# Screening SelectedSolanum Plants as Potential Rootstocks for the Management of Root-Knot Nematodes (Meloidogyne incognita)

Authors: Benjamin A. Okorley; Charles Agyeman; Naalamle Amissah; Seloame T. Nyaku
Journal: International Journal of Agronomy
 (2018)

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## Abstract

Root-knot nematodes (RKNs) (Meloidogyne spp.) represent agricultural pest of many economic crops, including tomatoes and potatoes. They advance a complex parasitic relationship with roots of tomato plants leading to modification of host structural and physiological functions in addition to significant yield loss. Resistance in solanaceous plants to RKNs has been identified and associated with the possession of Mi gene. The reaction of four Solanum rootstocks (S. aethiopicum L., S. macrocarpon L., S. lycopersicum L.“Mongal F1,” and S. lycopersicum L. “Samrudhi F1”) was evaluated in pots and in a natural Meloidogyne spp.-infested field in a two-year trial (2015–2016), to identify RKN-resistant rootstock(s), which can be utilized in tomato grafting as a management measure against these nematodes. A rootstock’s reaction to RKNs was assessed using root gall scores (GSs), egg count/g of root, and reproductive factors (Rfs) at the end of 6 and 12 weeks after transplanting (wat) in infested fields, respectively. Solanum macrocarpon, S. aethiopicum, and Mongal F1 showed tolerant responses with reduced root galling and low to high reproductive factors in pot and field experimentation. Although Samrudhi F1 was resistant in both pot and field trials and consistently decreased nematode root galling (<1.00) and reproduction (Rf < 1.00), it failed to significantly increase yield, as compared with the highest yield obtained by the tolerant rootstock, Mongal F1 (870.3 and 1236.6 g/plant, respectively). Evaluation of the four rootstocks against four (0, 500, 1,000, and 5000) RKN inocula levels (Juveniles) showed no significant differences among the growth parameters (fresh and dry shoot and root weights). Root-knot nematode-susceptible tomato varieties, for example, Pectomech F1, a popular tomato variety in Ghana, can be grafted onto the RKN-resistant and RKN-tolerant rootstocks for increased yields.


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## Body

## 1. Introduction

Tomato (Solanum lycopersicum L.) is an annual crop of the Solanaceae family, and the second most widely consumed vegetable after potatoes [1]. Tomato fruits are highly nutritious, and constitute a rich source of vitamins A, C, and E, and essential minerals, namely, potassium, phosphorus calcium, magnesium, and iron [2, 3]. Tomato and its products contain the antioxidant lycopene, which reduces cancers and development of atherosclerosis [4, 5]. The large diversity in the crop makes it adaptable to different climatic conditions, ranging from temperate to tropical environments, production methods, and uses [6]. Ghana has a young tomato industry (production estimated at 366, 722 tonnes from 47,000 hectares of land [7]), with the potential to increase production in the savannah and transitional zones because of the derived income and nutritional benefits. Pectomech F1, Power Rano, and “Wosowoso” are popular varieties cultivated for high yield and quality fruits. However, domestic production levels over the years have been low, mainly due to its vulnerability to pest and diseases, including plant-parasitic nematodes [8].Plant-parasitic nematodes are economic pests of agriculture worldwide, with more than 3000 plants species as host [9–11]. Global losses associated to root-knot nematodes (RKNs) alone from 75 countries as at 2000 was valued at $121 billion [12] and cost about $500 million for their annual control [13, 14]. In Ghana, RKNs are responsible for 33% of vegetable losses per season [15], in which 73–100% are realized in tomato production alone [16]. The RKNs thus impact a key limitation to tomato production, as the crop is considered the most vulnerable in the tropics [17, 18]. Currently, crop rotation, soil solarization, nematode-free seedlings, and the use of nematicides are adopted by Ghanaian farmers for managing nematodes [16, 19]. The withdrawal of extensively used fumigants such as ethylene dibromide, because of its carcinogenic nature and its ability to contaminate groundwater [12, 20], has necessitated the adoption and worldwide use of tolerant/resistant rootstocks, as an alternative for nematode control because they are safe to both farmers and the environment [21].Nematode-resistant plants or rootstocks have the intrinsic ability, to be unaffected significantly upon nematode attack, and will greatly contribute to reducing nematode infestations in tomato fields [22–24]. Grafting utilizing resistant rootstocks has proven to effectively manage RKNs and improve yield in tomato and eggplant cultivated in naturally infested nematode soils [25]. Resistance to RKN species (M. incognita, M. javanica, and M. arenaria) is conferred by a single dominant Mi gene, introduced from the wild relative S. peruvianum accession (P.I. 128657), through embryo rescue technique, into commercially cultivated tomatoes [26, 27]. Dhivya et al. [28] reported resistant and susceptible reaction, in wild Solanum plants after assessing their response to RKN. In another study, 33 tomato genotypes were evaluated against RKN, and variable responses in gall development and nematode reproduction existed [29]. Plants with the Mi gene also effectively increased yields to about ten-fold, compared with susceptible plants at high nematode inoculum levels of about 200, 000 eggs/plant [24]. Solanaceous plants, for example, S. aethiopicum, S. macrocarpon, S. torvum, and S. lycopersicum cultivars, are commercially available, and common among local farmer seed stocks in Ghana. However, the use of resistant rootstocks in tomato cultivation remains less explored, due to the unknown response of this Solanum spp. to RKNs. Grafting of tomato scions with superior traits onto RKN-resistant rootstocks will help manage this biotic stress in a healthy and environmentally friendly manner [30], reduce production cost, and improve yields [31].The use of resistantSolanum plants in grafting experiments in Ghana is in its infancy; therefore, there is a need to identify sources of resistance in the available Solanum rootstocks for managing the RKN problem in tomato fields. This study was thus initiated to screen and identify potential rootstock(s) among four selected Solanum plants for resistance to M. incognita.


## 2. Materials and Methods

### 2.1. Plant Materials

Seeds of the test rootstocks,S. aethiopicum and S. macrocarpon “Gboma,” were obtained from the Department of Crop Science, University of Ghana. Solanum lycopersicum “Mongal F1” and S. lycopersicum “Samrudhi F1” were supplied by Agriseed Company and East-West Seed Ltd., respectively.


