Rhizospheric Bioweapons for Tuber Yield Enhancement in ChlorophytumBorivilianum against Meloidogyne Incognita Infestation

The plant genera Chlorophytum Santapau and Fernandez (vern. Safed Musli) comprising approximately 256 species is a medicinally important crop from family Liliaceae with potent medicinal properties used as a revitalizer, remedy for diabetes, arthiritis, as a curative for natal and post-natal problems, in the treatment of diarrhea, dysentery, gonorrhea and leucorrhea [1]. Because of its immense therapeutic properties, the plant is frequently symbolized as “divya aushadhi” in Ayurveda [2,3]. The genus is represented in India by 12 different species viz. C. heyneanum Wall, C. breviscapum Dalz., C. arundinaceum Baker, C. glaucum Dalz., C. tuberosum Baker, C. khasianum Hooker, C. attenuatum Baker, C. malabaricum Baker, C. undulatum Wall. syn. C. nepalense (Lindl) Baker, C. orchidastrum Lindl., C. laxum Br. and C. borivilianum Santapau and Fernand [2], grown in the hilly areas of Uttar Pradesh, Gujarat, Rajasthan and Madhya Pradesh. It is a tuber crop with plant tubers containing 2-15% saponin accountable for enhancing vitality and immunity in human beings [4].


Introduction
The plant genera Chlorophytum Santapau and Fernandez (vern. Safed Musli) comprising approximately 256 species is a medicinally important crop from family Liliaceae with potent medicinal properties used as a revitalizer, remedy for diabetes, arthiritis, as a curative for natal and post-natal problems, in the treatment of diarrhea, dysentery, gonorrhea and leucorrhea [1]. Because of its immense therapeutic properties, the plant is frequently symbolized as "divya aushadhi" in Ayurveda [2,3]. The genus is represented in India by 12 different species viz. C. heyneanum Wall, C. breviscapum Dalz., C. arundinaceum Baker, C. glaucum Dalz., C. tuberosum Baker, C. khasianum Hooker, C. attenuatum Baker, C. malabaricum Baker, C. undulatum Wall. syn. C. nepalense (Lindl) Baker, C. orchidastrum Lindl., C. laxum Br. and C. borivilianum Santapau and Fernand [2], grown in the hilly areas of Uttar Pradesh, Gujarat, Rajasthan and Madhya Pradesh. It is a tuber crop with plant tubers containing 2-15% saponin accountable for enhancing vitality and immunity in human beings [4].
Despite of the vast diversity of plant species, C. borivilianum Santapau and Fernandez is the most important owing to its highest tuber yield along with the highest saponin/ phytochemical constituents [5]. The dried tubers of C. borivilianum contain 42% carbohydrates, 8-9% proteins, 3-4% fibres and saponins (Yadav and Pandey, 2004) and therefore form an obvious choice for the commercial cultivation of the crop. Furthermore, plant tubers are also imperative as the plant primarily propagates through roots due to the poor seed setting and viability (<20%). Though the crop is affected by several diseases like leaf spot, anthracnose and wilt, the major threat to its commercial cultivation occurs due to the root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood infestation [5]. M. incognita is a sedentary endoparasitic nematode, infesting the plant roots, thereby affecting growth and yield of the host plant. The nematode causes great loss to the agricultural economic sector as against the annual requirement of 3500 tonnes, India is barely able to contribute its share as 500-600 tonnes [6,7]. Furthermore, the over-exploitation of the plant due to its growing demands world over has placed it in the category of threatened species, on the verge of extinction [8][9][10]. The Medicinal Plant Board of India (MPBI) has also documented the plant as the sixth most important herb to be protected and promoted [11].
Therefore to meet the current industrial requirement for Chlorophytum roots, alternate strategies for the quality and quantity yield management are urgently required. Agricultural practices such as the use of insecticides or pesticides though have promising effects, yet the concentrated and continuous use of these chemicals disturbs soil health, leading to acidification, micro nutrient depletion, soil degradation, reduction in the activity of soil micro flora and fauna, poor crop health and lower yields [12,13]. Besides, the excessive use of these fertilizers may contribute to the environmental risks like increase in global temperature, ground and surface water pollution, etc. In this regard, it is desirable to have ecofriendly and cost-effective measures for agricultural sustainability [14,15]. Rhizospheric microbes serve as a better option for effective and less resource demanding measures having plant growth promoting and biocontrol potentials. Therefore, in the present investigation, attempts were made to determine the comparative impact of different microbes viz. Bacillus megaterium (NCBI-KC978881), Pseudomonas fluorescens (ATCC No. 13525), Glomus intraradices, and Trichoderma harzianum CIMAP-RPN01 (NCBI-KF157964), alone and in combination on the growth/yield of C. borvillianum along with their efficacy for protection against M. incognita infestation.

