Research Article
Biological Control of Meloidogyne incognita on Eggplant (Solanum melongena)
Department of Research Administration and Development, University of Limpopo, Private Bag x1106 Sovenga, 0727, South Africa
LiveDNA: 98.36021
Eggplant (Solanum melongena L.) is one of the premier vegetables in India and is widely cultivated. Iran is one of the main countries for eggplant production1.
Meloidogyne is the main nematode problem in Iran2,3. Several Meloidogyne species are associated with eggplant4. The genus Meloidogyne represents over 100 species5, with over 3000 host plant species. Meloidogyne is the most dangerous plant-parasitic nematode, which causes yield loss of the crops6. The biological control agents, such as antagonistic bacteria, are up-and-coming to reduce plant diseases7.
Among a range of bacteria possessing this ability, Fluorescent pseudomonads8 have drawn attention worldwide because they produce secondary metabolites, such as siderophore, antibiotics, volatile metabolites (cyanide, ammonia) and lytic enzymes (protease) that may suppress the pathogen and phytohormones that may stimulate plant growth and induce resistance9,10.
In a study by Siddiqui et al.7, the result indicated that two bacterial isolates yielded a significant (>50%) mortality of juveniles of the root-knot nematode M. javanica in the laboratory condition. Their result also indicated that the selected isolates of the antagonistic rhizobacteria reduced nematode penetration and subsequent root-knot infection in mung bean8.
Three mechanisms involved in reducing nematode infection include (i) production of metabolites that reduce egg to hatch and attraction, (ii) degradation of specific root exudates which control nematode behaviour and/or (iii) development of the defence mechanism in plants which cause the induction of systemic resistance11,12. Therefore, this study aimed to evaluate the biocontrol potential of ten different Iranian isolates of P. fluorescens against M. incognita through in vitro and in vivo conditions.
Inoculum of the bacterium: The ten isolates of P. fluorescens used (Table 1) were used from the soil laboratory at Plant Protection Clinic, Shahrekord Lab, from 2016-2017. Each isolate's inoculum was prepared by transferring one loopful of the test isolate from a pure culture growing on nutrient agar into 100 mL of tryptic soy broth (TSB; pH 7.0-7.1; autoclaved twice for 20 min on successive days). Broth cultures were reared at room temperature (22-27°C; 16 hrs photoperiod) on an orbital shaker (Thermo ScientificTM MaxQTM 4000 Benchtop Orbital Shakers, USA) at 120 rpm for 36 hrs. Bacterial cells were removed from the broth by centrifugation (SorvallTM LegendTM 14 Personal Microcentrifuge, USA) at 6,000 g for 10 min and washed to remove any residual nutrients by re-suspending them in a sterile saline solution (0.15 M NaCl; pH 7.6-7.8). The cells were removed from the suspension with second centrifugation before being re-suspended in sterile distilled water. Then, bacterial cells were counted using a hemocytometer (Hausser ScientificTM Bright-LineTM and Hy-LiteTM Counting Chambers, USA) and the suspensions were adjusted to a concentration of 109 colony-forming units per mL (CFU mL1).
Nematode inoculum: The root-knot nematode was isolated from eggplant in Shahrekord and multiplied from a single egg mass of M. incognita, on tomato seedling cv. Moneymaker (Lycopersicon esculentum Karssen). Single egg masses of a pure culture of the M. incognita picked by fine forceps from infected tomato root that was surface sterilized in 1:500 (v/v) aqueous solution of "chlorax" (sodium hypochlorite) for five minutes13. Eggs were placed in sterilized distilled water and incubated for 24-48 hrs at room temperature for their hatching. Hatched juveniles were collected in a beaker and were used for in vitro experiment.
Nematocidal activity in vitro: To test the isolates' nematocidal activity, 100 μL of each bacterial suspension was transferred to wells of 96 well microtiter plates, to which 100 μL of the freshly hatched nematode juvenile suspension containing 50 surface-sterilized juveniles. Juveniles kept in sterile distilled water measured as a control. Each treatment was replicated four times in a microplate and was kept at room temperature. After 48 hrs of exposure, the number of dead juveniles per replicate was counted and the mortality percentage was calculated. The nematodes were considered dead if they did not move on probing with a fine needle14. One week after, the experiment was repeated with similar results.
