Management of Radopholus similis and Helicotylenchus multicinctus in Ratoon Banana Grown under High Density Planting Systems

ABSTRACT

Cultivation of banana in modified high density planting systems with more crop cycles is widely followed in Gujarath, Kerala, and Tamil Nadu states of India due to its higher productivity and lower cost of production. In this system, three suckers were planted in a pit spaced at 1.8 × 3.6 m accommodating 1542 mats or 4626 plants/ha. Nematode pests remain a threat to this modified system of banana cultivation especially in ratoon cycles. Investigations were undertaken in ratoon banana grown under a high density planting system on effect of cartap hydrochloride 4G, carbofuran 3G, Glomus mosseae, Pseudomonas fluorescens, Sunhemp, Coriander, Marigold, and neem cake on control mixed populations of burrowing nematode, Radopholus similis, and spiral nematode, Helicotylenchus multicinctus. Application of cartap hydrochloride @ 20 g/mat twice at the time of ratooning followed by 4 months later gave greater control of R. similis and H. multicinctus in soil and root with lesser damage in root and corm than untreated plants. The banana plants in this treatment were taller with a larger number of leaves and thicker pseudostem girth resulting in 33.4–37.3% more bunch yield than other treatments including control. The effect of cartap hydrochloride at 20 g/mat was similar at higher dose (40 g/mat) but significantly superior than 10 g/mat dose. Carbofuran 3 G @ 80 g/mat twice, Neem cake @ 250 g/mat twice and growing Marigold around the basin and incorporating before flowering were the next best treatments on nematode control and growth promotion. The P. fluorescens and G. mosseae applied were not established well and caused lesser nematode control in ratoon banana grown under high density planting system. Hence application of cartap hydrochloride @ 20 g/mat or neem cake @ 250 g/mat or growing Marigold around the basin and incorporating before flowering could rectify the damage caused by R. similis and H. multicinctus in ratoon banana crops growing under a high density planting system.

KEYWORDS:

• Banana
• high density planting
• ratoon crop
• nematode control

Introduction

Banana is a globally consumed fruit and the fourth most important food crop after rice, wheat, and maize in the world. It provides livelihood and nutritional security to millions of people across the globe. Banana is grown in 150 countries across the world in an area of 4.84 M ha producing 95.5 MT (Singh, 2010). India has remained the largest producer of banana in the world with 26.21 MT fruit production from an area of 0.709 million ha which is around 28% of the world production (Tiwari, 2014). The normal spacing provided for Cavendish (AAA group) banana is 2.1 × 2.1 m (2267 plants/ha) with a yield of 42–60 T/ha. Basically, the total yield of a banana plantation depends on the total harvest per unit of area and per unit of time. Hence, the emphasis is now being given to increase the productivity per unit area by adopting various means. High density planting (HDP) concept is one among them, which has evolved in West Africa (Simmonds, 1966) and is now becoming popular in India to increase banana productivity without affecting the quality of fruits. A modified HDP in banana with 4444 plants per ha accommodating 1542 mats/ha or 3 suckers/pit has been reported to increase the yield without loss of quality in India (Krishnakumary et al., 1995; Nalina et al., 2003). This modified HDP system of banana cultivation is now getting popular among banana growers in Tamil Nadu, Maharastra, Gujarath, and Kerala states of India (Biswas and Kumar, 2010). A second and subsequent ratoon cropping practice followed in a normal banana plantation is also possible under HDP and is now being followed by the planters (Navaneethakrishnan et al., 2013). However, any change in a cultivation system has a concomitant effect on the banana ecosystem and associated biological communities.

Plant-parasitic nematodes are an important component of the microfauna associated with the rhizosphere of banana plants. A total of 132 species of nematodes belonging to 54 genera is reported to be associated with rhizosphere of the banana (Cruz et al., 2005). Out of these, 71 species of nematodes belonging to 33 genera are recorded from banana in various states of India (Sundararaju, 1996). Among them, Burrowing nematode (Radopholus similis (Cobb) Thorne), the root-lesion nematode (Pratylenchus coffea Goodey), spiral nematode (Helicotylenchus multicinctus (Cobb) Golden.), and root knot nematode (Meloidogyne spp.) are the major species affecting banana production in India (Seenivasan et al., 2013). Preliminary investigations on population dynamics of nematodes under different planting systems, such as 1 sucker/pit, 2 suckers/pit, and 3 suckers/pit in ratoon crop, proved that the HDP system with spacing of 1.8 × 3.6 m having 3 suckers/pit is more conducive for multiplication of H. multicintus, R. similis, and P. coffeae and need a suitable nematode management strategy to obtain a targeted yield (Seenivasan et al., 2008).

Currently, soil application of granular nematicide Carbofuran is the only practical option available to banana growers for the control of nematode in India. However, it is only a short-term solution, as the nematode population increases disproportionately a few months later requiring repeated applications at higher dosages, which finally become hazardous and uneconomical (Seenivasan and Poornima, 2010). Hence, there is a huge demand for potential alternative nematode control strategy among banana growers. Cartap hydrochloride, a derivative of nereistoxin, is a naturally occurring insecticide isolated from marine segmented worms (Lumrineris spp.). The insecticidal activity of cartap hydrochloride is well known. Several researchers have also reported that the soil application of cartap hydrochloride was found to reduce nematode population density in certain crops (Das et al., 2011; Mohan and Kurien, 2014; Mohanty et al., 2004; Naik et al., 2010; Pathak and Khan, 2010).

From an ecological safety point of view, use of an eco-friendly nematode control technique is a better option to keep nematode population levels at a minimum, thereby preventing major loss to the bunch yield. Several efforts have been reported in India to contain the population density of banana nematodes over the last 10 years with varying levels of success. These results included 30% nematode control in inter-cropping banana with Sunhemp, Crotolaria juncea L. (Thammaiah et al., 2007), 25–35% control in Glomus mosseae (Nicol.&Gerd.) Gerd. & Trappe. application @ 200 g/plant (Shanthi and Rajendran, 2006), 30–40% reduction in Pseudomanas flourescens (Migula) treatment @ 80 g/plant (Shanthi and Rajendran, 2006), 20% control in inter cropping banana with Coriander, Coriandrum sativum L. (Seenivasan et al., 2013), 40% control in inter-cropping banana with Marigold, Tagetes erecta L. (Sundararaju, 2005), and 70% control in neem cake @ 500 g/plant treatment (Seenivasan et al., 2013). However, as there were no reports about efforts on nematode control in ratoon banana grown under HDP, this present study was undertaken to determine the same.