Plant materials and growth conditions
Healthy plant tubers of C. borivilianum were obtained from the National Germplasm Repository for Medicinal and Aromatic Plants at the Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, India. The tubers were surface sterilized by soaking in 1% (v/v) sodium hypochlorite solution for 5 min, washed with distilled water and were transplanted singly into clay pots (7.0 kg soil capacity), containing a mixture of autoclaved soil (76% sand, 8% silt, 16% clay, pH 7.7) and composted farm manure in 5:1 ratio. Tubers after six days were inoculated with 1000 J2-juveniles (Pi) of M. incognita. Plants were maintained in a glasshouse at 30°±2°C and 13 h day length until the experiment was terminated six months after inoculation. Table 1 depicts the various treatments of biocontrol agents used in the experiment. These microbes had earlier shown better potentials for plant growth promotion through the substantial management of phytonematodes in various pot and field trials and were therefore chosen for the present study. Three replicates were established per treatment, and arranged in a completely randomized block design. The following three treatments serving as controls were also maintained: untreated -uninoculated, untreated -M. incognita inoculated and chemical treated (carbofuran @ 2kg a.i. ha -1 ) -inoculated. The microbial agents were applied four days before M. incognita inoculation. The treated pots were irrigated as needed to maintain soil moisture. There were three replicates per treatment, and the pots were arranged in a completely randomized block design. After six months tubers were harvested and their length, diameter, fresh weight and tuber number were determined. The soil and root samples from each treatment were processed by sieving and decanting method [16,17] to access the populations of M. incognita in soil and tuber samples. Roots were rated for galling severity according to Krusberg and Nielson [18] on a scale of 0-4.

Nematode isolation
The population of M. incognita was maintained on brinjal (Solanum melongena L.) grown in sterilized loamy-sand soil in earthen pots under glasshouse conditions. Mature egg masses were hand-picked from the roots, allowed to hatch and then juveniles (J2) were separated into a tray [19]. Populations of M. incognita were obtained by isolating from soil using Cobb's decanting and sieving technique followed by Baermann funnel method [20].

Culture and maintenance of bio control agents
Microbial cultures viz. T. harzianum, B. megaterium, P. fluorescens and G. intraradices are regularly maintained in the Microbial Technology and Nematology Department, CSIR-CIMAP, Lucknow. The fungal biocontrol agent was cultured using sand maize media while the bacterial isolates were cultured in Luria broth. For multiplication, the fungal cultures were kept in a BOD incubator for 96 h at 30 ± 1˚C and the bacterial cultures were placed on a rotary shaker at 28 ± 1˚C for 48 h at 200 rpm orbital shaking conditions. The fungal mycelial mat, after incubation and mass multiplication in the previously mentioned media was homogenized and suspended in 500 mL of 0.1 M phosphate buffer (K 2 HPO 4 ; KH 2 PO 4 ) at 1.2 x 10 6 colony forming units (CFU) mL -1 . The bacterial cultures, B. megaterium and P. fluorescens, as luria broth suspensions were centrifuged at 6000g for 10 min. The supernatant was discarded and the pellet containing bacterial cells was suspended in 500 mL of 0.9% saline to a final density of 2.3 x 10 8 (CFU) mL -1 for B. megaterium and 2.9 x10 8 (CFU) mL -1 for P. fluorescens. G. intraradices inoculum was propagated on maize roots (Zea mays) for 10 weeks in a 1:1 (v/v) mixture of sterilized sand and soil (5 kg) of low phosphorus content (7.5 kg ha -1 ) and subsequently left to shade-dry for 2 weeks. The inoculum was based on root fragments colonized (70%) with G. intraradices and the sand-soil fraction with AM fungus propagules (spores and mycelium) from dry maize pot culture [21]. The roots in the pot culture were extracted from the soil, cut into 1 cm segments and thoroughly mixed with the sand-soil mix from the pot culture and stored at 5˚C until use. The inoculums potential (potential of a specific amount of inoculum to cause root infection under a standard set of conditions) of G. intraradices used in this study was 8.9 ± 1.3 infecting propagules g -1 sand-soil mixture. Bacterial agents were inoculated as 10 mL of 10 8 CFU/pot and the fungal isolate as 10 mL of 10 6 CFU/pot. Inoculums of arbuscular mycorrhizal fungi, G. intraradices used in the present experiment consisted of soil containing spores (8-10 spores g -1 ).

