked plant parasitic nematodes due to its indirect effect on the plants. Nematode diseases are difficult to control when it enter in the root system hence pre adaptive measures are more successful to control them rather than post adaptive management practices. Plant parasitic nematodes not only cause damage individually but form disease-complexes with other microorganism and increased the crop losses (Etesami and Adl 2020).

In rhizospheres several beneficial micro-organisms viz bacteria fungi actinomycetes etc are found which control the nematode population naturally. In natural rhizospheric zone many plant growth promoting bacteria having very good growth promotion activities and potential biocontrol ability have been found. Which have direct mechanism for plant growth with mineral solubilization, phytohormone production while indirect mechanism with production of hydrogen cyanide, siderophore, antibiotic production and also have induced systemic resistance activities. Rhizobacteria are reported to produce diverse range of bioactive chemical which promote growth of plant and provide protection against many phytopathogen including M. incognita (Khanna et al. 2019; Liu et al. 2019; Etesami and Adl 2020).

Tomato is one of the most susceptible vegetables against root knots, nematodes  mostly causing 30 to 40 % yield losses in tropical regions, and also  it has been also found that M. incognita is responsible in deteriorating the quality of fruit and yield of tomatoes, (El-Shawadfy 2021). Among the various microorganisms, plant growth promoting rhizobacteria have attracted considerable attention for their usefulness in biofertilizer and biological control ability.

Hence  the current research work  was focused  on their biofertilizer ability as well as  on  the eco-friendly management of M. incognita by plant growth promoting traits for enhancing plant defense mechanism of tomato seedlings, raised under nematode infested soil  as previously reported by El-Ashry et al. (2020) and  Zao et al. (2021).

MATERIAL AND METHODS

Rhizospheric soil samples were collected from agricultural field of rice, tomato, oat and barley growing at four districts viz Varanasi, Mirzapur, Chunar and Jaunpur of Uttar Pradesh in India. Collected soil samples were kept in sterilized plastic bags at 4⁰C. Serial dilution technique was used for screening of bacterial colonies on King’s B medium. Serial diluted plates were incubated at 30°C for 48-72 hours (h) and bacterial colonies were isolated, maintained as pure cultures on King’s B media. For the morphological and biochemical characterization of growth promoting bacteria, isolated bacteria were identified firstly by macroscopic observation i.e., gram reaction, colony morphology, pigmentation, mobility and cell shape followed by several biochemical and enzymatic test viz Catalase test, Oxidase, Chitinase, Amylase and Gelatinase (Hayward 1960; Blazevic and Ederer 1975; Collins 1980; Fadden 1980; Somasegaran and Hoben 1994; Kaur et al. 2012).

Qualitative assay of phosphate solubilizing activity of bacteria was measured by Pikovaskaya on Pikovaskaya’s Agar Medium and clear transparent zone around the bacterial colony confirmed phosphate solubilization activity within 24 to 72 h (Pikovaskaya 1948).Indole acetic acid (IAA) Production was detected as described by Bric et al. 1991. Appearance of pink colour in broth by adding of Salkowaski reagent indicated IAA production. For the quantification of IAA, absorbance was taken at 540 nm by using UV/visible spectrophotometer (Bric et al. 1991).

HCN production was evaluated on Whatman filter paper No.1 soaked with picric acid (0.05%) solution in 2% sodium carbonate and placed in the test tube sealed with parafilm and incubated. A colour change of the filter paper from deep yellow to reddish-brown colour at 30°C for 48-72 h indicated HCN production and quantified (Baker et al. 1987). Siderophore production of bacterial isolates was detected by observing orange halo zone around the bacterial colony on chrome azurol S agar media after 72 h of growth. Quantification of siderophore was calculated in µg/ml (Schwyn and Neilands 1987).

For the preparation and screening of bacterial strains against M. incognita, nematode inoculum was prepared from large numbers of egg masses by hand-picked using a sterilized forceps from heavily infected tomato roots. These egg masses were washed in distilled water and placed in 10-cm diameter 15 mesh sieves. The hatched juveniles were collected and incubated in a micro-well plate containing 3 ml sterile distilled water for at 25⁰C. For the screening of bacterial isolates having ability to kill M. incognita in vitro, four bacterial isolates were screened for their nematode killing ability against M. incognita in microwell plate (25 wells). Heavy cell suspension for each bacterial isolates was prepared (1×10⁶cfu/ml) in sterile distilled water using a UV/Visual spectrophotometer. Each well contained 100 (J₂) of M. incognita in 1ml of sterile distilled water with 10µl/ml of bacterial suspension. Mortality of M. incognita was evaluated at 3, 6, 12 and 24 h time interval.