Materials and methods

Experimental site

The experiments were conducted at Research Orchard, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India, which lies at 11.01° N latitude and 76.93° E longitude. The field contained natural infestation of R. similis and H. multicinctus. Soil texture is sandy clay loam (22% clay, 31% silt, 47% sand); pH: 7.6; CEC: 10.1 c mol (p⁺)/kg; organic carbon: 3.8 g/kg; electrical conducutivity: 0.26 dS/m; available N: 170 kg/ha; P: 18 kg/ha; K: 479 kg/ha; Ca: 46 μg/g; Mg: 15 μg/g; and Zn: 5 μg/g.

Planting material

The banana cv. Grand Naine (Coimbatore selection), which belongs to the triploid genome group ‘AAA’ was used in field experiments because of its high popularity throughout the world. To achieve homogeneity among plants, the variety propagated through tissue culture at M/s. Spic Agro Biotech, Coimbatore and was used in two field trials.

Experimental design

Two field experiments were conducted: Experiment I during Oct. 2009–Dec. 2011 and Experiment II during Feb. 2012–Apr. 2014. Experiment I consisted of the following 10 treatments: (1) cartap hydrochloride 4G @ 10 g/mat soil application first at ratooning and second at 3 months after ratooning; (2) cartap hydrochloride 4G @ 20 g/mat soil application first at ratooning and second at 3 months after ratooning; (3) cartap hydrochloride 4G @ 40 g/mat soil application first at ratooning and second at 3 months after ratooning; (4) Glomus mosseae @ 200 g/mat soil application first at ratooning and second at 3 months after ratooning; (5) Pseudomonas fluorescens @ 80 g/mat first at ratooning and second at 3 months after ratooning; (6) growing sunhemp around the basin and incorporating before flowering; (7) growing Coriander around the basin and incorporating before flowering; (8) growing Marigold around the basin and incorporating before flowering; (9) neem cake @ 250 g/mat first at ratooning and second at 3 months after ratooning; and (10) untreated control. Experiment II consisted of nine treatments, which is similar to Experiment I excluding the treatments, such as cartap hydrochloride 4G @ 10g/mat soil application first at ratooning and second at 3 months after ratooning; cartap hydrochloride 4G @ 40 g/mat soil application first at ratooning and second at 3 months after ratooning; and including a standard chemical check Carobofuran 3 G @ 80 g soil application first at ratooning and second at 3 months after ratooning. The experiments were laid out in randomized block design with five replicates. The individual plot size was 14 × 12 m². The banana plantlets were planted at 1.8 × 3.6 m spacing in 45 × 45 × 45 cm pits with 3 suckers/pit and accommodated 21 mats/plot. However, treatments were imposed on the middle and the remaining 12 mats have served as buffer.

Treatments were imposed on ratoon crop after cutting the plant crop on 370 days after planting. The Caldan 4G (Dhanuka Agritech Limited, Gurgaon, India) was used for cartap hydrochloride and Furadan 3G (FMC India Private Limited, Bangalore, India) was used for Carbofuran. Vermiculite-based commercial formulation of G. mosseae (100 spores/g) used in this study was obtained from the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, India. Talc-based commercial formulation of P. fluorescens strain Pf 1 (6 × 10⁸ colony forming units/g) used in this study was obtained from the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, India. Talk-based commercial formulation of P. lilacinus strain Kk (6 × 10⁸ colony forming units/g) used in this study was obtained from Horticultural Research Station, Ooty, India. For inter-crop treatments, seeds of Sunhemp cv. Coimbatore Local, Coriander cv. CO1, and Marigold cv. Alangulam Local was sown in between banana mats. Then, 45 days after sowing Sunhemp, Coriander and Marigold plants were uprooted and cut into 5–10-cm pieces. Soil around the banana mats at 15-cm diameter was plowed at 30 cm deep using a hand hoe without damaging the root system. Then the pieces of Sunhemp, Coriander, and Marigold plants were incorporated around the root zone. Further, earthing up was done around the mats. Neem cake (Azadiractin-A content 4000 ppm) used in this study was obtained from M/s. Kovai vegetable oil expeller unit, Coimbatore, India. Cultural practices were followed as per Crop Production Techniques of Horticultural Crops (Anonymous, 2013). The mats were irrigated through the drip system @ 25 L per day. The plots were not irrigated on rainy days. Plants were fertilized with 300 N:90 P₂O₅:450 K₂O g per mat in the ratoon cycle. N in the form of urea, P₂O₅ in the form of super phosphate, and K₂O in the form of muriate of potash was used. A full dose of P₂O₅ was applied during ratooning. The N and K₂O dose were applied in four splits at 3rd, 5th, 7th, and 9th month after ratooning. The fertilizers were applied in a circular band at a distance of 0.5 m from the mats. Manual digging of soil and earthing up with a hand hoe was done at bimonthly intervals. The side suckers were removed at monthly intervals. Male flowers were removed a week after opening of the last hand. Floral remnants were removed a week after opening of the last hand. The mats at flowering were propped. The incidence of spiraling whitefly, Aleurodicus disperses, and scale insect, Aspidiotus destructor, was noticed in Experiments I and II. Control measures were not taken up since their incidence occurred at low (<5 number plant^–1). No other pest or disease was recorded in Experiment I and II plots. All of the plants were given uniform practices, except the nematode management treatments.