Statistical analysis
Analysis of variance (ANOVA) techniques were used for the statistical analysis of data. Duncan multiple tests applicable to RCBD were performed by ASSISTAT software version 7.6 beta. Duncan's test was performed to test the significant differences among treatments. Least Significant difference (LSD) was calculated at 5% probability level (P=0.05) for comparing the significance of difference between any two treatment mean (15).

Results
The incidence of root knot nematode disease caused by M. incognita has earlier been reported [6]. The infested fleshy roots were generally deformed and large numbers of eggs and larvae were detected inside the fleshy roots. Figure 1A-1E depicts the devastating effect of M. incognita populations on C. borivilianum crop. Since tubers are the plant parts of economic interest, a definite awareness and serious efforts are required to manage M. incognita problem for economic stability of this crop.

Growth promoting parameters
To estimate the beneficial effects of the symbiotic association (rhizospheric microbes and C. borivilianum), several plant growth promoting parameters viz. number of fleshy tubers, tuber length, tuber diameter and fresh tuber weight (g) were observed. Except for G. intraradices treatment, all the treatments, both singly and in  The combined treatment of T. harzianum with P. fluorescens (T9) was found to be effective for most of the observed parameters, significantly enhancing all the growth parameters. The treatment (T9) depicted best potentials for enhancing the number of tubers, tuber length and fresh tuber weight, recording an overall increase of 47.39, 43.61 and 62.94 percent respectively against the untreated-M. incognita inoculated control (Table 2). Furthermore, the same treatment was also found to be the most potent for the management of M. incognita populations. The combination treatment of T. harzianum and P. fluorescens showed maximum enhancement of growth parameters along with better management of M. incognita which suggests for the use of microbial consortia rather than single inoculation. When the main effects were significant, differences among factor levels were tested for significance using the mean sum of square statement at P = 0.05. All the treatments were statistically significant for the observed growth parameters as is evident by the MSS (mean sum of square) values ( Table 2). The treatments exhibited a variable effect in single and combined treatments and the combination treatments were found to be more effective. A lower least significant difference (LSD) value at 5% probability (P=0.05) for all the observed growth parameters confirms the affectivity of the bio-inoculants for plant growth promotion as well as for M. incognita management (Figure 2a). Figure 2a represents the total M. incognita population (root + soil) in various treatments. All the treatments showed good reduction in M. incognita populations as compared to untreated-M. incognita inoculated control with the best reduction results observed in the combination treatment of T. harzianum with P. fluorescens. The single treatment of G. intraradices, although found to be effective for growth promotion effect, did not respond well for the control of M. incognita populations. Figure 2b shows the effect of microbial treatments on the reproduction factor (Rf) of the root knot nematode. The results show that microbial agents completely counteract the negative impact of the nematode and reduce nematode population and root knot index, and were equally effective to carbofuran treatment.

Discussion
Medicinal and aromatic plants constitute a large segment of the flora, which provide raw materials for, cosmetic, fragrance and pharmaceutical industries. C. borivilianum, commonly christened as "Safed Musli" because of the white milky texture of its tubers after peeling, is a threatened/ endangered plant species in India. The fibrous roots of the plant are modified into fascicular roots (tubers) comprising the part of economic importance since its tubers are widely used as an important ingredient in the herbal Ayurvedic tonics. C. borivilianum is an important medicinal plant well known for its therapeutic potentials as aphrodisiac, immuno-modulator, adaptogen and antidiabetic.