### 2.2. Sowing of Rootstock Seeds

The seeds were sown in trays containing oven-sterilized soil.Solanum macrocarpon and S. aethiopicum were sown ten days earlier than the other tomato rootstocks because of their slow growth rate. A week after seedling emergence, N-P-K: 15-15-15 (6 g/L) nutrient solution was applied once every week by immersing in a bath for 5-6 minutes. A shade cover was placed over the seedlings to minimize the solar radiation on the leaves. Suncozeb 80 WP at 20 g/L of H20 was sprayed, until runoff close to the crown of the seedlings, to prevent damping-off on seedlings. Two weeks before transplanting the seedling, the shade cover was completely removed to allow them harden-off.


### 2.3. Experimental Area (Field Trials)

The field experiments were conducted at the Biotechnology and Nuclear Agricultural Research Institute (BNARI) of Ghana Atomic Energy Commission, Legon-Accra on a natural RKN-infested field. The experimental site had a sandy loam soil, humid tropical climate with average low-high day temperatures of 25–34°C (dry season) and 25–29°C (rainy season), pH ≈ 6.4, and a history of being used continuously for growing vegetables, thus making the site a hot spot for RKNs. Laboratory and nursery activities were carried out in the Department of Crop Science, University of Ghana.


### 2.4. Experimental Design (Field Trials)

The experimental field was slashed and debris collected; then, three beds each measuring 23.2 m × 2.0 m were raised and pegged at a planting distance of 80 cm × 40 cm. A randomized complete block design (RCBD) with three replicates partitioned by two alleys of 0.5 m each was used. A block contained five plots, each measuring 4.0 m × 1.6 m. Thirty (30) plants constituted a plot, with ten plants in a row and three plants between rows. Eight middle row plants were used as record plants, from which data were taken. The experimental setup was repeated for the succeeding trial in 2016. The layout for the pot experiments was a 4 × 4 factorial, arranged in a split plot design with three replicates.


### 2.5. Agronomic Practices and Parameters Taken on Tomato Plants (Field Trials)

Four-week-old seedlings were transplanted (one per stand), and a starter solution (N-P-K: 15-15-15 at 6 g/L of water) applied at 100 mL/plant. Two (2) kg N.P.K was mixed with 1 kg NH3 and was also applied at 15 g/plant as a side dressing. Agricombi (fenitrothion 30% + fenvalerate 10%) were sprayed twice, at a rate of 50 mL/L of H2O, to control whiteflies, aphids, and red spider mites. The plants were cared for by regular watering and weed control. Plant height, plant girth, and chlorophyll contents were taken 5, 7, and 9 weeks after seedling transplant.


### 2.6. Experimental Design (Pot Trials)

The layout for the pot experiment was a 4 × 4 factorial experiment, with a control arranged in a split plot design with three replicates, each experimental plot consisting of 30 plants. Root-knot nematode inoculums (Juveniles) were prepared by extracting nematode eggs from the roots of infested plants, and hatched into juveniles [32]. These were then concentrated into the various RKN inoculum levels (0, 500, 1,000, and 5,000). The plants (rootstocks) were then inoculated with the juveniles, by making a small hole or depression in the soil, at the base plants after which the required inoculum level was gently poured into the hole and covered.


### 2.7. Nematode Analysis

Soil samples taken from each plot were used to determine the RKN populations, before transplanting (initial nematode population Pi), six weeks after transplanting (P6), and after harvesting (Pf) in each trial. Six random soil samples per plot were taken with a soil augur, and bulked together into a well-labeled plastic bag. A subsample of 200 cubic centimeter (cc) was used in soil extraction, through the sieving and sucrose centrifugation method [33]. Reproductive factors (Rfs) for each treatment plot, which is a ratio of the final to initial population (Pf/Pi), were used to determine the rate of nematode multiplication in the soil. Gall scoring and nematode egg extraction were carried out 6 and 12 weeks after transplanting rootstocks, to estimate RKN gall development and egg production on the host. Sampled roots were washed separately and air-dried for 5 minutes. Galls on each root system were scored on a scale of 0–10 (no damage to severe damage), using the severity rating chart by Bridge and Page [34]. Roots were then cut into pieces, and nematode eggs extracted using 10% sodium hypochlorite (NaClO) [32]. Eggs obtained were counted under the compound microscope (Exacta–OptechBiostar B5P, Germany) (magnification x100) and recorded. The mean gall scores (GSs), egg count per gram of root, and reproductive factors (Rfs) obtained from the two field trials were used as the basis to evaluate the resistance status of the rootstocks. Rootstocks were classified as resistant when their root GS < 2 and Rf < 1, tolerant when GS < 2 and Rf ≥ 1, or susceptible when GS ≥ 2 and Rf > 1 [35, 36].


### 2.8. Statistical Analysis

Data collected on agronomic parameters (plant height and plant girth), chlorophyll content, yield parameters, and plant biomass content were subjected to analysis of variance (ANOVA), using Genstat 12th edition software [37], and significant means separated, using least significant difference (LSD) at 5%. Where necessary, data were transformed using the equation Log (x + 1), for normality.


## 2.1. Plant Materials

Seeds of the test rootstocks,S. aethiopicum and S. macrocarpon “Gboma,” were obtained from the Department of Crop Science, University of Ghana. Solanum lycopersicum “Mongal F1” and S. lycopersicum “Samrudhi F1” were supplied by Agriseed Company and East-West Seed Ltd., respectively.


## 2.2. Sowing of Rootstock Seeds

The seeds were sown in trays containing oven-sterilized soil.Solanum macrocarpon and S. aethiopicum were sown ten days earlier than the other tomato rootstocks because of their slow growth rate. A week after seedling emergence, N-P-K: 15-15-15 (6 g/L) nutrient solution was applied once every week by immersing in a bath for 5-6 minutes. A shade cover was placed over the seedlings to minimize the solar radiation on the leaves. Suncozeb 80 WP at 20 g/L of H20 was sprayed, until runoff close to the crown of the seedlings, to prevent damping-off on seedlings. Two weeks before transplanting the seedling, the shade cover was completely removed to allow them harden-off.