Production of antifungal metabolites: Hydrogen cyanide was assessed on the plate (9 cm diameter) include King, B (KB: Protease Peptone, 20 g; K2HPO4, 1/5 g; MgSO4, 7H2O, 1/5 g; Glycerol, 15 mL; Agar, 15 g; Water, 1000 mL ) medium comprising 4.4 g L1 glycines with indicator paper (Whatman soaked in 0.5% picric acid and 2% sodium carbonate) and 100 μL of each bacterial cells opaque suspension was poured on medium, then inoculated plates by a parafilm band and incubated at 27°C for 48-72 hrs in dark condition. Any positive response caused the indicator paper to turn from yellow to cream; light brown, dark brown and brick were scored using a 0-4 scale (0, none (yellow); 1, little (yellow to cream); 2, mediocre (light brown) 3, intense (dark brown); 4, solid (brick)15.
Protease was estimated as described by Maurhofer et al.16. Bacterium isolates were spotted on Skim Milk Agar (SMA) on the Petri dish plates (9 cm in diameter) at 27°C for 36 hrs in dark situations. Semi quantification of protease was done by measurement of the diameter (mm) of the halo zone around the bacteria colonies.
Effect of P. fluorescens isolates on plant growth parameters and Suppression of M. incognita: A factorial experiment was completed with 12 treatments and five replications in a completely randomized design. Bacterial factor had 11 levels (without bacteria, P. fluorescens in 10 levels including M1-M10) and M. incognita factor had two levels (zero population and 3500 eggs and larvae per kilogram of soil). Eggplant (cv Royal), were sown in sterilized soil. Three weeks after cultivation, 4-leaved seedlings were transferred to the pots comprising a 1.5 kg 1:1 mixture of sterilized soil and sand. Before planting, the seedlings were inoculated by 1 mL of 109 CFU mL1 suspension of each isolate of P. fluorescens. After transplanting, 3500 eggs and J2 larvae of M. incognita were added to every treatment17. The pots were kept at 18-32°C and 16:8 day/night photoperiod for 56 days. The nutrients were added to each pot by the Hygromix solution every two weeks. Plant growth parameters such as shoot height, root length, the fresh and dried weight of shoot and root and variables relevant to the nematode, including the mean gall number and egg masses per the plant root, number of eggs per egg mass and number of J2 in soil were calculated.
Statistical analysis: Data were tested for the normal distribution utilizing the Shapiro-Wilk test for normality before applying the parametric tests and then analyzed by ANOVA using SAS (V9.1). Mean comparison of the variables was performed by Least Significant Difference (LSD) at 0.05 level.
To evaluate the different isolates of P. fluorescens (M1-M10) on the nematode reproductive index and plant growth parameters in the laboratory and greenhouse conditions, Principal Component Analyses (PCA) were used. PCA analyses were done through XLSTAT. The data were normalized using XLSTAT before their analysis18.
All isolates of P. fluorescens killed J2 of M. incognita while no juvenile was found dead in control (Table 1). However, the highest percent of juvenile mortality occurred in the presence of isolate M1 (89.7%), which gave a nematode juvenile mortality similar to that caused by isolates M3 (84.6%), M6 (82%) and M7 (79.4%). High mortality was also achieved with isolate M2 (76.9%), which did not significantly (p<0.05) differ from the previous isolates nor isolate M5 (66.6%). The isolates M4, M8 and M10 had an intermediate effect (56.4-64%), while isolate M9 was the least influential (38.4%).
On KB medium, the isolates M3 and M5 produced more hydrogen cyanide than isolates M1, M4, M6, M7 and M8. No hydrogen cyanide was produced by isolates M2, M9 and M10 (Table 1).
Nine isolates produced protease on an SMA medium (Table 1). The largest halo zone was observed around the isolate M3 (4 mm), which was significantly larger than the halo zones around isolates M4 (8 mm) and M5, M6 and M7 (7 mm). Much less protease was produced by isolates M2 (4 mm), M3 (4 mm), M9 (3 mm) and M10 (5 mm) and isolate M8 produced no protease. Our results confirmed that P. fluorescens cause mortality in juveniles of root-knot nematodes, Meloidogyne spp., in laboratory conditions. The principal component analysis (Fig. 1) indicated an accumulated variability of 86.53% was detected in the in vivo based experiment (70.28% in the F1 and 16.26% in the F2). The result of PCA indicated that M1 isolate produces more hydrogen cyanide and protease and cause more mortality than control (Fig. 1). Our isolates did produce protease and hydrogen cyanide. However, although a clear positive relationship between the effects of the isolates on nematode mortality and its ability to produce metabolites could not be observed nevertheless, isolate M1 killed not only more nematode juveniles (89.7%) but also produced more hydrogen cyanide and protease.
