Induction of defence-related proteins by selected plant growth regulators and biocontrol agents against guava root knot nematode, Meloidogyne enterolobii

The population of M. enterolobii was maintained in Guava (Psidium guajava) seedlings under glasshouse conditions. Matured egg masses were collected and kept in distilled water at room temperature (25°C) for two to three days. The total number of hatched second-stage juveniles was counted in four aliquots. The nematode suspension was diluted approximately to about 100 infective juveniles/100g of soil and stored at 4°C for further experiments.

In total, 100 ppm of 1 litre salicylic acid solution was prepared by adding 0.1g of salicylic acid with 500ml of water and few pellets of sodium bicarbonate (to dissolve salicylic acid with water). Final volume of one litre was prepared by adding water to the dissolved solution.

Methyl jasmonate solution was prepared by diluting 0.1g of methyl jasmonate in 20ml of ethanol. In total, 980ml of water was added after complete dilution of methyl jasmonate in ethanol and make up to 1 litre of the total solution.

In total, 1,000 ppm of 1 litre Indole 3 Butyric acid solution was prepared by weighing 1g of crystalline Indole 3 Butyric acid and dissolved in 20 milliliters of alcohol. The prepared mixture was then added with 980 milliliters of water to make 1,000 ppm of 1 litre Indole 3 Butyric acid solution.

In total, 0.5g of solid Mepiquat chloride was weighed and dissolved in 1,000ml of water to prepare 1 litre of 500 ppm Mepiquat chloride solution.

One litre of 500 ppm Chloromequat chloride solution was prepared by dissolving 0.5g of solid Chloromequat chloride in 1 litre of water.

The effect of plant growth regulators on the growth of guava seedlings inoculated with root knot nematode, M. enterolobii was investigated under greenhouse conditions with three replications for each treatment. Different plant growth regulators viz., Indole 3-Butyric Acid (IBA), Salicylic acid, Jasmonic acid, Mepiquat chloride, Chlormequat chloride were applied alone and in combinations to detect their effect against the root-knot nematode, Meloidogyne enterolobii infested guava plants. One month old seedlings were dipped in growth regulators for 3 sec. The treatment details includes T₁ – Salicylic acid (100 ppm), T₂ – Jasmonic acid (100 ppm), T₃ – Indole3-Butyric Acid (1,000 ppm), T₄ – Salicylic acid + Jasmonic acid + Indole 3-Butyric Acid (100 ppm + 100 ppm + 1,000 ppm), T₅ – Salicylic acid + Jasmonic acid (100 ppm + 100 ppm), T₆ – Jasmonic acid + Indole 3-Butyric Acid (100 ppm + 1,000 ppm), T₇ – Salicylic acid + Indole 3-Butyric Acid (100 ppm + 1,000 ppm), T₈ – Mepiquat chloride (500 ppm), T₉ – Chlormequat chloride (500 ppm), and T₁₀ – Untreated control. One week after treatment with Plant Growth Regulators, the treated plants were inoculated with infective juveniles at the rate of 1 J₂/g of soil. The morphological characters Plant height, Root length, Fresh root biomass and Dry root biomass) physiological aspects (Chlorophyll index, Photosynthetic rate, Transpiration rate, Stomatal conductance, and Chlorophyll fluorescence (F_v/F_m ratio), biochemical parameters such as Total phenols, Peroxidase, Polyphenol oxidase, Acid phosphatase, and Phenylalanine ammonia lyase and nematode population in soil and root were recorded at 45, 75, and 105 days after planting (DAP). The effect of such plant growth regulators was evaluated under greenhouse conditions against M. enterolobii using guava cultivar ‘Lucknow-49’.

The nematode Meloidogyne enterolobii infested fields in two different locations: Location-I was laid out at a farmer’s field in Thondamuthur village of Coimbatore district, which was geographically situated in the western Agro-climatic Zone of Tamil Nadu at 11.0250481 N, 76.8839245 E at an altitude of 450m above MSL. Location-II was laid out in a farmer’s field at Avoor village of Thiruvannamalai district, which was geographically situated in the north eastern Agro-climatic Zone of Tamil Nadu at 12.1468353 N, 79.2209262 E at an altitude of 171 m above MSL were selected to test the effect of different biocontrol agents. The treatment parameters included T1 – Pseudomonas fluorescens (Pf1) (2.5 kg/ha), T2 – Bacillus subtilis (Bs1) (2.5 kg/ha), T3 – Trichoderma viride (Tv1) (2.5 kg/ha), T4 – Pochonia chlamydosporia (TNAUPc001) (2.5 kg/ha), T5 – Purpureocillium lilacinum (TNAUPl001) (2.5 kg/ha), T6 – Glomus mosseae (100 g/m²), and T7 – Untreated control. The observations were recorded at 30, 60, 90, 120, 150, 180, and 210 days after the application of bio agents. The effect of bio-control agents on root-knot nematode infested guava plants was determined based on different variables viz., Nematode population in soil and root, Morphological characters (Tree height (m), Girth of stem (cm) and Leaf area (cm²)), Nutrient status (Nitrogen content (%), Phosphorous content (%), and Potassium content (%)), Yield characters (Number of fruits per tree, Fruit length (cm), Shoots diameter (cm), Average fruit weight (g), Fruit yield (kg tree-1), and Estimated fruit yield (t/ha)).