## 2.3. Experimental Area (Field Trials)

The field experiments were conducted at the Biotechnology and Nuclear Agricultural Research Institute (BNARI) of Ghana Atomic Energy Commission, Legon-Accra on a natural RKN-infested field. The experimental site had a sandy loam soil, humid tropical climate with average low-high day temperatures of 25–34°C (dry season) and 25–29°C (rainy season), pH ≈ 6.4, and a history of being used continuously for growing vegetables, thus making the site a hot spot for RKNs. Laboratory and nursery activities were carried out in the Department of Crop Science, University of Ghana.


## 2.4. Experimental Design (Field Trials)

The experimental field was slashed and debris collected; then, three beds each measuring 23.2 m × 2.0 m were raised and pegged at a planting distance of 80 cm × 40 cm. A randomized complete block design (RCBD) with three replicates partitioned by two alleys of 0.5 m each was used. A block contained five plots, each measuring 4.0 m × 1.6 m. Thirty (30) plants constituted a plot, with ten plants in a row and three plants between rows. Eight middle row plants were used as record plants, from which data were taken. The experimental setup was repeated for the succeeding trial in 2016. The layout for the pot experiments was a 4 × 4 factorial, arranged in a split plot design with three replicates.


## 2.5. Agronomic Practices and Parameters Taken on Tomato Plants (Field Trials)

Four-week-old seedlings were transplanted (one per stand), and a starter solution (N-P-K: 15-15-15 at 6 g/L of water) applied at 100 mL/plant. Two (2) kg N.P.K was mixed with 1 kg NH3 and was also applied at 15 g/plant as a side dressing. Agricombi (fenitrothion 30% + fenvalerate 10%) were sprayed twice, at a rate of 50 mL/L of H2O, to control whiteflies, aphids, and red spider mites. The plants were cared for by regular watering and weed control. Plant height, plant girth, and chlorophyll contents were taken 5, 7, and 9 weeks after seedling transplant.


## 2.6. Experimental Design (Pot Trials)

The layout for the pot experiment was a 4 × 4 factorial experiment, with a control arranged in a split plot design with three replicates, each experimental plot consisting of 30 plants. Root-knot nematode inoculums (Juveniles) were prepared by extracting nematode eggs from the roots of infested plants, and hatched into juveniles [32]. These were then concentrated into the various RKN inoculum levels (0, 500, 1,000, and 5,000). The plants (rootstocks) were then inoculated with the juveniles, by making a small hole or depression in the soil, at the base plants after which the required inoculum level was gently poured into the hole and covered.


## 2.7. Nematode Analysis

Soil samples taken from each plot were used to determine the RKN populations, before transplanting (initial nematode population Pi), six weeks after transplanting (P6), and after harvesting (Pf) in each trial. Six random soil samples per plot were taken with a soil augur, and bulked together into a well-labeled plastic bag. A subsample of 200 cubic centimeter (cc) was used in soil extraction, through the sieving and sucrose centrifugation method [33]. Reproductive factors (Rfs) for each treatment plot, which is a ratio of the final to initial population (Pf/Pi), were used to determine the rate of nematode multiplication in the soil. Gall scoring and nematode egg extraction were carried out 6 and 12 weeks after transplanting rootstocks, to estimate RKN gall development and egg production on the host. Sampled roots were washed separately and air-dried for 5 minutes. Galls on each root system were scored on a scale of 0–10 (no damage to severe damage), using the severity rating chart by Bridge and Page [34]. Roots were then cut into pieces, and nematode eggs extracted using 10% sodium hypochlorite (NaClO) [32]. Eggs obtained were counted under the compound microscope (Exacta–OptechBiostar B5P, Germany) (magnification x100) and recorded. The mean gall scores (GSs), egg count per gram of root, and reproductive factors (Rfs) obtained from the two field trials were used as the basis to evaluate the resistance status of the rootstocks. Rootstocks were classified as resistant when their root GS < 2 and Rf < 1, tolerant when GS < 2 and Rf ≥ 1, or susceptible when GS ≥ 2 and Rf > 1 [35, 36].


## 2.8. Statistical Analysis

Data collected on agronomic parameters (plant height and plant girth), chlorophyll content, yield parameters, and plant biomass content were subjected to analysis of variance (ANOVA), using Genstat 12th edition software [37], and significant means separated, using least significant difference (LSD) at 5%. Where necessary, data were transformed using the equation Log (x + 1), for normality.


## 3. Results

### 3.1. Root-Knot Nematode Gall Development, Egg Formation, and Reproduction among Selected Rootstocks Six and 12 Weeks after Transplanting Rootstocks