Assessment of nematode population

The nematode population in soil was assessed during ratooning (pretreatment) and 4, 8, 12 months after ratooning. In each plot, five holes of 15-cm width and 30-cm depth were dug adjacent to pseudostem of randomly selected mats at ratooning, 4, 8, and 12 months after ratooning and soil samples were taken with a hand trowel at 5–25-cm depth from adjacent to the pseudostems. These five cores were pooled together into a composite sample (250 g for each plot). Samples were processed by Cobb’s sieving and decanting method (Cobb, 1918) and centrifugal floatation technique (Barker and Niblack, 1990). Nematode populations were expressed as numbers per 250 g soil. While collecting soil samples, primary roots of 5–10-mm thickness weighing 40–60 g were also collected from each sampling hole. To extract nematodes, roots were cut into 2-cm pieces, mixed thoroughly, and a sub-sample of 5 g was macerated in 250 mL of water in a blender for 10 s three times separated by 5-s intervals (Fallis, 1943). The macerated suspension was poured through 250-, 125-, 80-, 50-, and 20-µm pore size nested sieves. The residues collected on the last three sieves were processed by centrifugal floatation technique (Barker and Niblack, 1990) to separate nematodes. The nematode species were identified and counted using a compound stereomicroscope at 40× magnification.

Assessment of nematode damage in root and corm

Extent of nematode damage to roots in terms of root lesion index and corms in terms of corm grade were assessed according to Speijer and De Waele (1997) and Das et al. (2014) after harvesting of ratoon crop. A sucker was detached from each harvested plot and five functional primary roots were selected and were reduced to 10 cm long. Then roots were sliced lengthwise and percentage of root lesions were assessed in one half of each of the five roots. The maximum root lesion index given per root half was 20, giving a maximum root lesion index of 100 (percent) for all five together. Corm grade was assessed after thoroughly shaking off all soil and washing the corms with water. The outward half of the corm was assessed for damage after trimming the roots off. The number of roots showing black-purple lesions around their bases on the selected outward half of the corm was counted. The numbers of small lesions (lesions smaller in diameter than the root bases) and large lesions (lesions of equal or large diameter than the root bases) was counted and scoring was given as: 0—no lesions; 1—one small lesion; 2—several small lesions; 3—one large lesion; 4—several large lesions.

Assessment of growth and yield

Observations on growth and yield parameters were recorded at the time of harvest. Height of the plant was measured from the base of the pseudostem to the axils of the youngest leaf and expressed in cm. Pseudostem girth was measured from the ground level at 15 cm height and expressed in cm. Total number of leaves were counted and bunch weight was recorded.

Estimation of root colonization by biocontrol agents

To assess introduced P. fluorescens colonization, root samples along with adhering soil samples (rhizosphere) were collected from five plants from each plot in both trials at harvest and brought to the lab. The P. fluorescens was isolated by placing one gram root with adhering soil in a 250-mL Erlenmeyer flask containing nine mL of 0.1 M MgSO₄ solution (pH 6.5) plus 0.02% Tween 20 (Seenivasan et al., 2012). Ten-fold serial dilutions of the suspension were prepared and 100-µL aliquots from the appropriate dilution were plated onto King’s B Medium supplemented with rifampicin (190 µg mL^–1), actinomycin D (100 µg mL^–1), penicillin (90 µg mL^–1), and streptomycin (30 µg mL^–1). Three plates were maintained for each treatment. Inoculated plates were incubated at 25 ± 2 °C for 3 days in an incubator. After incubation, P. fluorescens colonies emitting a pale green fluorescent light under UV at 302 nm were counted.

Glomus mosseae root infection level was assessed from randomly selected root material after clearing in KOH and stained in tryphan blue (Phillips and Hayman, 1970). Percent root colonization was determined by the method of Giovannetti and Mosse (1990). Glomus mosseae spores in 50 g of soil from each replicate were estimated by recovering spores by wet sieving and decanting method (Gerdemann and Nicholson, 1963).

Statistical analysis

The data were analyzed using SPSS 16.0 for Windows software (SPSS Inc., Chicago, IL, USA). Data of nematode populations, growth, and yield parameters were tested for normal distribution by Shapiro-Wilk test and then subjected to analysis of variance (ANOVA). Nematode population count data were log transformed and the percentage values of the root lesion index were arcsine transformed before analysis. The treatment means were compared by Duncan’s multiple range test (Gomez and Gomez, 1984).

Results

The results of the Shapiro-Wilk test revealed that the original data of variables, such as corm grade, plant height, pseudostem girth, number of leaves, and bunch weight, followed normal distribution whereas R. similis and H. multicinctus population counts in soil/root and root lesion index did not follow normality. However, the log transformed soil/root population data and arcsine transformed percent root lesion index data were found to fit a normal distribution in Shapiro-Wilk test. Hence, prior to analysis, nematode population count data and root lesion index data were log and arcsine transformed to make normality. The transformed nematode count and root lesion index data were back transformed for presentation.

Effect on nematode density in soil

The population density of R. similis and H. multicinctus in soil was statically uniform at all the plots before imposing treatments in Experiments I and II (Tables 1 and 2). Four months after ratooning, the nematode population density was significantly differed among treatment plots (P < 0.001). Soil population was significantly less in cartap hydrochloride 4G @ 40 g/mat and cartap hydrochloride 4G @ 20 g/mat treatment plots in Experiment I. Cartap hydrochloride 4G @ 40 g/mat caused 87.9% and 88.5% reduction of R. similis and H. multicinctus than the untreated control. Cartap hydrochloride 4G @ 20 g/mat caused 87.0% and 88.3% reduction of R. similis and H. multicinctus than the untreated control. The effect of these treatments was statistically uniform. In Experiment II, the plots receiving cartap hydrochloride 4G @ 20 g/mat had a significantly lower nematode population density. It effected 87.0% reduction of R. similis and 88.4% reduction of H. multicinctus than untreated plots. The next best treatments were cartap hydrochloride 4G @ 10 g/mat, neem cake @ 250 g/mat, and growing Marigold around the basin, which provided 83.3%, 83.4%, and 82.5% reductions of R. similis; and 84.7%, 85.8%, and 86.0% reductions of H. multicinctus populations in Experiment I. In Experiment II the performance of neem cake, Marigold, and carbofuran treatments was next to cartap hydrochloride 4G @ 20 g/mat with nematode control efficacy of 83.4–83.8% of R. similis and 83.0–85.9% of H. multicinctus than the untreated control. In terms of nematode population reduction, P. fluorescens @ 80 g/mat, growing Sunhemp around the basin, G. mosseae @ 200 g/mat, and growing Coriander around the basin were the consecutive best treatments providing 49.2–67.4% of R. similis and 54.2–70.6% of H. multicinctus control, respectively, in Experiment I; and 49.8–67.7% of R. similis and 52.4–70.0% of H. multicinctus control, respectively, in Experiment II (Tables 1 and 2). Eight months after ratooning, there was no change in the effect of the treatments in reducing nematode population. At this time also, a smaller soil nematode population density occurred in the plots that received cartap hydrochloride 4G @ 40 g/mat (88.1% reduction of R. similis and 88.7% reduction of H. multicinctus) and cartap hydrochloride 4G @ 20 g/mat (87.2% reduction of R. similis and 89.1% reduction of H. multicinctus) in Experiment I; and cartap hydrochloride 4G @ 20 g/mat (87.7% reduction of R. similis and 87.1% reduction of H. multicinctus) in experiment II, relative to other treatment plots including control. At harvest, the application of cartap hydrochloride 4G @ 20 g/mat was the most effective treatment in reducing the nematode population density in both Experiments I and II with 87.5% reduction of R. similis and 87.6% reduction of H. multicinctus. It was equally as effective as that of the higher dosage cartap hydrochloride 4G @ 40 g/mat in Experiment I. Neem cake treatment was the next best with 83.6–84.8% of R. similis and 79.1–83.7% H. multicinctus density reduction in soil. The lowest reduction in nematode density (48.4–50.3% of R. similis and 52.0–55.9% of H. multicinctus) was observed in growing Coriander around the basin treatment.

