Enhanced tolerance of Mentha arvensis against Meloidogyne incognita (Kofoid and White) Chitwood through mutualistic endophytes and PGPRs

Abstract

The use of herbs in pharmaceutical preparation is ever increasing, and the demand for pesticides free material by the concern industries is on the rise. Consequently the need to grow disease-free plants using non-chemical fertilizers and pesticides is the need of the hour. Mentha arvensis cv. kosi is highly infested with Meloidogyne incognita (Kofoid and White) Chitwood, and severe oil yield loss occurs due to this nematode pest. Employing ecofriendly ways of nematode management, the mutualistic endophytes (Trichoderma harzianum strain Thu, Glomus intraradices) and plant growth promoting rhizobacteria (Bacillus megaterium and Pseudomonas fluorescens) were assessed individually and in combination on plant biomass, oil yield of menthol mint (M. arvensis cv. kosi), reproduction potential and population development of root knot nematode, M. incognita under glasshouse conditions. These microbes enhanced the plant biomass and percent oil yield both with and without M. incognita inoculation. Dual application of mutualistic fungal endophytes and Plant Growth Promoting Rhizobacteria (PGPRs) may be a wise option for enhancing the oil yield and tolerance of menthol mint against M. incognita infection.

Keywords:

• Bacillus megaterium
• Glomus intraradices
• Pseudomonas fluorescens
• Meloidogyne incognita
• Mentha arvensis
• Trichoderma harzianum
• management

Introduction

Menthol mint (Mentha arvensis L.), an important essential oil bearing herb, is a major source of natural menthol, menthyl acetate, menthone, and terpenes. These constituents of menthol mint oil are continuously used in pharmaceutical, perfumery, cosmetic, and food industries all over the world. Root knot nematode is one of the most important limiting factors for successful cultivation of menthol mint (Pandey 2005a). Although a large number of plant parasitic nematodes have been associated with menthol mint, the root knot nematode (Meloidogyne spp.) is a major pest highly prevalent in tropics and subtropics and causes more than US$100 billion loss/year worldwide (Luc et al. 2005). Menthol mint, a commercial crop in India, suffers severely with the infestation of root knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood. Due to endoparasitic nature of this nematode the distribution is facilitated by infected suckers/runners/roots, which are used for propagation of this crop. This nematode species has become a major bottleneck for the successful cultivation of medicinal and aromatic plants in India (Pandey 2005b; Pandey and Kalra 2010). Chemical nematicides were the major weapons to fight against this nematode all around the world but due to their adverse effects on human health, environment, and on non-target organisms, its use has not only been restricted but also effective chemical nematicides have already been withdrawn from the world market. Because of this concern, there has been a worldwide swing toward the use of ecofriendly tactics which are bio-efficacious, economical, biodegradable, and environmentally safe and could be ideal for use as reliable agents to manage nematode diseases. The use of mutualistic fungi and plant growth promoting Rhizobacteria (PGPRs) to manage nematode disease offers an attractive alternative to synthetic nematicides. The growth or activity of pest and pathogen can be inhibited by several beneficial mutualistic endophytes and rhizobacteria (Bruehl 1987; Becker et al. 1988; Lynch 1990; Glick 1995; Pandey et al. 1997, 1999; Weller et al. 2002; Howell 2003; Lucy et al. 2004; Somers et al. 2004; Kloepper and Ryu 2006; Khan et al. 2008; Raaijmakers et al. 2009; Pandey 2010). Mutualistic endophytes and PGPRs are major occupants of rhizosphere, and their extensive microbial activity may provide major defense against nematode pest. Therefore, in the current experimentation the mutualistic fungi (Trichoderma harzianum, Glomus intraradices) and plant growth promoting bacteria (Bacillus megaterium, Pseudomonas fluorescens) and their different combinations were used to examine the impact on menthol mint biomass, oil yield, and population development of M. incognita.

Materials and methods

Nematode inoculums

Root knot nematode, M. incognita race-1 was obtained from pure cultures maintained on brinjal plant roots (Solanum melongena pusa purple long) using a single egg mass in glasshouse. Whenever inoculums were required, plants were uprooted and the entire root system was dipped in water to remove adhering soil. Egg masses were handpicked using forceps and placed in 9 cm diameter sieves of 1 mm pore size, which had been lined with cross-layered tissue paper. The sieves were placed in petri dishes with distilled water for hatching and incubated at 28°C. Five thousand freshly hatched second-stage juveniles were applied to each plant. Entire experiment was conducted in earthen pots (30 cm diameter) filled with autoclaved soil, sand, and compost mixture (7:2:1).