FourSolanum rootstocks were evaluated for resistance to RKNs in a naturally infested field, in two consecutive trials (dry season of 2015 and rainy season of 2016). Data on the RKN gall development and reproduction among the rootstocks for the two-year trial were highly comparable. Six weeks after transplanting the rootstocks, lower gall scores ranging from 0.33 to 0.67 were observed on S. macrocarpon, Samrudhi F1, and Mongal F1 (Table 1). In year 2, S. macrocarpon had the least root gall score (0.00), with a slight increase for Samrudhi F1 (0.17) compared with the others (>0.50), six weeks after transplanting the rootstocks. Significant differences were absent among the rootstocks for eggs/g of root, six weeks after transplanting in both years; however, differences were observed among the eggs numbers twelve weeks after transplanting in both years. The recovered number of eggs/g of root ranged from 2 to 14,714 in Samrudhi F1 and S. aethiopicum, six and twelve weeks after transplanting the rootstock seedlings for year 1. In year 2, however, eggs/g of root ranged from 20 to 11,280 in Samrudhi F1 and S. aethiopicum, six and twelve weeks after transplanting the rootstock seedlings. Nematode reproductive factors (Rfs) on the various rootstocks also ranged from 0.07 to 1.57 for Mongal F1 and S. aethiopicum, 6 and 12 weeks after rootstock transplant, respectively, in year 1. In year 2, however, Rf ranged from 0.45 to 2.37 for Samrudhi F1 and S. aethiopicum six and twelve weeks after rootstock transplant, respectively.Table 1
Initial and final nematode count, mean gall scores, egg count/g of root, and reproductive factors (RFs) of RKNs after 6 and 12 weeks of transplanting rootstocks in 2015 (year 1) and 2016 (year 2) for field experiments.
Treatment
Nematode count/200 cm3 of soil
Mean gall scores (0–10)
Egg count/gram of root (transformed)∗
Reproductive factor (Pf/Pi)
yReaction
Weeks
Initial(Pi)
Six (Pf)
Twelve (Pf)
Six
Twelve
Six
Twelve
Six
Twelve
Year 1 (dry season)
Sam
133.00
85.33
48.67
0.50a∗
0.33a
16.30 (1.06)a
54.33 (1.23)a
0.64
0.36
R
Mon
839.33
59.67
99.00
0.67a
3.00ab
14.30 (0.90)a
1047.67 (2.33)ab
0.07
0.12
T
S. M
197.67.00
46.67
24.00
0.33a
0.67a
2.00 (0.43)a
1438.33 (3.06)bc
0.24
0.12
T
S. A
255.33
112.67
400.00
1.67a
5.17b
17.00 (0.86)a
14714.00 (4.07)c
0.44
1.57
T
Year 2 (rainy season)
Sam
397.00
178.00
227.00
0.17
0.67
20.00a
900.00 (2.66)a
0.45
0.57
R
Mon
220.00
176.00
331.00
0.50
1.67
50.00a
5145.00 (3.68)b
0.80
1.50
T
S. M
472.00
244.00
515.00
0.00
1.33
33.00a
4267.00 (3.58)b
0.52
1.09
T
S. A
239.00
286.00
567.00
0.50
2.17
48.00a
11280.00 (3.91)c
1.20
2.37
T
Gall scores: 0 = no knots on roots; 10 = all roots severely knotted, plant usually dead; rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M =S. macrocarpon; S. A = S. aethiopicum. yReaction of rootstocks derived from 6 to 12 weeks after transplanting rootstocks (R = resistance and T = tolerance). ∗Means having different letters in a column differed significantly (P≤0.05). ∗Log (x + 1) transformed.Within the pot experiments, significant differences did not exist among the rootstocks in relation to the various RKN inocula levels (500, 1000, and 5000), for mean gall scores and egg counts six weeks after treatment application (Table2). However, significant differences were noted for mean gall scores and egg counts, twelve weeks after treatment application. The RFs for all rootstocks were below 1.Table 2
Response of four rootstocks to four RKN inoculum levels on gall scores, egg count per gram of root, and reproductive factors 6 and 12 weeks after inoculation in pot experiments.
Rootstocks
Initial inoculum
Mean gall score (1–10)
Nematode count per 200 cc of soil (Pf)
Egg count per gram of root
Reproductive factor (Pi/Pf)
Reaction
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
Mon
500
0.51a∗
0.00a
48.30
41.28
6.50a
5.94a
0.09
0.08
T
SA
500
0.22a
0.20ab
125.60
60.72
9.60a
5.11a
0.25
0.12
T
Sam
500
0.02a
0.10a
46.60
4.00
2.60a
5.44a
0.09
0.22
R
SM
500
0.38a
0.00a
107.90
108.56
9.60a
3.00a
0.22
0.01
T
Mon
1000
1.11a
0.10a
62.20
20.56
12.8a
3.89a
0.06
0.02
T
SA
1000
0.67a
0.00a
192.30
93.89
12.2a
8.56b
0.19
0.09
T
Sam
1000
0.50a
0.00a
124.30
2.16
7.00a
0.15c
0.09
0.11
R
SM
1000
0.44a
0.00a
86.10
108.44
15.3a
6.33a
0.12
0.00
T
Mon
5000
0.33a
0.70c
91.60
9.39
7.20a
4.33a
0.02
0.00
T
SA
5000
0.72a
0.20ab
158.40
90.33
11.9a
5.00a
0.03
0.02
T
Sam
5000
1.00a
0.10a
84.70
13.66
8.50a
6.33a
0.04
0.01
R
SM
5000
0.55a
0.00a
206.00
71.00
9.60a
1.15c
0.02
0.00
T
Sam =Solanum lycopersicum “Samrudhi F1,” Mon = Solanum lycopersicum “Mongal F1,” SM = Solanum macrocarpon, and SA = Solanum aethiopicum (R = resistance and T = tolerance). ∗Means having different letters in a column differed significantly (P≤0.05).There were no significant differences among the various plant growth parameters for weeks 6 and 12, among the rootstocks for the four RKN inocula levels applied (Table3). Generally, dry shoot and root weights were lower compared with the fresh shoot and root weights.Table 3
Fresh and dry shoot and root weights in four rootstocks to three RKN inocula levels (0, 500, 1000, and 5000) 6 and 12 weeks after treatment application.
Rootstocks
Initial RKN inocula levels
Fresh root weight (g)
Fresh root weight (g)
Dry root weight (g)
Dry root weight (g)
Fresh shoot weight (g)
Fresh shoot weight (g)
Dry shoot weight (g)
Dry shoot weight (g)
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
Mon
0
5.58a∗
6.32a
1.11a
1.69a
50.10a
53.78a
5.05a
7.91a
SA
0
7.19a
6.06a
1.82a
2.34a
45.70a
55.69a
5.34a
7.69a
Sam
0
6.82a
6.40a
0.94a
3.91a
43.30a
42.30a
7.09a
8.69a
SM
0
13.70a
9.40a
4.57a
3.68a
40.60a
52.16a
11.09a
6.21a
Mon
500
8.12a
4.34a
2.13a
2.55a
48.80a
57.73a
6.16a
7.40a
SA
500
9.21a
8.62a
2.64a
2.26a
48.80a
55.99a
6.96a
7.05a
Sam
500
5.23a
5.36a
1.21a
4.17a
47.40a
34.62a
6.18a
6.82a
SM
500
12.20a
12.85a
4.38a
3.92a
62.00a
51.32a
6.73a
6.75a
Mon
1000
3.28a
7.13a
1.36a
1.94a
37.70a
42.94a
9.56a
9.12a
SA
1000
15.65a
13.21a
3.48a
2.78a
46.70a
45.62a
7.49a
4.91a
Sam
1000
3.08a
10.97a
0.73a
3.21a
26.20a
34.07a
10.29a
7.78a
SM
1000
15.19a
15.24a
5.13a
3.10a
36.80a
43.68a
7.18a
8.79a
Mon
5000
7.50a
10.08a
1.94a
2.43a
50.70a
38.73a
7.28a
7.81a
SA
5000
16.52a
15.70a
11.46a
4.47a
54.40a
52.32a
8.08a
7.14a
Sam
5000
8.68a
15.12a
1.31a
2.72a
49.20a
46.15a
4.54a
6.75a
SM
5000
12.60a
15.26a
6.56a
3.59a
45.70a
41.58a
8.71a
7.29a
∗Means having different letters in a column differed significantly (P≤0.05).


