Abstract: Background and Objective: Tuta absoluta is a devastating pest of tomato crops. Currently, T. absoluta control is mainly based on chemicals control. However, these products have a harmful impact on environment. This work aims to evaluate the preventive and curative effect of P. fluorescens Q036B, P. fluorescens Q110B and P. putida (Q172B and Q110B) against T. absoluta. Materials and Methods: The three strains were tested directly on mortality of Tuta absoluta adult. Indeed, the effect on larva was done on both sides as a preventive and curative trials. Each bacteria suspension was tested at 108 CFU mL–1. The mortality rate was recorded 24-120 h after application. Results: The results indicated that all tested bacterial isolates has a toxic effect on T. absoluta larva and adults. Within the 24-120 h of exposition period, T. absoluta mortality rates up to 81% for adults and to 100% for larva for Q110B and Q036B isolates, respectively. The Q036B isolates induced higher mortality rates than Q172B and Q110B in both preventive and curative control. Regarding the mechanism of action, all isolates produced hydrogen cyanide, siderophores in addition to the exhibited protease and cellulose activities, while only Q036B and Q172B possess chitinase activity. Conclusion: Our results concluding that the P. fluorescens Q110B and P. fluorescens Q036B were a promising candidates bio-agent for biological control of T. absoluta and has potential to be an efficient component in an integrated pest management program. Then the fruits produced will be qualified as safe for consumers and the environment.
INTRODUCTION
The tomato leafminer, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae), was originated from south America where it is considered as one of the most devastating pests of tomato crops1-3. This pest infests tomato plants and fruits destined to fresh market. The larvae causes severe damage to different part of the plants: Leaves, flowers, stems and fruits4,5. In 2008, T. absoluta was detected in Morocco, especially in the tomato growing regions, where it causes considerable economic losses6,7. The most common control practice of T. absoluta is based on chemical insecticides8. However, these products have negative impact on environment and effects on natural enemies which are based on biological control9,10. In addition, chemicals can also lead to insect resistance8,11,12, environmental pollution and risk for human health13. Therefore, integration of other control methods (cultural, biological and biotechnological methods) becomes more and more imperative. After the introduction of T. absoluta in the Mediterranean Basin, some indigenous parasitoids and predators have been reported to prey on this exotic pest14-17. Different botanicals with efficient insecticidal activities against T. absoluta has been known at length and more recent studies have also shown the efficacy of plant extracts against larva18,19. In the same context, the use of micro-organisms as insecticides has been widely known as well. The B. thuringiensis has been reported as efficient on T. absoluta larval stages in many countries20-22. Several reports about plant growth promoting rhizobacteria (PGPR) like Pseudomonas, Bacillus and Serratia indicated that these micro-organisms are efficient root colonisers and able to protect plants from different crop pests23-26. Most of the studies that focused on the effect of B. thuringiensis on T. absoluta have been performed and reported from originated region of T. absoluta27-29. Commercial formulates based on B. thuringiensis have been used for decades to control insect pests as an alternative to chemicals. Such formulates are ecofriendly, harmless to human and other vertebrates30-32 have shown high compatibility with the use of natural enemies33-35. Furthermore, the biological products are also recommended when insect populations have developed resistance to other products or when treatment is required just before harvest36. Thomas and Ellar37 reported that B. thuringiensis involve an insecticidal mechanism in which interaction of toxin with specific plasma membrane lipids causes a detergent-like rearrangement of the lipids, leading to disruption of membrane integrity and eventual cytolysis. Pseudomonas fluorescens isolates were found effective in killing or causing morphological defects in a widely used laboratory pests26,38. The ingestion of P. fluorescens bacteria by D. melanogaster larvae causes both lethal and non-lethal phenotypes, including delay in the onset of metamorphosis and morphological defects in surviving adult flies, which can be decoupled39. Accordingly, few studies have evaluated the efficacy of Pseudomonas on T. absoluta. The aim of this study is to evaluate the preventive and curative efficacy of three Pseudomonas isolates as bio-insecticides against T. absoluta along with the establishment of the mechanisms involved in the control of this pest.
MATERIALS AND METHODS
All experiments were done in the plant protection laboratory of INRA-Agadir Morocco from March and the end of July on 2018.