Mass culture of T. harzianum strain Thu

Pure culture of T. harzianum strain Thu (ATCC No. PTA 3701) was obtained from MTED, CIMAP, Lucknow, and maintained on potato dextrose agar (PDA) slants by sub-culturing periodically on fresh media at an interval of three months (Elad et al. 1981). For mass culture of T. harzianum, the mint distilled waste was air dried for 3–4 consecutive days, then ground to a fine powder. Powder of this distilled mint waste weighing 100 g each was filled into polypropylene bags to which 10 ml of sterilized distilled water was added. The bags were plugged with non-absorbent cotton. The medium was then autoclaved for 30 minutes at 121°C. Mycelial disc (3 mm diameter) from PDA slants was added to each polypropylene bag containing 100 g mint distilled waste with the help of inoculating needle under aseptic condition. The polypropylene bags were then incubated at 28±1°C for 7 days in an incubator for its multiplication.

Maintenance of bacterial culture and its multiplication

Pure cultures of P. fluorescens (ATCC No. 13525) and B. megaterium (ATCC No. 14581) were grown on King's B agar and nutrient agar media, respectively, and incubated at 30±1°C for 48 hours. The slants were then covered with sterilized mineral oil and preserved in refrigerator at 4°C for further use. For multiplication of P. fluorescens and B. megaterium, the actively growing cultures of P. fluorescens and B. megaterium were grown in nutrient broth for 48 hours in an incubated shaker at 30±1°C. The cultures were then centrifuged at 5500 RPM for 7 minutes, supernatant discarded, and the pellets containing bacterial cells were suspended in 100 ml of 0.01 M MgSO₄ solution in sterile distilled water, and concentration of suspensions was adjusted to 10⁸ CFU/ml. A quantity of 10 ml of bacterial suspension was used as inoculum per plant.

Maintenance, isolation, and observation of % infection of Glomus intraradices Schenck & Smith

The inoculum of G. intraradices (Gi) was originally obtained from MTED, CIMAP, Lucknow, and maintained in a glasshouse with palmarosa (Cymbopogon martinii, Roxb.JF Watson). A 30 g soil/pot containing 10 chlamydospore/g soil was placed along with suckers at the time of transplanting. The chlamydospores were extracted from the soil by wet sieving and decanting techniques (Gerdmann and Nicolson 1963). Rhizosphere soil samples with root from mycorrhizal treatments were collected. Roots were separated, washed and cut into 1–1.5 cm pieces, cleaned with KOH at 90°C for 1 hour in an oven and stained with trypan blue (Phillips and Haymann 1970; Giovannetti and Moss 1980). G. intraradices infection of each plant was determined by estimating the percent root colonization as described by Biermann and Lindermann (1981). A minimum of 50 root segments were determined each time.

Isolation of nematode population from soil and roots

A 200 g sub-sample of well-mixed soil from each treatment was processed by Cobb's sieving and decanting method followed by Baermann's funnel extraction to extract the nematode population (Southey 1986). For estimating the number of juveniles, eggs, and females inside the roots, a 1 g sub-sample of roots was macerated in a Waring blender, and nematodes in the resulting suspension were counted. Nematodes counting in roots were carried out by multiplying the number of nematodes in 1 g of root by the total root fresh biomass.

Plants were inoculated with mutualistic endophytes viz. Glomus intraradices Schenk & Smith 40 g soil inoculum (containing 10 chlamydospores/g soil), T. harzianum Rifai isolate Thu 2 g waste materials (containing 2.0×10⁸ CFU/g waste materials), and PGPRs (P. fluorescens, B. megaterium) 10 ml of each/plant (containing 10⁸ CFU/ml at the time of transplanting menthol mint suckers. Five centimeter menthol mint suckers bearing single bud were sterilized with 0.01% mercuric chloride solution for one minute and rinsed three times with sterile distilled water and planted singly in each earthen pot. Carbofuran was incorporated at 0.0015 g a i/kg soil. In addition, an untreated-inoculated pot was kept as nematode control. There were five replicates for each treatment.

After two weeks of transplantation each pot was inoculated with 5000 freshly hatched second-stage larvae of M. incognita by pipetting nematode larvae into 1 cm holes around the base of plant and filling the holes with moist soil. After inoculation, pots were arranged in glasshouse in complete randomized complete block design. The plants were watered frequently for maintenance of proper moisture as the plant grows well in sufficient moisture levels. Daily observations were made for recording the development of symptoms. After 90 days of inoculation, the experiment was terminated, and data of different growth parameters viz. fresh and dry root/shoot weight, percent oil yield were determined by hydro distillation of shade dried herbs using Clevenger apparatus (Clevenger 1928), and nematode population in soil and root was isolated (Southey 1986). The root gall indices were calculated according to Krusberg and Neilson (1958) on 0–4 scales, where 0 = no infection or no root galling, 1 = slight infection (1–25%), 2 = moderate infection (26–50%), 3 = severe infection (51–75%), and 4 = very severe infection (76–100%).