Table 1. Effect of different treatments on Radopholus similis population in soil at 4th, 8th, and 12th month after ratooning.

Treatments	Radopholus similis population numbers ± SE in 250 g soil at different months after rationing
	Experiment I				Experiment II
	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning	Pretreatment Population	4th month after ratooning	8th month after ratooning	12th month after ratooning
CH @ 10 g	286.4 ± 11.3	76.2 ± 3.8b^z	117.6 ± 6.2b	170.3 ± 8.1b	—	—	—	—
CH @ 20 g	276.7 ± 12.1	61.2 ± 3.8a	95.3 ± 4.6a	130.6 ± 6.4a	320.7 ± 15.6	67.6 ± 3.7a	100.3 ± 5.3a	146.1 ± 7.6a
CH @ 40 g	282.7 ± 14.2	57.3 ± 3.3a	88.3 ± 3.7a	126.3 ± 7.1a	—	—	—	—
G. mosseae	275.7 ± 13.1	214.4 ± 12.1d	329.6 ± 17.1d	465.2 ± 21.3d	306.6 ± 14.1	221.1 ± 9.2d	346.8 ± 17.1d	487.6 ± 28.1e
P. fluorescens	297.2 ± 14.3	154.3 ± 9.3c	230.1 ± 12.6c	337.7 ± 16.3c	337.5 ± 16.8	168.6 ± 7.4c	267.4 ± 14.7c	367.3 ± 18.3d
Sunhemp	286.4 ± 16.7	155.3 ± 7.8c	245.6 ± 11.7c	342.1 16.3c	346.4 ± 15.7	174.3 ± 8.1c	265.2 ± 13.6c	295.7 ± 16.4c
Coriander	272.3 ± 13.1	240.4 ± 13.6e	370.5 ± 16.7e	542.8 ± 28.1e	333.2 ± 18.1	262.1 ± 14.1e	394.3 ± 19.6e	599.4 ± 29.3f
Marigold	288.6 ± 16.6	82.2 ± 3.7b	126.4 ± 5.7b	176.6 ± 8.2b	322.5 ± 16.1	84.5 ± 5.1b	135.6 ± 7.1b	193.2 ± 8.1b
Neem cake	291.6 ± 14.7	78.2 ± 4.1b	117.8 ± 5.1b	171.9 ± 7.8b	326.6 ± 14.1	86.8 ± 4.7b	122.4 ± 7.5b	182.3 ± 9.7b
Carbofuran @ 80 g	—	—	—	—	328.3 ± 12.7	83.5 ± 5.1b	133.4 ± 6.7b	214.6 ± 10.2b
Untreated control	294.0 ± 16.2	473.6 ± 28.3 f	747.2 ± 35.6f	1052.8 ± 42.6f	329.5 ± 13.7	523.1 ± 27.3f	817.6 ± 40.3f	1199.6 ± 42.6g

Note. Mean of five replications, nine mats/replication. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according to Duncan’s multiple range test.

Table 2. Effect of different treatments on Helicotylenchus multicinctus population in soil at 4th, 8th, and 12th month after ratooning.

Treatments	Helicotylenchus multicinctus population numbers ± SE in 250 g of soil at different months after rationing
	Experiment I				Experiment II
	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning
CH @ 10 g	89.7 ± 4.6	26.8 ± 1.7b^z	43.7 ± 2.6b	59.7 ± 2.5b	—	—	—	—
CH @ 20 g	102.3 ± 5.7	20.4 ± 1.3a	28.6 ± 1.7a	48.1 ± 2.1a	113.2 ± 5.2	21.3 ± 1.4a	34.9 ± 2.1a	48.1 ± 3.2a
CH @ 40 g	89.3 ± 5.1	20.1 ± 1.3a	29.4 ± 2.1a	42.1 ± 2.3a	—	—	—	—
G. mosseae	92.1 ± 6.3	64.0 ± 3.4d	98.5 ± 4.8d	146.8 ± 8.3d	116.4 ± 6.8	81.3 ± 3.8d	120.2 ± 7.1d	180.1 ± 10.6e
P. fluorescens	88.4 ± 5.6	51.5 ± 3.1c	85.0 ± 3.7c	112.5 ± 6.1c	106.5 ± 5.1	56.1 ± 2.6c	79.6 ± 4.2c	122.6 ± 7.2d
Sunhemp	105.9 ± 7.4	54.7 ± 4.2c	77.8 ± 4.1c	120.2 ± 7.4c	103.6 ± 6.2	55.0 ± 3.1c	83.8 ± 5.1c	109.3 ± 6.7c
Coriander	95.7 ± 6.2	80.1 ± 5.3e	129.3 ± 7.1e	171.4 ± 8.3e	110.9 ± 7.1	87.3 ± 4.6e	145.7 ± 7.8e	179.0 ± 9.3f
Marigold	91.1 ± 5.3	24.4 ± 1.7b	39.7 ± 2.1b	62.0 ± 2.8b	113.3 ± 6.8	31.2 ± 1.4b	44.9 ± 2.7b	67.6 ± 3.1b
Neem cake	97.4 ± 6.4	24.8 ± 1.6b	39.1 ± 2.3b	51.3 ± 2.1b	120.7 ± 7.4	25.9 ± 1.8b	45.6 ± 2.3b	60.8 ± 3.2b
Carbofuran @ 80 g	—	—	—	—	112.6 ± 7.7	23.1 ± 1.6b	44.1 ± 1.9b	76.2 ± 4.1b
Untreated control	88.1 ± 5.2	175.2 ± 9.6f	262.4 ± 15.3f	389.4 ± 20.6f	109.8 ± 6.4	183.7 ± 10.2f	272.5 ± 16.1f	373.3 ± 18.3g