Regarding the greenhouse study, the results of this research presented that Pseudomonas isolates sponsored not only the plant growth parameters but also reduced the damage produced by M. incognita. However, there were insignificant differences among the Pseudomonas treatments. The plants inoculated only by M. incognita had the lowest shoot length (39 cm). This can be predictable because of nematode damage in the absence of any prohibiting treatment. Results of plant growth parameters also specified that P. fluorescens isolate M1 improved the fresh shoot weight (81 g).
Fig. 1: | Biplot of principal component analysis of in vitro assay of P. fluorescens activity against M. incognita |
Table 1: Effect of ten isolates of P. fluorescens on the mortality of M. incognita, after 48 hrs and their production of protease and hydrogen cyanide | ||||
Isolates | Egg hatch (%) | Mortality (%) | Protease1 | Hydrogen cyanide2 |
P. fluorescens M1 | 14b | 89.7a | 11a | 2 |
P. fluorescens M2 | 19b | 76.9ab | 4c | 0 |
P. fluorescens M3 | 23c | 84.6a | 4c | 1 |
P. fluorescens M4 | 16b | 59c | 8b | 1 |
P. fluorescens M5 | 17.5b | 66.6bc | 7b | 2 |
P. fluorescens M6 | 22c | 82a | 7b | 1 |
P. fluorescens M7 | 14b | 79.4a | 7b | 1 |
P. fluorescens M8 | 25c | 64c | 0c | 1 |
P. fluorescens M9 | 19.8b | 38.4d | 3c | 0 |
P. fluorescens M10 | 14.5b | 56.4c | 5c | 0 |
Check | 50.6a | 0e | ||
Table 2: Mean comparison of eggplant growth parameters in the plants inoculated with P. fluorescens and M. incognita, under greenhouse conditions | ||||||
Treatment | Shoot length (cm) | Shoot fresh weight (g) | Shoot dry weight (g) | Root length (cm) | Root fresh weight (g) | Root dry weight (g) |
Control | 61d | 42d | 10.5c | 17a | 70a | 12.2a |
P. fluorescens M1 | 100.5a | 85a | 21a | 24.5a | 81b | 20.4a |
P. fluorescens M2 | 89b | 71b | 14b | 22a | 54c | 11b |
P. fluorescens M3 | 98a | 83a | 21a | 24a | 69a | 19.5a |
P. fluorescens M4 | 92ab | 83.5a | 20.5a | 24.5a | 97d | 23a |
P. fluorescens M5 | 91ab | 71b | 19.5a | 20a | 58c | 19.5a |
P. fluorescens M6 | 71c | 57c | 17ab | 19.5a | 56c | 16ba |
P. fluorescens M7 | 93.5ab | 78.5ab | 19.5a | 19a | 57c | 22a |
P. fluorescens M8 | 92ab | 79ab | 19.5a | 20.25a | 77ab | 18.9a |
P. fluorescens M9 | 83b | 58c | 15b | 19a | 30e | 10b |
P. fluorescens M10 | 81b | 59c | 14.5b | 21a | 29.5e | 8.2b |
M. incognita | 39e | 31e | 9c | 11b | 52c | 25a |
Similar letter (s) in a column are non-significant statistically at p<0.05 |
P. fluorescens M1's also increased the shoot fresh (85 g) and dry weight (21 g) and root dry weight (20.4 g) significantly compared with the control (Table 2). In contrast, P. fluorescens M2 and M10 yielded the lowest shoot dry weight, 14 and 14.5 respectively (Table 2). P. fluorescens M1 and M3 had more effect of increasing shoot dry weight (21 g). Results of fresh root weight exhibited that this parameter in inoculated plants with P. fluorescens M4 and M1 (97 and 81 g, respectively) had a significant difference with the control. In this regard, root growth was improved significantly by P. fluorescens isolates. P. fluorescens M1 had the highest P. fluorescens M10 had the lowest percentage of increase in mean dry root weight with 20.4 and 8.2 g, respectively than the control treatment (Table 2).
Fig. 2: | Biplot of principal component analysis of in vivo assay of P. fluorescens activity against M. incognita |
The principal component analysis indicated that there is an accumulated variability of 89.03% for the in vivo based experiment (69.89% in the F1 and 19.14% in the F2). The PCA result indicated that M1 isolate had more effect on dry and fresh root weight than control (Fig. 2).