### 3.2. Mean Plant Height, Plant Girth, and Chlorophyll Contents in Rootstocks to Root-Knot Infection Five, Seven, and Nine Weeks after Transplanting Rootstocks

Growth performances varied greatly among the rootstocks and between the dry and wet season trials. Significant increases (P≤0.05) in plant height, girth, and chlorophyll contents were recorded for S. aethiopicum during the dry season (Figures 1(a1), 1(b1), and 1(c1)). Conversely, the vegetative growth of the three tomato rootstocks were robust in the rainy season, relative to S. aethiopicum and S. macrocarpon, but began diminishing during fruiting or after the seventh week (Figures 1(a2), 1(b2), and 1(c2)). Aside having the thickest stem, S. macrocarpon’s chlorophyll content was also significantly increased (P≤0.05), compared with the other rootstocks screened against RKNs (Figure 1(c2)).Figure 1
Growth parameters of rootstocks to RKN infection at five, seven, and nine weeks after transplanting rootstocks. (a) The mean plant height, (b) the mean plant girth (c) the mean chlorophyll content in 2015 (1) and 2016 (2). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M =S. macrocarpon; S. A = S. aethiopicum. Data were analyzed with two-way ANOVA and significant means separated with the least significant difference (LSD) test. LSD bars represent the least significant difference at P≤0.05.
(a1)
(a2)
(b1)
(b2)
(c1)
(c2)


### 3.3. RKN Infestation on the Different Rootstocks Yield Parameters

Overall,S. macrocarpon and S. aethiopicum showed significantly (P≤0.05) increased number of fruits (10 and 12/plant) and high yield performances (227.50 g and 147.61 g/plant, respectively) in the dry season (year 1). Higher fruit yields were obtained for the three tomato rootstocks in the rainy season (year 2). The number of fruits/plant increased significantly (P≤0.05), in both Mongal F1 (20) and S. aethiopicum (19). Fruit yield/plant for Mongal F1 (1236.60 g) greatly increased (P≤0.05) relative to other three rootstocks screened against RKNs. Furthermore, the weight of individual fruits/plant for Samrudhi F1 (66.95 g) was on the average heavier compared with the other rootstocks (Table 4).Table 4
Mean yield parameters of the rootstocks after RKN infestation in 2015 (year 1) and 2016 (year 2).
Treatment
Days to 50% flowering
Number of fruits/plant
Fruit yield/plant (g)
Fruit yield (kg/ha)
Mean fruit weight/plant (g)
Year 1 (dry season)
Sam
28.00a
2.33a
23.70a
2961a
8.89a
Mon
27.00a
5.00ab
44.62a
5581a
7.43a
S. M
39.00b
10.33bc
227.50c
28442b
22.74b
S. A
51.00c
12.00c
147.61b
18448ab
12.30b
Year 2 (rainy season)
Sam
27.00b
13.00ab
870.30b
27196.87b
66.95c
Mon
29.00b
20.00b
1236.60c
38643.75c
61.82c
S. M
41.00c
9.00a
339.10a
10596.88a
37.68b
S. A
58.00d
19.00b
204.40a
6387.50a
10.75a
Means having different letters in a column differ significantly (P≤0.05). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M = S. macrocarpon; S. A = S. aethiopicum.


### 3.4. Effects of Root-Knot Nematode Infestation on Fresh and Dry Weights of Shoot and Roots of the Rootstocks

Twelve weeks after transplanting,S. aethiopicum and S. macrocarpon accumulated significant (P≤0.05) amounts of plant biomass, with respect to its fresh shoot (331.40 g and 159.10 g), and root weight (39.72 g and 14.05 g), obtained in the dry season trial (Table 5). In the subsequent trial, however, the tomato rootstocks picked up steadily the fresh shoot weight of S. macrocarpon (357.39 g) and Mongal F1 (325.28 g), which were significantly increased (P≤0.05), compared with the other rootstocks (Table 5). Significant differences also existed among the dry shoot and root weights for both year trials, and S. aethiopicum produced the highest dry weights (16.81 g and 19.43 g) for years 1 and 2, respectively.Table 5
Mean fresh and dry weights of shoot and roots of rootstocks after 12 weeks of root-knot nematode infestation.
Treatment
Fresh shoot weight (g)
Dry shoot weight (g)
Fresh root weight (g)
Dry root weight (g)
Year 1 (dry season)
Sam
40.60a
8.74a
3.74a
0.96a
Mon
53.70a
11.13a
5.17ab
1.42ab
S. M
159.10ab
33.78ab
14.05b
5.20b
S. A
331.40b
76.73b
39.72c
16.81c
Year 2 (rainy season)
Sam
257.74a
47.74a
25.13a
10.65a
Mon
325.28ab
65.02a
27.09a
12.09a
S. M
357.39b
62.14a
24.74a
12.67a
S. A
255.44a
63.51a
32.31a
19.43b
Means having different letters in a column differ significantly (P≤0.05). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M = S. macrocarpon; S.A = S. aethiopicum.