Note. Mean of five replications, nine mats/replication. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according to Duncan’s multiple range test.

Effect on nematode infestation in roots

All of the treatments evaluated in the two experiments were capable of significantly reducing the R. similis and H. multicinctus infestation on banana roots throughout the ratoon crop cycle (P < 0.001). However, the degree of nematode infestation on roots significantly varied among the treatments (Tables 3 and 4). Cartap hydrochloride 4G @ 40 g/mat and cartap hydrochloride 4G @ 20 g/mat treatments resulted in the lowest nematode infection until 12 months after ratooning. In these treatments, R. similis population was 5.3- to 6.2-fold less and H. multicinctus population was 4.7- to 6.6-fold less than the control in Experiment I. In Experiment II, cartap hydrochloride 4G @ 20 g/mat treatment had 5.4- to 7.7-fold less population of R. similis and 5.1- to 8.2-fold less population of H. multicinctus in roots at 4, 8, and 12 months after treatment. Neem cake and Marigold were the next best treatments recorded 3.6- to 4.2-fold less population of R. similis and 3.5- to 4.0-fold less population of H. multicinctus in Experiments I and II. Carbofuran 3G @ 80 g had 3.4–3.9-fold less population of R. similis and 2.7–4.1-fold less population of H. multicinctus, which was on par with neem cake and Marigold treatments in Experiment II. P. fluorescens @ 80 g/mat and growing Sunhemp around the basin gave intermediate root protection but was significantly better than G. mosseae @ 200 g/mat and growing coriander around the basin treatments.

Table 3. Effect of different treatments on Radopholus similis population in banana roots at 4th, 8th, and 12th month after ratooning.

Treatments	Radopholus similis population numbers ± SE in 5 g root at different months after ratooning
	Experiment I				Experiment II
	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning
CH @ 10 g	25.5 ± 1.7	9.7 ± 0.7b^z	15.2 ± 0.7c	20.1 ± 1.3b	—	—	—	—
CH @ 20 g	21.3 ± 1.6	7.1 ± 0.4a	11.7 ± 0.9ab	16.8 ± 1.1a	25.8 ± 1.1	7.9 ± 0.6a	7.9 ± 0.4a	16.1 ± 1.1a
CH @ 40 g	24.0 ± 1.7	6.8 ± 0.5a	9.8 ± 0.6a	13.5 ± 1.2a	—	—	—	—
G. mosseae	21.7 ± 1.3	15.3 ± 0.7d	7.3 ± 0.4a	36.6 ± 2.1d	27.5 ± 1.6	19.5 ± 1.3e	27.5 ± 1.8d	43.0 ± 2.3d
P. fluorescens	25.6 ± 2.1	12.5 ± 0.8c	19.6 ± 1.6d	25.8 ± 1.7c	23.9 ± 1.7	13.6 ± 1.6c	20.3 ± 1.1c	30.6 ± 1.6c
Sunhemp	22.0 ± 1.7	14.4 ± 0.6c	22.0 ± 1.8d	32.8 ± 1.9c	25.2 ± 1.6	16.2 ± 1.7d	21.0 ± 1.4c	31.8 ± 1.8c
Coriander	24.3 ± 2.6	18.1 ± 1.1e	26.5 ± 1.4e	41.1 ± 2.6d	27.4 ± 2.1	18.1 ± 1.4e	29.8 ± 2.3e	43.1 ± 2.1d
Marigold	22.2 ± 1.8	8.6 ± 0.5b	14.9 ± 0.8c	19.6 ± 1.6b	25.6 ± 1.8	10.6 ± 0.7b	16.0 ± 0.9b	24.1 ± 1.7b
Neem cake	23.8 ± 2.1	9.6 ± 0.7b	14.3 ± 0.7c	22.6 ± 1.8b	28.3 ± 2.2	10.9 ± 0.8b	18.3 ± 0.7b	22.6 ± 1.4b
Carbofuran @ 80 g	—	—	—	—	26.7 ± 2.1	10.3 ± 1.1b	17.6 ± 1.7b	28.4 ± 1.8bc
Untreated control	23.9 ± 1.7	42.0 ± 2.6f	65.3 ± 3.7f	79.6 ± 4.1e	25.6 ± 2.1	40.7 ± 2.3f	65.3 ± 2.8f	98.0 ± 5.2e

Note. Mean of five replications, nine mats/replication. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according to Duncan’s multiple range test.

Table 4. Effect of different treatments on Helicotylenchus multicinctus population in banana roots at 4th, 8th, and 12th month after ratooning.