For the pot experiment, all the four promising bacteria were inoculated on tomato seeds (variety Kashi Abhiman) by seed biopriming. Seeds were soaked with bacteria containing 10⁹ CFU/ml for overnight. Bioprimed seeds were sown in pots containing I kg sterilized soil followed by 2000J₂ population of M. incognita in each pot. Subsequently single plants per pot was maintained at 60% water holding capacity and plants were uprooted after 21 days. Seed germination and vigour index were observed by towel method. Leaf area was calculated by using following formula (Yashida et al. 1972; International Seed Testing Association 1993). The experiment was performed in completely randomized block design. Statistical significance between the treatments was compared by least significant difference test at P < 0.05 probability level on different growth promoting parameters using SPSS16.0 software.

RESULTS AND DISCUSSION

Four potential bacteria viz BHU1, BHU2, BHU and BHU4 were isolated from different rhizospheric locations (Table1) and their morphological characteristics of rhizobacterial isolates BHU1, BHU2, BHU3 and BHU4 showed variation in colony, shape, colour, pigmentation, colony surface and margin.

Table 1. Isolation of bacteria from rhizospheric soil from different locations

S.No.	strains	Location	Rhizospheric soil associated with Crops
1	BHU1	Varanasi  (25.3176 N, 82.9739 E)	Rice (Oryza sativa)
2	BHU2	Mirzapur (25.32N,  83.33E)	Tomato (Lycopersicon esculentum)
3	BHU3	Chunar (25.1037 N, 82.8721 E)	Oat (Avena sativa)
4	BHU4	Jaunpur(24.240N, 82.70E)	Barley (Hordeum vulgare)

Bacterial strain was identified on the basis of morphological appearance of bacterial colony and their biochemical performance which is mention in table 1. BHU1 showed green pigmentation while BHU3 isolates showed yellow pigmentation on YEMA agar media with rough and shiny surface (Table2).

Table 2. Morphological characterization of selected isolate

Ss.No.	Bacteria	Strain	Gram stain	Colony Shape	Colony Colour	Pigmentation	Colony Surface	Colony Margin
1	Pseudomonas fluorescens	BHU1	-ve	Round	Yellow	Green	Smooth	Entire
2	Enterobacter spp	BHU2	-ve	Biconcave	White	No pigmentation	Rough	Entire
3	Bacillus spp	BHU3	-ve	Circular	Creamy white	Yellow	Rough	Undulate
4	Burkholderia spp	BHU4	-ve	Circular	Yellow	No pigmentation	Rough	Entire

Bacterial strain shows various type of biochemical characterization like phosphate solubilization, indole acetic acid, HCN and siderophore production (Table 3). Selected bacteria showed phosphate solubilization activities on Pikovaskaya’s media by forming clear transparent zone around the bacterial colony. Among four isolates highest phosphorus was solubilized by BHU4 strain (Figure1&2A). Phosphorus is a major nutrient required for plant growth and development but plants are unable to utilize phosphate because 95-99% phosphate found in the soil in insoluble form. Making phosphorous in available form for plants is an important trait for PGPR selection (Shahzad et al. 2008).

Some PGPR have been reported to produce citrate, lactate, succinate activity that dissolve the mineral phosphates as a result of anion exchange or chelation of Fe and Al ions associated with phosphate. This leads to an increase in the soluble P in the rhizosphere resulting its more uptake by the plants. IAA is important phytohormones which stimulatory effect on plant growth under biotic stress condition. Among four bacteria, three bacteria produced IAA in tryptophan yeast broth. Highest IAA production was recorded by BHU1 (89μg/ml) followed by BHU4 (82 μg/ml) and minimum IAA production by BHU2 (5 μg/ml) production (Figure 1&2B) (Shahzad et al. 2008).