Root diameter (mm)
Fresh tuber weight (g)   T1  T2  T3  T4  T5  T6  T7  T8  T9 T1  T2  T3  T4  T5  T6  T7  T8  T9  The infestation of root knot nematode, M. incognita is a major yield constraint and visible threat to the effective commercialization of this valuable crop. Therefore, reliable and advanced information on the economic importance of this plant is needed to ensure that the developmental policies and investments take account on full costs and benefits of alternative land uses for economical purposes. The awareness for pests and pathogens, cultivation practices and proper orientation of this plant should be given major priority.
Plant parasitic nematodes cause great losses to the agricultural economy (estimated to be approximately 12% of the agricultural production), representing about 100 billion dollars in annual damage worldwide [22,23]. The intricate association of plant parasitic nematodes causes significant damage to almost all the medicinal plants studied till date [24,25]. The magnitude of crop damage due to nematode-plant interactions has established M. incognita as the most important nematode parasites limiting production, with infestations reported on Mentha arvensis, Ocimum sanctum, Papaver somniferum, Bacopa monnieri, Withania somnifera and Artemesia annua [26,27]. The present study provides an insight to the extent of damage caused due to M. incognita in C. borivilianum. The study also evaluates the rhizospheric microbes for the successful and efficient management of this phytopathogen. M. incognita, being a sedentary endoparasite of root, causes maximum damage to the plant roots, thereby having a significantly negative impact on the agricultural economy of the crop.
C. borivilianum, an important medicinal herb, is reported to be a susceptible host for M. incognita [28,6]. The root knot infested plants are stunted with smaller and chlorotic leaves, fewer rootlets and roothairs and the roots bear small knots/galls. M. incognita infestation results in unthrifty growth and yellowing of leaves due to the reduced uptake of nutrients such as P, K, Zn, Mn and Cu etc. [26]. The complete eradication of M. incognita from agricultural soils is nearly impossible due to the polyphagus nature of this pest. In the approaches to the management of nematodes, the use of chemical nematicides is prevalent, and carbofuran is the only effective nematicide available in market till date. These synthetic nematicides besides rising the costs of production also presents risks to the environment and non-target organisms [29]. For this reasons, alternative methods of control are the need of the hour [30]. In this sense, the application of microbial inoculants as biocontrol agents can represent the substitution of conventional pesticides and become an alternative method to the nematodes control [14].
Rhizospheric microorganisms prove to be a prospective source of new natural products for exploitation in agriculture and are recognized as a promising group in requisites of diversity and pharmaceutical potential [31]. In the present study, rhizospheric microorganisms with potent plant beneficial activities were used in single/ combined treatments for the crop boost-up in C. borivilianum. The microbes were also evaluated for their biocontrol potentials against M. incognita and T. harzianum was found to be the most efficient single treatment in controlling the M. incognita populations. The combination treatments for T. harzianum with P. fluorescens (T9) and T. harzianum with B. megaterium (T10) however, were having a better biocontrol potential than single treatment inoculations (Figure 2a and 2b). Biocontrol activity or the reduction of inoculum density of a pathogen by the microorganisms is achieved by mechanisms viz. competition, hyperparasitism, induced resistance etc. In this study, Trichoderma sp. chiefly operate by mechanisms such as mycoparasitism and production of volatile and non-volatile antibiotics. At CIMAP, earlier our group has identified a strain of T. harzianum that has been used to produce a disease-free, healthy nursery with a reduction in root-knot nematode infection and improving the growth and yields of M. arvensis [32].
Rhizospheric microbes destroy nematodes continuously in virtually all soils because of their constant association with nematodes in the rhizosphere. A large number of bacteria viz. Rhizobium sp., Bradyrhizobium japonicum, Pseudomonas (P. fluorescens and P. aeruginosa) and Bacillus (B. subtilis) have shown antagonistic effects against nematodes. Several microbial mechanisms such as phenolic compounds, organic acids and secondary metabolites [28,33] may be involved in the suppression of nematode soil populations and enhancement of plant growth and yield. This study revealed a drastic reduction in RKI and increase in total weight of the plant. Except for the treatments having G. intraradices, either in single or dual combinations, all the treatments were able to reduce the disease incidence in C. borivilianum. The combination treatment of T. harzianum with P. fluorescens (T9) showed the best biocontrol effect, suggesting for the use of microbial consortia for better management of M. incognita and plant growth promotion.
The present experimentation evaluates the effect of microbial bioinoculants on the growth and yield attributes of Safed Musli (C. borivilianum). The study demonstrates that microorganisms greatly alter the growth/ yield parameters and also possess effective root-knot nematode suppression potentials. Results from the present experiment demonstrate the nematicidal potential of the biocontrol agents tested, especially T. harzianum and P. fluorescens, which resulted in increased tuber yield of C. borivilianum. Rhizospheric microbes, especially in combination treatments were found to be efficient in the management of M. incognita populations and at the same time were also beneficial in enhancing the plant growth parameters in C. borivilianum. It was observed that all the microbial treatments, except for the G. intraradices treatment, were capable enough for the suppression of M. incognita populations. Our study emphasizes the use of rhizospheric microbes which apart from providing benefits of supporting better plant growth/ yield also reduces losses from root-knot nematode infestation in crop plants. Present results presume potential importance in developing new biocontrol based root-knot nematode management technology in the era of environment friendly organic farming. With this experiment as primary step, further study is needed to develop microbial consortium for commercially grown medicinal plants.