The data generated from various experiments of the present study were analyzed following Gomez and Gomez (1984) method. The package used for analysis was IRRISTAT version 91-1 developed by International Rice Research Institute, Biometrics unit, Philippines.

The results showed that plant height measured at 45, 75, and 105 days after planting showed a significant increase in all the treatments and the maximum plant height was recorded at 105 days. The plant height measured for plants treated with two plant growth regulators was higher than the single plant growth regulator treatment. Comparatively, the combination of three plant growth regulators (T4) showed a statistically higher value compared to all treatments and untreated control (T10) (Fig. 1A) (Plate 1). The root length in all the treatments from T1 to T9 was significantly higher than the untreated control. Root length of all the treated and untreated plants increased from 45 to 105 days. The results were statistically significant compared to the untreated control (Fig. 1B). Fresh and dry root biomass of all treatments weighed in 45, 75, and 105 days after planting were statistically and significantly lower than the plants in the untreated control (T10). The fresh root biomass of T10 plants increased from 11.17 g in 45 days to 17.67 in 105 days. Similarly, the dry root biomass of T10 plants increased from 2.69 to 3.81 grams (Fig. 1C, D).

Plate 1:

All plant growth regulators used in this study significantly influenced the physiological parameters such as chlorophyll index, photosynthetic rate, transpiration rate, stomatal conductance, and Chlorophyll fluorescence in all treatment and untreated control. The chlorophyll index measured at 105 days was high in T3 compared to T1 and T2. The chlorophyll index was high in T7 compared to T5 and T6 (p ≤ 0.05). T4 plants showed higher values when compared to the single and combined plant growth regulator application and the untreated control (Fig. 1E). Plant growth regulators in single and combined applications enhanced the photosynthetic rate of all the treated plants were statistically significant compared to the untreated control. The highest photosynthetic rate was recorded in T4 followed by T7, T6, T3, T5, T1, T2, T8, and T9 (Fig. 1F). The transpiration rate of T4 plants applied with the combination of three plant growth regulators was found to be maximum (4.96, µmol H₂O m-2 s-1) when compared to other treatments and untreated control (Fig. 1G). Similar results were obtained for stomatal conductance and chlorophyll fluorescence and the highest value was obtained for T4 statistically significant when compared to other treatments and untreated control T10 (Fig. 1H, I).

The induction of peroxidases, polyphenol oxidase, phenylalanine ammonia-lyase, acid phosphatase, and total phenols linking to the defence mechanism of the nematode infected plants treated with plant growth regulators were analyzed (Fig. 1J–N). Treatments from T1 to T7 significantly produced a high percentage of peroxidase compared to T8, T9 and untreated control T10 (Fig. 1J). The analysis of polyphenol oxidase (PPO) activity of plant growth regulator treated plants indicated that the result of T3 plant was higher than T5, similar to T6 and lower than T4 and T7. All the single and combined plant growth regulator treated plants showed higher PPO activity than T8, T9 and untreated control (p ≤ 0.05) (Fig. 1K). Among the plants treated with a single plant growth regulator, T3 plants treated with IBA at a concentration of 1,000 ppm showed higher PAL. T3 result was lower than the plants treated with combined plant growth regulators and higher than T8, T9 and untreated control T10 (Fig. 1L). The total phenol of T3 plants was higher than T1, T2 and T5 and lower than T4, T6, and T7. Plants treated with single as well as combined treatments showed higher values for total phenol production (Fig. 1N).

All the biochemical parameters tested were on the raising side when the treated plants were analyzed 45, 75, and 105 days after application of plant growth regulators. The highest value was recorded by T4 followed by T7 and significantly influenced the growth, physiological, and biochemical parameters compared to other treatments and untreated control.