Additionally, the impact of P. fluorescens isolates on the nematode reproductive factor indices, including gall number, egg mass, egg number in each egg mass, reproduction factor and J2 per 100 g soil, displayed significant differences in the plants treated with P. fluorescens isolates M1-M10 (Fig. 3). In the treatment, solely inoculated by M. incognita, 35 gal g1 of root and 31.75 egg mass g1 of root were detected. On the other hand, all ten P. fluorescens isolates significantly reduced nematode variables indices. The lowest gall number, 10.2 gal g1 of the root, was recorded in the plants treated with P. fluorescens M1 (Fig. 3), while the lowest number of egg mass was detected in P. fluorescens M7 treated plants (Fig. 3). In these circumstances, P. fluorescens M1 reduced the gall number by 51% compared with the control. Plants treated with P. fluorescens M1 also had the least egg number inside every egg mass, as low as 51% of the infected M. incognita inoculated control (Fig. 3). Bacterial isolates also meaningfully affect the reproductive factor of M. incognita, so that P. fluorescens M1 reduced the reproductive factor from 108.3-21.2 (Fig. 3).
We have tested the effects of the ten isolates of P. fluorescens on M. incognita in vitro. The biological control of soil-borne pathogens by rhizosphere bacteria is susceptible to environmental circumstances19 This study did not reveal whether our best isolates of P. fluorescens can successfully colonize a crop plant's rhizosphere or how much and for how long they could control M. incognita.
However, significant differences were observed in different isolates' effectiveness, thus warranting screenings to identify the most suitable isolate to control a given nematode. Some isolate of P. fluorescens, such as CHA0, produces the antimicrobial phl (polyketides, 2,4-diacetylphloroglucinol) and plt (pyoluteorin) that protect plants from several phytopathogenic fungi12,20. This is in agreement with the result obtained in the present study. The mechanism of antagonism of rhizosphere bacteria against plant-parasitic nematodes might be due to toxic metabolites or antibiotics21,22. Besides, proper management of fungal pathogens by fluorescent pseudomonads is due to the production of toxic metabolites, antibiotics, or siderophores23,24.
The same result obtained by Pseudomonas fluorescens isolate Wayne 1R for control of M. incognita in watermelon25 However, those Pseudomonas isolated did not enormously improve the growth parameters for watermelon. Additionally, P. fluorescens presented inhibitory effects on M. incognita related to cowpea26 and eggplant27. A previous study indicated that two isolates of Paenibacillus castaneae and two isolates of Mycobacterium immunogenum were promising biocontrol agents for managing M. incognita28 Plant growth-promoting bacteria can inhibit egg hatching in Meloidogyne species. However, reducing the number of galls in the plants inoculated by bacteria and nematodes could be due to the absence of hatched eggs. This reduction may improve the plant defense systems' through the plant chitinases production29. While chitin layers in nematode eggs require development, the chitinases can interrupt this process and prevent egg hatching. However, several isolates of P. fluorescens did not reduce the gall number and J2 in the soil for M. incognita associated with watermelon25. In contrast, P. fluorescens significantly reduced the gall number on eggplant root infected with M. incognita27.
Fig. 3: | In vivo assay of variables for M. incognita related with eggplant exposed to P. fluorescens Similar letter(s) are non-significant statistically at p<0.05 |
Additionally, P. fluorescens Pf1 and Pf2 showed a 69.8 and 62.3% reduction in cowpea infected with M. incognita26. Several bacterial species exhibited an inhibitory effect on Meloidogyne species management30. Therefore, more investigation using many crop species is needed to find out the best isolate for the biological management of M. incognita. The limitation of using Pseudomonas is the geographic region where the isolates recovered. The isolates aiming to use in the biological control of the pest and disease should be native and specific to a particular geographical region. Therefore, the results of this study become significant in the biological control program of the nematodes. As the Pseudomonas isolated from the same region applied to the nematode, their establishment and effectiveness will be boosted.
The chemical effectiveness against certain nematodes such as Meloidogyne and the hazardous effect on the environment causes a significant reason to use the friendly-environment biological agent to maintain the environment safe. Additionally, the use of chemicals in Iran is a reason for contaminating the underground water sources. Therefore, native bacterial isolates can be used as a biological control agent for plant-parasitic nematode management to achieve better yield and organic production. The present study results suggest that P. fluorescence M1 seems an appropriate bacterium for the management of M. incognita, which is auspicious for a forward way to achieve sustainable agriculture and environment.
This study discovers the potential effect of the native isolates of Pseudomonas on M. incognita that can be beneficial for the management of the plant-parasitic nematode. This study will help the researcher to uncover the critical areas of biological control of root-knot nematodes that many researchers were not able to explore. Thus a new theory on these microbes and possibly other microorganisms, may be arrived at.
ACKNOWLEDGMENT
The author thanks the University of Limpopo, South Africa.