Treatments	Helicotylenchus multicinctus population numbers ± SE in 5 g root at different months after ratooning
	Experiment I				Experiment II
	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning	Pretreatment population	4th month after ratooning	8th month after ratooning	12th month after ratooning
CH @ 10 g	69.2 ± 2.6	27.7 ± 1.7b^z	43.6 ± 2.8c	64.3 ± 3.1b	—	—	—	—
CH @ 20 g	71.4 ± 3.1	21.1 ± 1.3a	31.9 ± 1.4ab	45.6 ± 2.2a	77.6 ± 3.2	22.8 ± 1.3a	25.3 ± 1.8a	51.2 ± 2.6a
CH @ 40 g	72.1 ± 3.3	19.6 ± 1.6a	29.4 ± 1.8a	42.8 ± 2.6a	—	—	—	—
G. mosseae	73.0 ± 3.7	51.3 ± 2.7d	23.5 ± 1.7a	109.8 ± 5.7d	78.6 ± 3.8	53.6 ± 2.7e	74.5 ± 3.8d	116.5 ± 7.6d
P. fluorescens	69.5 ± 2.8	37.7 ± 1.8c	58.8 ± 3.4d	86.6 ± 4.1c	80.4 ± 4.6	41.1 ± 3.1c	64.3 ± 3.1c	91.8 ± 4.2c
Sunhemp	69.7 ± 3.1	41.2 ± 1.9c	62.7 ± 3.7d	88.4 ± 4.7c	79.9 ± 4.1	43.8 ± 2.8d	70.6 ± 3.8c	100.7 ± 6.8c
Coriander	69.3 ± 3.4	54.3 ± 2.3e	84.1 ± 4.6e	117.1 ± 6.2d	74.3 ± 3.7	60.8 ± 3.9e	89.6 5.1e	129.3 ± 7.3d
Marigold	70.5 ± 3.9	29.0 ± 1.7b	42.5 ± 2.1c	62.4 ± 3.6b	77.1 ± 4.3	30.3 ± 1.8b	45.9 ± 1.8b	65.2 ± 3.1b
Neem cake	71.6 ± 4.1	30.6 ± 1.4b	48.7 ± 2.7c	67.8 ± 3.9b	76.8 ± 3.6	32.7 ± 2.1b	49.7 ± 1.7b	75.9 ± 3.9b
Carbofuran @ 80 g	—	—	—	—	78.3 ± 4.0	31.4 ± 1.9b	47.8 ± 2.1b	86.3 ± 4.7bc
Untreated control	68.4 ± 3.7	113.8 ± 5.3f	176. 9 ± 8.2f	266.5 ± 14.3e	77.1 ± 4.6	129.1 ± 6.7f	196.2 ± 9.6f	279.3 ± 15.6e

Note. Mean of five replications, nine mats/replication. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according to Duncan’s multiple range test.

Effect on root and corm damage

Nematode damage to the banana plants was severe in untreated plots that had 56.4–65.4 root lesion index and 3.7 corm grade (Table 5). The least lesion index (12.3–16.2) and corm grade (1.3–1.7) was observed in cartap hydrochloride 40-g and 20-g treated plants. The next improved protection to root and corm damage was observed in carbofuran, neem cake, and Marigold treatment plots. In other treatment plots, the root and corm damage were significantly more severe as lesion index ranged from 29.5 to 54.1 and corm grade from 2.7 to 3.3.

Table 5. Effect of different treatments on root and corm damage in first ratoon cycle banana cv. Grand Naine during harvest.

Treatments	Root lesion index (%)		Corm grade
	Experiment I	Experiment II	Experiment I	Experiment II
CH @ 10 gc	24.3 ± 1.3b^z	—	2.1 ± 0.15b	—
CH @ 20 g	14.1 ± 0.7a	16.2 ± 0.7a	1.3 ± 0.1a	1.7 ± 0.15a
CH @ 40 g	12.3 ± 0.7a	—	1.3 ± 0.1a	—
G. mosseae	46.7 ± 2.3d	38.6 ± 1.3d	3.1 ± 0.15d	3.1 ± 0.2d
P. fluorescens	36.3 ± 1.7c	29.5 ± 1.1c	2.7 ± 0.15c	2.7 ± 0.15c
Sunhemp	38.7 ± 1.7c	32.1 ± 1.1c	2.7 ± 0.15e	2.7 ± 0.15c
Coriander	54.1 ± 2.7e	44.7 ± 1.7e	3.3 ± 0.2b	3.3 ± 0.2de
Marigold	25.7 ± 1.3b	22.7 ± 0.7b	2.3 ± 0.1b	2.1 ± 0.15b
Neem cake	22.7 ± 1.1b	23.5 ± 1.1b	2.0 ± 0.15b	2.1 ± 0.15b
Carbofuran @ 80 g	—	22.3 ± 1.5b	—	2.2 ± 0.13b
Untreated control	65.4 ± 2.7f	56.4 ± 2.1f	3.7 ± 0.2f	3.7 ± 0.2f

Note. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according

to Duncan’s multiple range test.

Effect on growth and yield

Growth parameters, such as plant height, pseudostem girth, and number of leaves, were increased significantly following cartap hydrochloride, carbofuran, G. mosseae, P. fluorescens, Sunhemp, Coriander, Marigold, and neem cake treatments reducing the nematode infestation on banana roots throughout the ratoon crop cycle (P < 0.001). The untreated plants produced smaller plants with a thin pseudostem and reduced number of leaves (Table 6). The cartap hydrochloride 40 g and 20 g had a significantly higher effect in plant growth promotion than all other treatments. These treatments produced significantly taller plants (186.3–213.8 cm height) with thick pseudostem (60.3–66.8 cm pseudostem girth) and increased number of leaves (7.7–9.3) than the other treatments. The increased plant growth in different treatment plots ultimately resulted in a significant increase in bunch yield (P < 0.001) than control. Cartap hydrochloride 40-g and 20-g treated plants produced heavier bunches (25.2–36.3 kg bunch weight) than other treatments including control. Among biological treatments, neem cake and Marigold produced significantly taller plants (177.3–196.5 cm height) with thick pseudostem (56.7–62.3 cm pseudostem girth), increased number of leaves (6.9–8.1), and heavy bunches (22.9–34.2 kg bunch weight).

Table 6. Effect of different treatments on plant height, plant girth, number of leaves, and bunch weight of first ratoon cycle banana cv. Grand Naine during harvest.