## 3.1. Root-Knot Nematode Gall Development, Egg Formation, and Reproduction among Selected Rootstocks Six and 12 Weeks after Transplanting Rootstocks

FourSolanum rootstocks were evaluated for resistance to RKNs in a naturally infested field, in two consecutive trials (dry season of 2015 and rainy season of 2016). Data on the RKN gall development and reproduction among the rootstocks for the two-year trial were highly comparable. Six weeks after transplanting the rootstocks, lower gall scores ranging from 0.33 to 0.67 were observed on S. macrocarpon, Samrudhi F1, and Mongal F1 (Table 1). In year 2, S. macrocarpon had the least root gall score (0.00), with a slight increase for Samrudhi F1 (0.17) compared with the others (>0.50), six weeks after transplanting the rootstocks. Significant differences were absent among the rootstocks for eggs/g of root, six weeks after transplanting in both years; however, differences were observed among the eggs numbers twelve weeks after transplanting in both years. The recovered number of eggs/g of root ranged from 2 to 14,714 in Samrudhi F1 and S. aethiopicum, six and twelve weeks after transplanting the rootstock seedlings for year 1. In year 2, however, eggs/g of root ranged from 20 to 11,280 in Samrudhi F1 and S. aethiopicum, six and twelve weeks after transplanting the rootstock seedlings. Nematode reproductive factors (Rfs) on the various rootstocks also ranged from 0.07 to 1.57 for Mongal F1 and S. aethiopicum, 6 and 12 weeks after rootstock transplant, respectively, in year 1. In year 2, however, Rf ranged from 0.45 to 2.37 for Samrudhi F1 and S. aethiopicum six and twelve weeks after rootstock transplant, respectively.Table 1
Initial and final nematode count, mean gall scores, egg count/g of root, and reproductive factors (RFs) of RKNs after 6 and 12 weeks of transplanting rootstocks in 2015 (year 1) and 2016 (year 2) for field experiments.
Treatment
Nematode count/200 cm3 of soil
Mean gall scores (0–10)
Egg count/gram of root (transformed)∗
Reproductive factor (Pf/Pi)
yReaction
Weeks
Initial(Pi)
Six (Pf)
Twelve (Pf)
Six
Twelve
Six
Twelve
Six
Twelve
Year 1 (dry season)
Sam
133.00
85.33
48.67
0.50a∗
0.33a
16.30 (1.06)a
54.33 (1.23)a
0.64
0.36
R
Mon
839.33
59.67
99.00
0.67a
3.00ab
14.30 (0.90)a
1047.67 (2.33)ab
0.07
0.12
T
S. M
197.67.00
46.67
24.00
0.33a
0.67a
2.00 (0.43)a
1438.33 (3.06)bc
0.24
0.12
T
S. A
255.33
112.67
400.00
1.67a
5.17b
17.00 (0.86)a
14714.00 (4.07)c
0.44
1.57
T
Year 2 (rainy season)
Sam
397.00
178.00
227.00
0.17
0.67
20.00a
900.00 (2.66)a
0.45
0.57
R
Mon
220.00
176.00
331.00
0.50
1.67
50.00a
5145.00 (3.68)b
0.80
1.50
T
S. M
472.00
244.00
515.00
0.00
1.33
33.00a
4267.00 (3.58)b
0.52
1.09
T
S. A
239.00
286.00
567.00
0.50
2.17
48.00a
11280.00 (3.91)c
1.20
2.37
T
Gall scores: 0 = no knots on roots; 10 = all roots severely knotted, plant usually dead; rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M =S. macrocarpon; S. A = S. aethiopicum. yReaction of rootstocks derived from 6 to 12 weeks after transplanting rootstocks (R = resistance and T = tolerance). ∗Means having different letters in a column differed significantly (P≤0.05). ∗Log (x + 1) transformed.Within the pot experiments, significant differences did not exist among the rootstocks in relation to the various RKN inocula levels (500, 1000, and 5000), for mean gall scores and egg counts six weeks after treatment application (Table2). However, significant differences were noted for mean gall scores and egg counts, twelve weeks after treatment application. The RFs for all rootstocks were below 1.Table 2
Response of four rootstocks to four RKN inoculum levels on gall scores, egg count per gram of root, and reproductive factors 6 and 12 weeks after inoculation in pot experiments.
Rootstocks
Initial inoculum
Mean gall score (1–10)
Nematode count per 200 cc of soil (Pf)
Egg count per gram of root
Reproductive factor (Pi/Pf)
Reaction
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
Mon
500
0.51a∗
0.00a
48.30
41.28
6.50a
5.94a
0.09
0.08
T
SA
500
0.22a
0.20ab
125.60
60.72
9.60a
5.11a
0.25
0.12
T
Sam
500
0.02a
0.10a
46.60
4.00
2.60a
5.44a
0.09
0.22
R
SM
500
0.38a
0.00a
107.90
108.56
9.60a
3.00a
0.22
0.01
T
Mon
1000
1.11a
0.10a
62.20
20.56
12.8a
3.89a
0.06
0.02
T
SA
1000
0.67a
0.00a
192.30
93.89
12.2a
8.56b
0.19
0.09
T
Sam
1000
0.50a
0.00a
124.30
2.16
7.00a
0.15c
0.09
0.11
R
SM
1000
0.44a
0.00a
86.10
108.44
15.3a
6.33a
0.12
0.00
T
Mon
5000
0.33a
0.70c
91.60
9.39
7.20a
4.33a
0.02
0.00
T
SA
5000
0.72a
0.20ab
158.40
90.33
11.9a
5.00a
0.03
0.02
T
Sam
5000
1.00a
0.10a
84.70
13.66
8.50a
6.33a
0.04
0.01
R
SM
5000
0.55a
0.00a
206.00
71.00
9.60a
1.15c
0.02
0.00
T
Sam =Solanum lycopersicum “Samrudhi F1,” Mon = Solanum lycopersicum “Mongal F1,” SM = Solanum macrocarpon, and SA = Solanum aethiopicum (R = resistance and T = tolerance). ∗Means having different letters in a column differed significantly (P≤0.05).There were no significant differences among the various plant growth parameters for weeks 6 and 12, among the rootstocks for the four RKN inocula levels applied (Table3). Generally, dry shoot and root weights were lower compared with the fresh shoot and root weights.Table 3
Fresh and dry shoot and root weights in four rootstocks to three RKN inocula levels (0, 500, 1000, and 5000) 6 and 12 weeks after treatment application.
Rootstocks
Initial RKN inocula levels
Fresh root weight (g)
Fresh root weight (g)
Dry root weight (g)
Dry root weight (g)
Fresh shoot weight (g)
Fresh shoot weight (g)
Dry shoot weight (g)
Dry shoot weight (g)
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
6 weeks
12 weeks
Mon
0
5.58a∗
6.32a
1.11a
1.69a
50.10a
53.78a
5.05a
7.91a
SA
0
7.19a
6.06a
1.82a
2.34a
45.70a
55.69a
5.34a
7.69a
Sam
0
6.82a
6.40a
0.94a
3.91a
43.30a
42.30a
7.09a
8.69a
SM
0
13.70a
9.40a
4.57a
3.68a
40.60a
52.16a
11.09a
6.21a
Mon
500
8.12a
4.34a
2.13a
2.55a
48.80a
57.73a
6.16a
7.40a
SA
500
9.21a
8.62a
2.64a
2.26a
48.80a
55.99a
6.96a
7.05a
Sam
500
5.23a
5.36a
1.21a
4.17a
47.40a
34.62a
6.18a
6.82a
SM
500
12.20a
12.85a
4.38a
3.92a
62.00a
51.32a
6.73a
6.75a
Mon
1000
3.28a
7.13a
1.36a
1.94a
37.70a
42.94a
9.56a
9.12a
SA
1000
15.65a
13.21a
3.48a
2.78a
46.70a
45.62a
7.49a
4.91a
Sam
1000
3.08a
10.97a
0.73a
3.21a
26.20a
34.07a
10.29a
7.78a
SM
1000
15.19a
15.24a
5.13a
3.10a
36.80a
43.68a
7.18a
8.79a
Mon
5000
7.50a
10.08a
1.94a
2.43a
50.70a
38.73a
7.28a
7.81a
SA
5000
16.52a
15.70a
11.46a
4.47a
54.40a
52.32a
8.08a
7.14a
Sam
5000
8.68a
15.12a
1.31a
2.72a
49.20a
46.15a
4.54a
6.75a
SM
5000
12.60a
15.26a
6.56a
3.59a
45.70a
41.58a
8.71a
7.29a
∗Means having different letters in a column differed significantly (P≤0.05).