Pseudomonas isolates: The three isolates of Pseudomonas, P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B were isolated from the rhizosphere of tomato plants and stored in glycerol at -20°C in the plant protection laboratory, INRA-Agadir26.
Insect rearing: A colony of T. absoluta was obtained from the Tuta-rearing glasshouse at the experimental farm-Belfaa (Agadir, Morocco). They were reared under glasshouse conditions, on tomato plants at 26±2°C, L: D 16:8 and 70±5% RH and no pesticides were applied during the process of mass rearing.
Effect of P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B on T. absoluta adults: The effect of bacterial isolates on T. absoluta adults was studied under laboratory conditions using a leaf-dip bioassay12. A leaf cage was prepared using vials (5 cm in diam. and 8 cm in h) containing Whatman paper soaked in sterile distilled water. A 1.5 cm diam. hole was made in the lid of bottles and covered with muslin tissue. Tomato leaflets were dipped in each isolate concentration (108 CFU mL–1) for 30 sec. The treated leaflets were dried under a laminar hood then transferred to leaf cages. The control leaflets were dipped only in sterile distilled water. Eleven T. absoluta adults (male to female sex ratio: 1-1.2) were then transferred to leaf cages. The cages were incubated in growth chamber (Ehret, Type KLT/04) at 25±2°C, 70% RH and 16 h: 8 h light-dark. Four replicates for each isolate were used and the bioassay was replicated thrice. The T. absoluta mortality rate was assessed 24, 48, 72 and 120 h after treatment and were corrected using Abbott’s equation40 (Eq.1):
| (1) |
Where:
| CrrM | = | Corrected mortality |
| DIN | = | Dead insect number |
| DINC | = | Number of dead insect in control |
| ITN | = | Total insect number |
Toxicological effect of P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B on T. absoluta larva
Preventive control: Larva of T. absoluta were collected from the Tuta-rearing greenhouses. Infested tomato leaves were transferred to the INRA Plant Protection Laboratory in Agadir. Bioassays were carried out on the same day. A leaf cage was prepared using Petri dishes (9 cm) containing Whatman paper soaked in sterile distilled water. A 1.5 cm diam. hole was made in the lid of Petri dishes and covered with muslin tissue. Tomato leaflets were dipped in each isolate concentration (108 CFU mL–1) for 30 sec. The treated leaflets were dried under a laminar hood then transferred to leaf cages. The control leaflets were dipped in sterile distilled water. Five T. absoluta larva (L2) were then transferred to cage leaflets. The cages were incubated at 25±2°C with a photoperiod of 16: 8 h (L: D) and T. absoluta mortality rate was assessed 24, 48, 72 and 120 h after treatment. Five replicates for each isolate were used and the bioassay was four times replicated.
Curative control: The T. absoluta infested tomato leaves were collected from tomato plants in an infested greenhouse. The leaves containing newly hatched eggs (5-7) were dipped in each bacterial isolate (108 CFU mL–1) for 30 sec. The treated leaflets were dried under a laminar hood then transferred to leaf cages. The control leaflets were dipped only in sterile distilled water. The cages were incubated at 25±2°C with a photoperiod of 16: 8 h (L: D). The rate of damage of T. absoluta larva was estimated 24, 48, 72 and 120 h after treatment. Four replicates (2 leaves/replicate) for each isolate were used and the bioassay was replicated thrice. After incubation period, the mesophyll area consumed was estimated and the anti-feeding rate (AFR) was calculated using the equation41 (Eq. 2):
| (2) |
Where:
| Dc | = | Area of mesophyll consumed in the control leaves |
| Dt | = | Area of mesophyll consumed in the treated leaves |
Mechanisms of action of P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B: The ability of an isolate to produce chitinase was determined as described by Cattelan et al.42. For the cellulase activity, M9 medium agar amended with cellulose and yeast extract was used to test the cellulase activity43. Protease activities of Pseudomonas were determined according to the method reported by Jha et al.44. The ability of volatile organic compounds production, hydrogen cyanide (HCN), was determined as described by Bakker and Schippers45. The phosphatase activity was determined by development of a clear zone in NBRIP medium46 while, Siderophore production was determined using the FeCl3 test47 and the chrome azurol S (CAS) assay48.