Treatments	Experiment I				Experiment II
	Plant height (cm)	Plant girth (cm)	No. of leaves at harvest	Bunch weight (Kg/mat)	Plant height (cm)	Plant girth (cm)	No. of leaves at harvest	Bunch weight (Kg/mat)
CH @ 10 g	195.6 ± 9.3b^z	61.7 ± 2.8b	7.8 ± 0.4b	34.1 ± 1.8b	—	—	—	—
CH @ 20 g	207.0 ± 11.4a	66.3 ± 3.3a	8.7 ± 0.5a	36.0 ± 2.1a	186.3 ± 7.3a	60.3 ± 3.8a	7.7 ± 0.5a	25.2 ± 1.8a
CH @ 40 g	213.8 ± 12.6a	66.8 ± 3.0a	9.3 ± 0.7a	36.3 ± 1.7a	—	—	—	—
G. mosseae	175.3 ± 10.1d	56.5 ± 2.5cd	6.9 ± 0.4cd	27.3 ± 1.6d	157.2 ± 8.3d	49.1 ± 2.7d	5.4 ± 0.4d	19.6 ± 1.1cd
P. fluorescens	183.3 ± 11.7c	59.1 ± 2.3c	7.1 ± 0.5c	31.7 ± 2.0c	168.7 ± 9.1c	52.1 ± 3.1c	6.4 ± 0.4c	20.8 ± 1.6a
Sunhemp	184.1 ± 10.6c	58.7 ± 3.1c	7.0 ± 0.3c	29.7 ± 1.6c	166.1 ± 7.4c	52.8 ± 3.9c	6.5 ± 0.5c	21.0 ± 1.9c
Coriander	166.4 ± 10.3e	54.1 ± 2.0e	6.2 ± 0.4e	25.3 ± 1.8e	146.3 ± 6.6e	45.1 ± 3.2e	5.4 ± 0.3e	19.0 ± 1.3d
Marigold	193.0 ± 11.2b	61.1 ± 2.7b	7.7 ± 0.5b	34.0 ± 1.7b	177.3 ± 8.2b	56.7 ± 4.3b	6.9 ± 0.6bc	22.9 ± 1.6b
Neem cake	196.5 ± 12.7b	62.3 ± 3.2b	8.1 ± 0.6b	34.2 ± 2.3b	175.1 ± 9.6b	57.0 ± 3.8b	7.0 ± 0.5bc	23.0 ± 1.7b
Carbofuran @ 80 g	—	—	—	—	176.7 ± 8.6b	56.9 ± 3.6b	7.0 ± 0.6bc	22.4 ± 1.8b
Untreated control	155.6 ± 9.2f	51.0 ± 2.8f	5.6 ± 0.3f	22.7 ± 1.8f	134.4 ± 7.2f	41.3 ± 2.8f	4.9 ± 0.3f	15.8 ± 1.2e

Note. CH: Cartap hydrochloride.

^zMeans followed by the same letter in a column are not significantly different at p ˂ 0.05 according to Duncan’s multiple range test.

Root colonization by introduced biocontrol agents

Root colonization by G. mosseae and P. flourescens was 12% and 4.7 ± 0.33 × 10⁵ CFU in Experiment I and 14% and 5.3 ± 0.27 × 10⁵ CFU in Experiment II, respectively. In G. mosseae treated plots the spore count in the soil was 44 spores/50 cm³ in Experiment I and 56 spores/50 cm³ in the soil in Experiment II (Table 7).

Table 7. Colonization of roots of banana cv. Grand Naine by the bio-control agents at harvest in ratoon cycle crop.

Treatments	Experiment I	Experiment II
Cartap hydrochloride @ 10 g/mat	—
Cartap hydrochloride @ 20 g/mat	—	—
Cartap hydrochloride @ 40 g/mat	—	—
AM @ 200 g/mat	24 spores/50 cm^3 soil (9% root colonization)	36 spores/50 cm^3 soil (11% root colonization)
Pf @ 80 g/mat	4.7 ± 0.33 × 10^5 CFU	5.3 ± 0.27 × 10^5 CFU
Growing Sunhemp around the basin	—	—
Growing Coriander around the basin	—	—
Growing Marigold around the basin	—	—
Neem cake @ 250 g/mat	—	—
Carbofuran @ 80 g/mat	—	—
Untreated control	—	—

Discussion

Experimental results of testing the comparative efficacy of cartap hydrochloride 4G at three dosages, such as 10 g, 20 g, and 40 g; Glomus mosseae @ 200 g/mat, Pseudomonas fluorescens @ 80 g/mat, intercropping and incorporation of Sunhemp Coriander, and Marigold; and neem cake @ 250 g/mat revealed that all of the above treatments were found to reduce nematode problems in ratoon banana grown under a high-density planting system as compared to untreated plots. However, soil application of cartap hydrochloride 4G @ 20 g/mat and @ 40 g/mat two times, one at the time of ratooning and subsequent at 90 days after first application, was the most effective approach as they could reduce the soil nematode density and root infestation to the extent of 87.1–88.4% and 81.4–83.8% as compared to control. Cartap hydrochloride is a systemic insecticide of Nereistoxin analoque group, which belongs to nicotinic acetylcholine receptor (nAChR) channel blockers and has stomach and contact action. Earlier findings established that the nematicidal potential of several carbamate group pesticides, such as Aldicarb (Sarah et al., 1988), Oxamyl (Chabrier et al., 2005), Aldoxycarb (Figueroa, 1980), and Carbofuran (Mateille et al., 1988); and organophosphorus group compounds, such as Thionazin (Whitehead, 1986), Fenamiphos (Mateille et al., 1988; Mulder, 2012), Ethoprophos (Bartholomew et al., 2014), Fensulfothion (Mcsorley and Parrado, 1986), Terbufos (Cabrera et al., 2010), Isazofos (Mateille et al., 1988; Speich, 1982), cadusafos (Queneherve et al., 1991), and fosthiazate (Chabrier et al., 2002) on the control of banana nematodes. However, many of these nematicides are more expensive and still not available in countries like India. In this situation, this study established a nematicidal potential of a dithiocarbamate pesticide cartap hydrochloride against banana nematodes. The other nematicides used in banana plantations are organobromide DBCB (Minz et al., 1960) and the dithiocarbamates metham sodium (Kai et al., 2011) and dazomet (Kim et al., 2013). Cartap hydrochloride has been previously reported in crops, such as rice against white tip nematode, Aphelenchoides besseyi (Mohanty et al., 2004); Coleus, Solenostemon rotundifolius (Poir) Morton against Meloidogyne sp. (Mohan and Kurien, 2014); tuberose, Polianthes tuberosa L. against Meloidogyne incognita (Naik et al., 2010) and A. besseyi (Pathak and Khan, 2010); and cotton against Rotylenchulus reniformis (Das et al., 2011). This is the first report known to the authors mentioning the use of one more dithiocarbamate group of pesticide cartap hydrochloride in the control of banana nematodes R. similis and H. multicinctus especially in ratoon cycle of high-density banana crop. Our results confirm the existence of R. similis and H. multicinctus control as well as pathogenicity reduction on banana roots after soil application of cartap hydrochloride. Results of this study also proved that cartap hydryrochloride 4G @ 20 g/mat is sufficient to achieve maximum nematode control since it was equally as good as that of cartap hydrochloride 4G @ 40 g/mat and superior to carbofuran 3 G @ 80 g/mat in suppressing the nematode density in soil, root infestation, root and corm damage.