HCN producing bacteria (BHU1, BHU3 and BHU4) showed antagonistic activities against phytopathogen three bacteria produced HCN with highest HCN production 13ppm by BHU1 (Figure 2C). HCN production is a type of volatile organic compounds (VOCs) toxic for phytopathogenic nematodes and provides protection from various biotic stresses. As the plants exposed to nematode infection grow weak in their defense system hence shielding them by HCN producing bacteria which can be helpful in overcoming the chances of pathogenic infection. Siderophore production was confirmed by the change of CAS broth colour to brown colour (Fig. 1C).

Three bacteria (BHU1, BHU3 and BHU4) were observed producing siderophore and highest siderophore production was observed by BHU1 (68μg/ml) (Figure 2D). Siderophores are low molecular weight iron chelating compound which restrict the growth of phytopathogen, indirectly by depriving fungal pathogens and plant parasitic nematodes from iron uptake and supported plant growth by iron supply resulting in increased dry biomass of plant and rootlet elongation (Cheng et al. 2017; Sahebani and Gholamrezaee 2021).

Figure 1: Biochemical characterization of PGPR Phosphate solubilization
(A), IAA production (B), Siderophore (C).

Figure 2: Biochemical characterization of growth promoting bacteria (A) P-solubilization, (B) IAA production, (C) HCN production and (D) Siderophore production. Significant of variance (p < 0.05)  has been  analyzed by Duncan Multiple Range Test.

Extracellular enzyme activities are important parameter for selection of growth promoting bacteria and also favour strong evidence for improve defense mechanism. Selected bacteria were showing positive catalase activity, maximum catalase production was observed by BHU1 and BHU4. The oxidase test used to identify bacteria that produce cytochrome C oxidase, an enzyme of the bacterial electron transport chain. The reagent N, N-dimethyl-p-phenylenediamine is a dark-blue to maroon color when oxidized and become colorless  when reduced. BHU4 was observed strongly positive for oxidase test. Chitinase enzyme serves various functions such as antagonistic activity against chitin-containing pests and in nutrient cycling. BHU4 strain produced chitin followed by BHU2. Gelatin hydrolysis test is used to detect the ability of an organism to produce gelatinase which is a type of proteolytic enzyme that liquefy gelatin hydrolysis of gelatin indicates the presence of gelatinase (Subedi et al. 2020).

Maximum gelatin was produced by BHU4 bacteria followed by BHU2 (Table3). Three bacterial isolates BHU1, BHU3 and BHU4 had greater efficiency to produced lytic enzyme like catalase, oxidase, chitinase amylase and gelatinase. Calalase and oxidase enzyme reduce the oxidative damage while α-amylase enzyme in the aleurone layer helping in hydrolyzing the endosperm starch into sugars, which provide the energy for vigour index parameter. Other important mechanisms include production of extracellular lytic enzymes, such as CHI, β-1,3-GLU and protease, which lyse chitin and glucan (present in the cell wall of fungi) and protein in the nematode cuticle (Lee et al. 2015; Gupta et al. 2017; Subedi et al. 2020; Sahebani and Gholamrezaee 2021).

Table 3. Extracellular enzyme production by selected bacteria

S. no	strains	Catalase	Oxidase	Chitinase	Amylase	Gelatinase
1	BHU1	+++	+	+	+	+
2	BHU2	+	–	++	–	–
3	BHU3	++	++	+	++	++
4	BHU4	+++	+++	+++	+++	+++

(-) no production, (+) weak, (++) moderate and (+++) strong

When these bacteria were examine for their antagonistic properties against M. incognita three bacteria BHU1, BHU3 and BHU4 kill nematode but BHU2 did not show ability to kill it (table 4 and 5). Bacteria BHU1 and BHU3 kill M. incognita 100% and 93% respectively after 12 hours. The bacterial mechanism of infection caused the lysis of nematode M. incognita particularly on the oesophageal region (BHU1) and intact body (BHU3) which is mention in table 4 and 5.

In many studies it has been found that growth promoting microbes produced various types of secondary metabolites and volatile organic compound (VOC) at infected region. Antagonistic bacteria tend to prohibit the invasion of RKNs into the roots and maintain the balance of nutrients and mineral status in plants moreover, growth promoting bacteria also control the gall formation and maintain the vascular integrity in order to boost the physiology and biochemistry of the plant. Consequently, the most vital mechanism to prevent gall formation through PGPR’s by production of nematicidal compound like hydrogen sulfide, ammonia, butyric acid and degradation of nematode attractants of root exudates. Furthermore, they bind to the root surface and produced hydrolytic enzyme that can kill the nematode (Safni et al. 2018; Sahebani and Gholamrezaee 2021).