Treatment with plant growth regulator viz., salicylic acid and Jasmonic acid effectively reduced the nematode population in the soil. A combination of three followed by two plant growth regulators showed high percentages of reduction in nematode population compared to single plant growth promotor application. Reduction of nematode population noted in T3 treated with Indole Butyric Acid at 1,000 ppm concentration was high compared to T1, T2, and untreated control T10. Application of Salicylic acid and Indole butyric acid in combination (T7) effectively reduced the nematode population in soil compared to other plant growth regulator combinations (T5 and T6). The result of the three plant growth regulators (Salicylic acid + Jasmonic Acid + Indole Butyric Acid) combined at a particular concentration specified in this study reduced the nematode population at a high percentage compared to single and combination of two plant growth regulators as well as untreated control. Among T8 and T9, Chlormequat chloride at 500 ppm concentration showed a high percentage of increase in nematode population in the soil in 105 days (Table 1).

Nematode population parameters such as number of females, number of eggs, and egg masses are calculated for 5 gram of root sample collected from Guava plants treated with plant growth regulators in single and in combinations of two and three. A reduction in the number of egg masses was noted from 45th day to 105th day. The highest reduction in egg masses was observed in T3 plants treated with IBA at a concentration of 1,000 ppm. When the plant growth regulators were used in combination (T5, T6, T7), SA + IBA showed a high percentage of reduction in egg mass.

The highest reduction in egg mass was noted in T4 (SA + JA + IBA) combination. The results of T8, T9, and T10 indicated an increase in egg masses. The highest increase was noted in T8 compared to T9 and T10 (Table 1).

Reduction in the female nematode population was noted in T3 in single plant growth regulator treatments and T7 in combined plant growth regulator treatments. Subsequently, an increase in the female population was noted in T8, T9, and T10. The analysis of eggs per egg masses showed that Salicylic acid reduced the number of egg per egg masses, while T2 and T3 resulted in an increased number of eggs/egg mass. The treatments T5, T6, T7, T8, and T9 showed an increase in the number of eggs per egg mass (Table 1).

All biocontrol agents significantly reduced the nematode population both in soil and plant roots compared to the untreated control. In total, 10 guava plants infested with the root-knot nematode, M. enterolobii in each treatment were treated with the Commercial biocontrol agents at regular intervals under natural field conditions in the Experiment. Soil and root samples were collected at 0, 30, 90, 150, and 210 days after the application of biocontrol agents.

A high percentage of reduction in nematode population was noted in the plants treated Pochonia chlamydosporia (T4) (TNAUPc001) applied at the rate of 2.5kg/ha, in Experiments 1 and 2. Followed by Purpureocillium lilacinum (TNAUPl001), Pseudomonas fluorescens (Pf1), Bacillus subtilis (Bs1), Trichoderma viride (Tv1), and Glomus mosseae effectively reduced the nematode population in soil compared to the untreated control. However, the lowest nematode population was observed in plants treated with Pochonia chlamydosporia. The percentage of reduction in nematode population was 64.91 in 210 days in Experiment I and 76.14 in Experiment II (Table 2).

The analysis of the plant roots in 0, 30, 90, 150, and 210 days after the application of biocontrol agents showed that Pochonia chlamydosporia recorded the lowest egg mass of 2.85 per 5 gram of the root, analyzed in 210 days in both Experiments I and II. The results did not differ significantly within T4 and T5 and between T2, T3, and T4 in Experiment I. The results obtained in Experiment II revealed that the plants of T4 and T5 treated with Pochonia chlamydosporia and Purpureocillium lilacinum differed significantly.

The untreated control plants showed a increase trend for the same parameters tested, i.e. the number of egg masses increased from 16.94 to 23.48 egg masses/5g of the root, similarly increase in female population from 29.26 to 36.42 females was observed. The number of eggs per egg mass increased from 158.23 to 175.53 in Experiments I and II (Table 3).

All the plants treated with commercial biocontrol agents at regular intervals during the year 2018 significantly enhanced the plant growth and yield of guava plants under natural field conditions. Application of Pochonia chlamydosporia (T4) (Plate 2) and Purpureocilium lilacinum (T5) enhanced the tree height girth of the stem and leaf area in both vegetative and fruiting stages of nematode infested guava plants chosen in Experiments I and II. Pseudomonas fluorescens (Pf1) Bacillus subtilis (Bs1) showed statistically similar results. Comparatively all the biocontrol agents used in this study have improved both vegetative and fruiting stage parameters of guava plants. Glomus mosseae was found to be least effective in both Experiments I and II (Table 4).

Plate 2:

Further, this study showed that treating the M. enterolobii infested guava plants with biocontrol agent Pochonia chlamydosporia reduced the nematode population and showed greater improvement on plant growth and yield parameters. Next to Pochonia chlamydosporia, P. lillacinum also showed a greater impact in reducing the nematode population by increasing the plant growth and yield parameters.