Among biological treatments, neem cake and Marigold treatments are superior on nematode control than P. fluorescens, G. mosseae, and Coriander treatments. Neem cake application @ 500 g/plant at planting and growing Marigold (Tagetes errecta) around the basin and incorporation of their bio-mass 60 days after sowing was proved to be the most effective treatments against R. similis and H. multicinctus in first cycle banana crop (Seenivasan et al., 2013). This study supports the earlier finding and validated the feasibility of neem cake and Marigold treatments on nematode control in ratoon banana grown under the HDP system. The significant nematicidal efficacy of neem cake is inferred as a result of nemato-toxic fractions, such as azadirachtin, nimbin, salannin, limonoids, steroids, and terpenoid glycosides (Oka, 2010). The significant nematicidal efficacy of Marigold observed in the present study could be inferred due to the release of biologically active toxic principles like α-terthienyl and 5-(3-buten-1-enyl)-2,2′–bithienyl from roots, generation of singlet oxygen by photoactivated α-terthienyl, dodecanoic acid, myristic acid, palmitic acid, and steric acid from flowers (Seenivasan et al., 2013).

Field efficacy of P. fluorescens and G. mosseae against banana nematodes is established earlier (Selvaraj et al., 2014; Shanthi and Rajendran, 2006). However, the results of this study revealed that bio-efficacy of these bio-agents are low against R. similis and H. multicinctus in ratoon banana grown under a high-density planting system. In this study, the root colonization of P. fluorescens was 4.7–5.3 × 10⁵ CFU and G. mosseae was 9–11% at harvest stage. But in earlier studies the P. fluorescens establishment was 6.2 × 10⁷ CFU g^–1 root (Selvaraj et al., 2014) and G. mosseae root colonization was 44% (Shanthi and Rajendran, 2006). The biocontrol potential of P. fluorescens and G. mosseae had directly related to their root colonization potential (Seenivasan, 2011; Seenivasan and Devrajan, 2008). The strains, inoculums level in the formulations, and dosages of P. fluorescens and G. mosseae used in the earlier studies were only used in this study too. Hence, it clearly indicates that P. fluorescens and G. mosseae were not able to establish well in ratoon banana grown under a high density planting system and resulted in a reduction of bio-efficacy against banana nematodes. In this study, all of the treatments, including P. fluorescens and G. mosseae, were applied in ratoon crop planted in HDP. The bio-control agents, P. fluorescens and G. mosseae, colonize and establish well on banana roots when they are applied at an early stage of the crop either as soil application, sucker treatment, or bio-priming of plantlets (Jaizme-Vega et al., 1997; Rodriquez-Romero et al., 2008). Selvaraj et al. (2014) also reported that repeated application of P. fluorescens formulation for three times from planting up to 4 months after planting is needed to get sufficient control of H. multicinctus populations in banana. Ratoon banana grown under HDP was reported to cause necrosis or rotting on 55.5% of roots due to nematode damage (Seenivasan et al., 2008). The P. fluorescens and G. mosseae are obligatorily dependent on the live roots for better colonization and establishment. Hence, the delayed inoculation on damaged banana roots might be the reason for the reduced bio-efficacy of P. fluorescens and G. mosseae in ratoon banana crop grown under HDP.

It is well proven that R. similis and H. multicinctus are one of the causes of growth and yield decline of banana (Barekye et al., 2000; Rajendran and Sivakumar, 1996; Sarah et al., 1993). The damage was especially higher when they occur as mixed population than the damage caused by H. multicinctus alone (Barekye et al., 2000). Fogain and Gowen (1997) also confirmed that growth and yield decline in banana is a consequence of severe destruction of root systems by nematodes. Hence, the growth and yield improvement of banana plants as reported in cartap hydrochloride, neem cake, and Marigold-treated plots might be attributed to superior nematode control and nematode damage reduction achieved in these plots.

In India, the availability of nematicides is a concern due to the fact that nematodes are a rather difficult target and the cost of research and registration is enormous for prospecting a new synthetic nematicide. Currently, application of granular nematicide carbofuran is considered to be the only solution for the control of banana nematodes in India. However, it is only a short-term solution, as the nematode population increases disproportionately a few months after application, requiring repeated use at higher dosages, which may finally become hazardous and uneconomical (Seenivasan et al., 2013; Shanthi and Rajendran, 2006). In this situation, the findings of this study, which is application of cartap hydrochloride @ 20 g/mat, could be a novel technique to achieve nematode control and sustain the bunch yield in ratoon banana grown as a high density crop. Different neem formulations have been used against R. similis on banana with promising results (Kosma et al., 2011; Seenivasan et al., 2013) and application of neem was now well established as an alternative to synthetic nematicides to control R. similis (Bartholomew et al., 2014). Hence, biological approaches, such as neem cake @ 250 g/mat followed by 250 g after the 3rd month or growing Marigold (Tagetus erecta) around the basin and incorporating before flowering might also be recommended for ratoon high-density banana plantations.

Acknowledgments

The author wishes to thank the anonymous reviewers for providing valuable comments to improve this article. The author also extends thanks to Dr. B. Augustine Jerard, Senior Scientist (Horticulture), Central Plantation Crops Research Institute, Kerala for technical and language correction of the manuscript.

Funding

The author gratefully acknowledges the Indian Council of Agricultural Research, New Delhi, India for the financial support through the All India Co-ordinated Research Project on Tropical Fruits.

Additional information

Funding

The author gratefully acknowledges the Indian Council of Agricultural Research, New Delhi, India for the financial support through the All India Co-ordinated Research Project on Tropical Fruits.