Experimental design

The experiment was set up in a complete randomized block design with 17 treatments. These treatments were tested in the presence and absence of M. incognita. Each treatment was replicated five times. The experiment was performed in a glasshouse and repeated twice. Data from both the experiments were almost identical, and therefore pooled.

Analysis of data

Data were analyzed by one way ANOVA and significant differences among the treatments were tested by critical difference (CD) test at the 5% probability level (Cochran and Cox 1957).

Results and discussion

Root knot disease caused by M. incognita on menthol mint was suppressed with application of mutualistic fungi and bacteria as soil applications. Root knot indices were much lower, thereby indicating the effectiveness of the treatments in inhibiting the development of M. incognita. Root knot nematode was substantially managed by individual and combined application of T. harzianum, B. megaterium, and G. intraradices. The combination of T. harzianum with G. intraradices, B. megaterium, and P. fluorescens caused 63.7%, 45.4%, and 54.6% reduction in root knot indices and 67.8%, 56.3%, and 60.4% reduction in total M. incognita population whereas the combined application of G. intraradices + B. megaterium and G. intraradices+P. fluorescens reduced only 52.7 and 54.0% of the total nematode population, respectively (Figure 1). The combination of both the PGPRs also reduced the nematode population at significant level (48.5%). The individual application of T. harzianum, G. intraradices, B. megaterium, and P. fluorescens reduced root knot indices by 45.4%, 27.3%, 18.0%, and 27.3%, respectively, and total root knot nematode population reduction by 48.9%, 38.7, 20.1%, and 26.7% whereas the reduction of nematode population in carbofuran treated pots was 31.6% and root knot indices was 36.3%, respectively (Figure 2). The total nematode population in roots of Carbofuran B. megaterium and P. fluorescens treated soils become high as compared to control-Mi inoculated treatment, may be due to enhancement in the total root biomass, whereas their population in soil is significantly reduced (Table 1).

Figure 1.  Effect of different mutualistic fungi and PGPRs on percent reduction of root-knot indices in menthol mint.

Figure 2.  Effect of different mutualistic fungi and PGPRs on percent reduction in total nematode population development in menthol mint.

Table 1. Effect of mutualistic endophytes and PGPRs on growth/yield of menthol mint, root-knot, and mycorrhizal population development.

	Plant dry weight (g)					Total nematode population			Gi spore population and% root infection
Treatments	Root	Shoot	Total	Oil yield (%)	RKI	In total root	In total soil	Total	Gi spore/100g soil	Percent infection
Control–Uninoculated	13.2	20.8	34.0	0.43	–	–	–	–	–	–
Control –Mi	9.3	16.4	25.7 (–24.4)^a	0.32 (–25.6)^a	3.66	21068	29400	50468 (10.1)^b	–	–
T. harzianum alone	16.1	26.0	42.1 (+23.8)	0.52 (+20.9)	–	–	–	–	–	–
G. intraradices alone	14.2	23.3	37.5 (+10.3)	0.47 (+9.3)	–	–	–	–	860	89
B. megaterium alone	13.6	21.6	35.2 (+3.5)	0.45 (+4.7)	–	–	–	–	–	–
P. fluorescens alone	14.1	22.7	36.8 (+8.2)	0.46 (+7.0)	–	–	–	–	–	–
Carbofuran + Mi	12.3	19.1	31.4 (–7.6)	0.41 (–4.7)	2.33	24100	10400	34500 (6.9)	–	–
T. harzianum+Mi	12.7	22.1	34.8 (+2.4)	0.42 (–2.3)	2.00	16164	9600	25764 (5.2)	–	–
G. intraradices+Mi	11.8	21.5	33.3 (–2.1)	0.40 (–7.0)	2.66	20530	10400	30930 (6.2)	380	65
B. megaterium+Mi	10.5	20.4	30.9 (–9.1)	0.39 (–9.3)	3.00	29126	11200	40326 (8.1)	–	–
P. fluorescens+Mi	11.3	22.1	33.4 (–1.8)	0.40 (–7.0)	2.66	26848	10141	36989 (7.4)	–	–
T. harzianum + Gi+Mi	16.3	24.5	40.8 (+20.0)	0.44 (+2.3)	1.33	10244	6000	16244 (3.2)	560	78
T. harzianum + Bm+Mi	13.6	22.4	36.0 (+5.9)	0.42 (–2.3)	2.00	13240	8824	22064 (4.4)	–	–
T. harzianum + Pf+Mi	14.2	24.7	38.9 (+14.4)	0.45 (+4.7)	1.66	12200	7800	20000 (4.0)	–	–
G. intraradices + Bm+Mi	12.6	21.8	34.4 (+1.2)	0.41 (–4.7)	2.00	14600	9280	23880 (4.8)	440	70
G. intraradices + Pf + Mi	12.8	22.9	35.7 (+5.0)	0.42 (–2.3)	2.00	14400	8800	23200 (4.6)	500	74
B. megaterium + Pf +Mi	11.8	22.7	34.5 (+1.5)	0.40 (–7.0)	2.66	15600	10400	26000 (5.2)	–	–
Grand mean	13.086	22.061	–	0.4253	1.667	13218	8147	–	548.00	75.20
Standard error	0.3467	0.2046	–	0.0050	0.2580	756.7	493.5	–	13.073	2.110
LSD at 5%	0.998	0.5894	–	0.01449	0.7433	2179.9	1421.7	–	29.051	4.321
^a=Percent increase (+) or decrease (–) over untreated-uninoculated control, ^b=Reproduction factors (Rf) = Final nematode population (Pf)/Initial nematode population (Pi).
Note: Inoculated with 5000 freshly hatched juveniles of Meloidogyne incognita (Mi)/pot. Gi, Glomus intraradices; Pf, Pseudomonas fluorescens; Bm, Bacillus megaterium.