Table 4. Mechanism of antagonistic bacterial infection inside the body of M. incognita

Table 5. Mortality percentage of M. incognita hours after inoculation

S.No.	Treatments	Mortality percentage of M. incognita hours after inoculation
		3 h	6 h	12 h	24 h
1	Control	0±0a	0±0a	0±0a	0±0a
2	BHU1	33.33±3.9b	66.66±4.5c	100±0d	100±0b
3	BHU2	0±0a	0±0a	0±0a	0±0a
4	BHU3	23.33±2.8b	46.66±3.4b	93.3±6.9cd	100±0b
5	BHU4	23.33±2.8ab	56.66±4.8bc	76.66±6.4b	100±0b

Values are mean of three replicates SE. Significant of variance (p < 0.05) is analyzed by Duncan Multiple Range Test

Biopriming tomato seed with bacteria showed higher root length, shoot length, vigour index and reduced gall/ knot formation after 21 days of seed germination. In the current study, it has been found that M. incognita cause root knot disease in tomato seedling resulted in declined root length, shoot length, fresh weight and dry weight, leaf area, leaf number of tomato seedlings.  This reduction is due to the formation of root knot in the root leads to the reduction in the growth promotion due to disruption of root system of tomato plant and inhibition of nutrient uptake from the root system (Safni et al. 2018; Sahebani and Gholamrezaee 2021).

Biopriming tomato seed with bacteria showed higher root length, shoot length, vigour index and reduced gall/ knot formation after 21 days of seed germination. Plants inoculated with BHU1, BHU2, BHU3 and BHU4 strain exhibited significant increase in root and shoot length compared to the un-inoculated control. Maximum root length and shoot length of tomato plant was observed by BHU1 strain followed by BHU4 bioprimed bacteria as compare to control plant i.e. 475% root length increment and enhanced shoot length by 6.74cm. Highest fresh root weight as compare to untreated plant infested with M. incognita and shoot weight was enhanced upto 2.36g by BHU1strain over the control plant (Figure 3).  Similarly seed germination and viour index reduced in tomato crop under M. incognita infection which is a type of biotic stress (Khanna et al. 2019; Sahebani and Gholamrezaee 2021).

Growth promoting attributes like nutrient mobilization and hormone production maintained even under stressed condition by BHU1, helped in enhancing the root growth, nutrient and water uptake and augmenting the dry weight. Highest root and shoot dry weight were observed by BHU1 and BHU3 strain as compare to control plant. High phosphate solubilizing activity of BHU1 more than 4.8cm zone of clearance must have favored the plant growth directly and depicted as enhancement of fresh and dry root weight and shoot weight respectively and the leaf area had significantly increased and the treated plant with BHU1 and BHU4 was observed to impart more leaf area with 124% increment as compare to control plant. Biopriming of seed with selected bacteria significantly enhanced percentage of seed germination ranged from 57-100%. Maximum vigour index 3880 as compare to untreated plant was observed  in BHU1 treated seed which is a very important parameter for promotion of plant growth (Table 6). While almost similar type of result was also observed in tomato by bacterial induction of resistance in tomato against root-knot nematode M. javanica with biocontrol agents (Zhai et al. 2018; Dey and Raghuwanshi 2020; Mazrou et al. 2020).

Values are mean of three replicates SE. Significant of variance (p < 0.05) is analyzed by Duncan Multiple Range Test

Figure 3: Effect of selected isolates in tomato plants under nematode infection.

CONCLUSION

The finding of the present study suggests that BHU1 bacterial strain have a very good potential to augment plant growth activity and potential biological control managing root knot nematodes in tomato and in feature leads to incorporate compatible other microorganism enhancing their positive attributes to replace chemical application with promising biocontrol agent. While studying the rhizospheric microorganism and their interaction with plant under biotic stresses there are chances to recognized some microorganism which can enhance directly or indirectly defense immunity in the plant. The performance of individual bacterial strain will always lead to screen potential microorganism for harnessing them to mitigate losses occurred due to biotic stresses.

ACKNOWLEDGEMENTS

This study was financially supported by Banaras Hindu University, Varanasi, Uttar Pradesh, India.

Conflict of Interests: Authors have no conflict of interests to disclose.

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