## 3.2. Mean Plant Height, Plant Girth, and Chlorophyll Contents in Rootstocks to Root-Knot Infection Five, Seven, and Nine Weeks after Transplanting Rootstocks

Growth performances varied greatly among the rootstocks and between the dry and wet season trials. Significant increases (P≤0.05) in plant height, girth, and chlorophyll contents were recorded for S. aethiopicum during the dry season (Figures 1(a1), 1(b1), and 1(c1)). Conversely, the vegetative growth of the three tomato rootstocks were robust in the rainy season, relative to S. aethiopicum and S. macrocarpon, but began diminishing during fruiting or after the seventh week (Figures 1(a2), 1(b2), and 1(c2)). Aside having the thickest stem, S. macrocarpon’s chlorophyll content was also significantly increased (P≤0.05), compared with the other rootstocks screened against RKNs (Figure 1(c2)).Figure 1
Growth parameters of rootstocks to RKN infection at five, seven, and nine weeks after transplanting rootstocks. (a) The mean plant height, (b) the mean plant girth (c) the mean chlorophyll content in 2015 (1) and 2016 (2). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M =S. macrocarpon; S. A = S. aethiopicum. Data were analyzed with two-way ANOVA and significant means separated with the least significant difference (LSD) test. LSD bars represent the least significant difference at P≤0.05.
(a1)
(a2)
(b1)
(b2)
(c1)
(c2)


## 3.3. RKN Infestation on the Different Rootstocks Yield Parameters

Overall,S. macrocarpon and S. aethiopicum showed significantly (P≤0.05) increased number of fruits (10 and 12/plant) and high yield performances (227.50 g and 147.61 g/plant, respectively) in the dry season (year 1). Higher fruit yields were obtained for the three tomato rootstocks in the rainy season (year 2). The number of fruits/plant increased significantly (P≤0.05), in both Mongal F1 (20) and S. aethiopicum (19). Fruit yield/plant for Mongal F1 (1236.60 g) greatly increased (P≤0.05) relative to other three rootstocks screened against RKNs. Furthermore, the weight of individual fruits/plant for Samrudhi F1 (66.95 g) was on the average heavier compared with the other rootstocks (Table 4).Table 4
Mean yield parameters of the rootstocks after RKN infestation in 2015 (year 1) and 2016 (year 2).
Treatment
Days to 50% flowering
Number of fruits/plant
Fruit yield/plant (g)
Fruit yield (kg/ha)
Mean fruit weight/plant (g)
Year 1 (dry season)
Sam
28.00a
2.33a
23.70a
2961a
8.89a
Mon
27.00a
5.00ab
44.62a
5581a
7.43a
S. M
39.00b
10.33bc
227.50c
28442b
22.74b
S. A
51.00c
12.00c
147.61b
18448ab
12.30b
Year 2 (rainy season)
Sam
27.00b
13.00ab
870.30b
27196.87b
66.95c
Mon
29.00b
20.00b
1236.60c
38643.75c
61.82c
S. M
41.00c
9.00a
339.10a
10596.88a
37.68b
S. A
58.00d
19.00b
204.40a
6387.50a
10.75a
Means having different letters in a column differ significantly (P≤0.05). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M = S. macrocarpon; S. A = S. aethiopicum.


## 3.4. Effects of Root-Knot Nematode Infestation on Fresh and Dry Weights of Shoot and Roots of the Rootstocks

Twelve weeks after transplanting,S. aethiopicum and S. macrocarpon accumulated significant (P≤0.05) amounts of plant biomass, with respect to its fresh shoot (331.40 g and 159.10 g), and root weight (39.72 g and 14.05 g), obtained in the dry season trial (Table 5). In the subsequent trial, however, the tomato rootstocks picked up steadily the fresh shoot weight of S. macrocarpon (357.39 g) and Mongal F1 (325.28 g), which were significantly increased (P≤0.05), compared with the other rootstocks (Table 5). Significant differences also existed among the dry shoot and root weights for both year trials, and S. aethiopicum produced the highest dry weights (16.81 g and 19.43 g) for years 1 and 2, respectively.Table 5
Mean fresh and dry weights of shoot and roots of rootstocks after 12 weeks of root-knot nematode infestation.
Treatment
Fresh shoot weight (g)
Dry shoot weight (g)
Fresh root weight (g)
Dry root weight (g)
Year 1 (dry season)
Sam
40.60a
8.74a
3.74a
0.96a
Mon
53.70a
11.13a
5.17ab
1.42ab
S. M
159.10ab
33.78ab
14.05b
5.20b
S. A
331.40b
76.73b
39.72c
16.81c
Year 2 (rainy season)
Sam
257.74a
47.74a
25.13a
10.65a
Mon
325.28ab
65.02a
27.09a
12.09a
S. M
357.39b
62.14a
24.74a
12.67a
S. A
255.44a
63.51a
32.31a
19.43b
Means having different letters in a column differ significantly (P≤0.05). Rootstocks: Sam = Samrudhi F1; Mon = Mongal F1; S. M = S. macrocarpon; S.A = S. aethiopicum.