The maximum enhancement of plant dry weight was recorded in T. harzianum alone (23.8%) followed by T. harzianum+G. intraradices (20.0%), T. harzianum+P. fluorescens (14.4%), G. intraradices alone (10.0%) P. fluorescens alone (8.2%), G. intraradices+P. fluorescens (5.0%), G. intraradices+B. megaterium (1.2%), B. megaterium+P. fluorescens (1.5%), respectively, as compared to untreated-uninoculated control (Table 1, Figure 3). Tolerance of menthol mint against root knot nematode can be, therefore, enhanced via combined application of mutualistic fungi and PGPRs. These results show that mutualistic fungi and plant growth promoting bacteria were more effective when they were combined in different manner as compared to alone but could not bring about complete inhibition of nematode population development. As reported by various workers (Duffy et al. 1996; Larkin and Fravel 1998; Nemec et al. 2003; Kokalis-Burelle and Kloepper, 2004; Raj et al. 2005), the use of combinations of multiple antagonistic organisms may provide improved disease control over the use of single organism. Multiple organisms may enhance the level and consistency of control by providing multiple mechanisms of action, more stable rhizosphere community, and be effective over a wider range of environmental conditions (Li et al. 2006; Pandey 2010). The primary aim of the study was to identify effective mutualistic fungi and PGPRs which are able to reduce the severity of root knot disease. However, their performance in the fields would greatly depend on the natural microflora of the particular environment and their capability to survive and proliferate under such condition. This study perhaps is the first step toward developing microbial consortia for effective root knot disease management and higher plant productivity. However, the microbes must be tested under field conditions before finally arriving at a logical conclusion. The trend in% increase (+) or decrease (–) in plant dry weight and oil yield was found to be similar with little differences (Figures 3 and 4). In particular, combinations of fungi and bacteria may provide protection at different times or under various conditions, and occupy different or complementary niches. Such combinations may overcome inconsistencies in the performance of individual isolates. There are reports where production of metabolites by rhizospheric bacteria causes lysis of nematode eggs (Westcott and Kluepfel 1993), reduces egg hatching (Oostendorp and Sikora 1989), affects vitality of second-stage juveniles (Becker et al. 1988) and degrades specific root exudates resulting in reduced attraction and penetration of nematodes (Oostendorp and Sikora 1990; Pandey and Kalra 2010). Siddiqui and Shaukat (2002) also reported that root colonization by rhizospheric bacteria reduced nematode invasion. The present study revealed that the tested strains of mutualistic fungi in combination with PGPRs had no adverse effect on plants but promoted significantly (p = 0.05)% oil yield of menthol mint (Table 1). Thus, the protective and nutritional properties of these microbes make them an ecofriendly useful tool for reducing deleterious impact of root knot disease caused by root knot nematode M. incognita in menthol mint.

Figure 3.  Effect of different bio-inoculants on percent increase (+) or decrease (–) of total dry weight of menthol mint.

Figure 4.  Effect of different bio-inoculants on percent increase (+) or decrease (–) in oil yield of menthol mint.

In conclusion, present results indicate that pre-inoculation with mutualistic fungi T. harzianum along with G. intraradices and P. fluorescens was highly beneficial for enhancing plant growth/yield by suppressing the development and reproduction of M. incognita. It is common practice in menthol mint growing areas of the world to transplant suckers and runners, and it is therefore feasible to incorporate different microbes during transplantation for obtaining higher oil yield/biomass of this commercial crop.

Acknowledgements

Authors are highly grateful to Prof. Ram Rajasekharan, Director, CIMAP, Lucknow, for his kind support and encouragement and to fellow scientists for their critical comments and suggestions.