## 4. Discussion

The reproductive factors (Rfs) of the various rootstocks together with their mean gall scores (GS) provided an estimate of host suitability, to support nematode reproduction and were used to verify host resistance [38]. A rootstock’s reaction to RKN resulting to GS < 2 and Rf < 1, GS < 2 and Rf ≥ 1, and GS ≥ 2 and Rf > 1 was identified as resistant, tolerant, and susceptible, respectively [35].There is evidence to show that fecundity increases in more vulnerable plant hosts than in resistant or tolerant hosts [39]. Sobczak et al. [40] also described a stalled response of the tomato Hero-gene, against invading cyst nematode, leading to the death of nematodes at the late J2 stage, rather than the average 12 hours postinfection response [26].Wehner et al. [41] recounted 12 weeks postinoculation nematode, counts as the best option for inferring the response of rootstocks, as compared with six weeks nematode counts. Nematodes cause severe damage depending on their population densities, host species, temperature, and the period of incubation, to allow significant differentiation between resistant and susceptible rootstocks. A remarkable increase in nematode damage, along with reproduction, was observed 12 weeks after transplanting in both trials in S. aethiopicum. However, some rootstocks (Samrudhi F1, Mongal F1, and S. macrocarpon) suppressed nematode activities, indicating the existence of genetic variability among the rootstocks tested. In a previous study, Rahman et al. [42] observed the response of S. torvum and S. sisymbriifolium as resistant to RKNs. Similarly, Dhivya et al. [28] described S. torvum and S. aethiopicum as poor hosts of RKNs, whereas the former was found tolerant by Ioannou [25]. In our study, S. aethiopicum could be classified as tolerant to RKNs, although had high root gall development (GS = 2.17–5.17), with increased reproduction (Rf = 1.58–2.37), the severest symptoms were observed during the dry season. This response could probably be due to predominant elevated soil temperatures, leading to the permanent breakdown of nematode resistance Mi gene [43, 44].Unlike the resistance found in Samrudhi F1 (GS = 0.33–0.67 and Rf = 0.36–0.57),S. macrocarpon and Mongal F1 were found promising because of their potential in reducing nematode damage (GS = 0.67–1.33 and 1.67–3.00, respectively). Based on nematode reproduction on a host, their response could best be described as tolerant because population levels of nematodes recovered were still high (Rf > 1) in field experiments, but below 1 in pot experiments. Similarly, Lopez-Perez et al. [45] documented a tolerant response in resistant Mi gene-bearing rootstock Beaufort, for supporting RKN reproduction, with higher yields compared with a susceptible genotype (Blitz). In another test, where 33 tomato genotypes were assessed by Jaiteh et al. [29], Mongal T-11 and Mongal F1 number 5 emerged resistant (GS = 3.25 and Rf = 0.71) and susceptible (GS = 7.25 and Rf = 2.47), respectively. Tzortzakakis et al. [46] also reported S. macrocarpon’s susceptibility to RKNs. However, in the current study, the observed tolerance in S. macrocarpon and Mongal F1 might be due to inactivation of the Mi gene arising from mutation, if present in the genotypes [45], or the possession of other genes (horizontal resistance) rather than Mi gene. Solanum macrocarpon’s hardy roots makes penetration relatively difficult for nematodes compared with tomato roots. Alternatively, Mi gene dosage effect may account for increased nematode reproduction in tolerant Mi heterozygous individuals than in homozygous individuals [47, 48].Apart from reducing nematode damage, resistant rootstocks are cultivated to help improve plant fitness and yield. Consequently, growth and yield performance of the test rootstocks was evaluated in the study in infected and controlled environments, to various inocula levels of RKN in pot experiments. The absence of significant differences in the rootstocks in relation to the growth parameters (fresh and dry shoot and root weights) shows the tolerance levels of the various rootstocks to the RKN, irrespective of the inocula level in the pot trials. Within the field experiments, significant differences in plant girth, height, and chlorophyll content were obtained among the tested rootstocks. These observed differences showed that although the rootstocks belonged to the sameSolanum genus, these had (3) different species origins. Among the three tomato rootstocks, Mongal F1, however, presented a more robust growth after transplanting. The plants were tall with thick stems and appeared dark green. Compared with Samrudhi F1, the plants stood more erect (even at flowering) with soft stem tissues that easily break upon fruit bearing (weight). The resistant response of host crops to RKNs is well known to correspond to increased yield [24, 42, 45]. The fruit yield of the tolerant rootstock (Mongal F1) was significantly increased, compared with the resistant Samrudhi F1. This suggests Mongal F1’s efficiency in converting resources into fitness and in bearing of fruit rather than controlling nematodes. Restif and Koella [49] detected the distribution of tolerant plant resources towards fitness, unlike in resistant plants where this is aimed at reducing pathogen infection. Within Samrudhi F1 genotypes, appreciable yields would be obtained in addition to suppressed nematode population, making it possible for the cultivation of susceptible crops in subsequent years [50].


## 5. Conclusion

Our investigation showed tomato Samrudhi F1 as resistant, whereas tomatoS. macrocarpon, S. aethiopicum, and Mongal F1 were tolerant to RKNs. These rootstocks, can serve as tomato rootstocks, in breeding programs targeted at nematode management. Tolerance mechanisms of plants against nematodes are less understood and should be further investigated.



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*Source: 6715909-2018-09-25.xml*