Statistical analysis: The percentage of mortality was calculated for each Pseudomonas isolate. To determine the efficacy of Pseudomonas on T. absoluta, mortality and anti-feeding rates were subjected to the analysis of variance test (ANOVA- one-way) with Duncan’s multiple range test at 1% level of significance using Statistica software (Ver 6).
RESULTS
Effect of Pseudomonas isolates (P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B on T. absoluta adults: The results indicated that P. fluorescens Q110B was most effective and caused 82% of mortality compared to P. putida Q172B and P. fluorescens Q036B with 55 and 34%, respectively at 120 h after treatment (Table 1).
Toxicological effect of P. putida (Q172B and Q110B) and P. fluorescens Q036B on T. absoluta larva
Preventive control: The three tested bacteria showed strong larvicidal effects with mortality rates ranging from 80-100% at 120 h after treatment for P. putida Q172B and P. fluorescens Q036B, respectively (Table 2).
Curative control: The results of curative control show that all these three tested Pseudomonas are highly efficient in reducing the damage caused by T. absoluta larva.
| Table 1: | Mortality rate caused by three Pseudomonas isolates (P. fluorescens Q036B, P. fluorescens Q110B and P. putida Q172B on T. absoluta adults |
| *By column, the rates followed by the same letters are not statistically different at p<1% | |
| Table 2: | Larvicidal effect of three Pseudomonas isolates (P. fluorescens Q036B, P. fluorescens Q110B and P. putida Q172B: preventive control |
| *By column, the rates followed by the same letters are not statistically different at p<1% | |
| Table 3: | Larvicidal effect of three Pseudomonas isolates (P. fluorescens Q036B, P. fluorescens Q110B and P. putida Q172B: curative control |
| *By column, the rates followed by the same letters are not statistically different at p<1% | |
| Table 4: | Mechanisms involved by three Pseudomonas isolates (P. fluorescens Q036B, P. fluorescens Q110B and P. putida Q172B |
| Hydrogen cyanide detected by discoloration of filter paper when incubated with reagent and siderophores detected by orange halo of CAS medium. The chitinase, protease and cellulase and phosphatase activity were detected by surrounding the colony. *By column, the rates followed by the same letters are not statistically different at p<1% | |
P. fluorescens Q036B showed highest toxicity on T. absoluta larva compared to control with an AFR of 91%.
Mechanisms of action of P. putida (Q172B and Q110B) and P. fluorescens Q036B: The results showed that all three Pseudomonas have a positive activity for cellulase and protease activity and produce hydrogen cyanide (Table 3). They produce siderophores in solid medium, as indicated by orange halo surrounding the colony in CAS medium. Regarding phosphatase enzyme, all three Pseudomonas produce phosphatase as indicated by clear halo surrounded the colony. The chitinase plate tests of both Pseudomonas isolates Q036B and Q172B manifested a clear halo surrounding the colony which, confirmed their ability to induce chitin degradation, however, no chitinase activity was involved for Q110B (Table 4).
DISCUSSION
The genus Pseudomonas is known to have biocontrol propriety against some pest species such as Cnaphalocrocis medinalis (Lepidoptera: Crambidae)49, two-spotted spider mites (Tetranychus urticae) and red spider mite (Oligonychus coffeae)26,50-52. The integration of micro-organisms as alternative to chemical pesticides shows an important interest53. Treatments based on B. thuringiensis provide a safe strategy to manage T. absoluta pest, as B. thuringiensis control larva54. To our knowledge, the present work is indeed the first report related to the high curative effect of Pseudomonas isolates against T. absoluta in the Mediterranean Basin. This study provides further evidence that P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B cause increased mortality of the adult and larvae of T. absoluta and induce potential preventive and curative effect against this pest. The tested bacteria reduce damage caused by T. absoluta larva at 100% with Q036B in laboratory conditions. These differences explain the mode of action involved by each bacterium against this insect. The achieved results are consistent with those found by Qessaoui et al.26, which showed that Pseudomonas isolates provided significant death and repellence to spider mite T. urticae adults.
The results obtained in this study have shown that it is possible to reduce the pest’s impact to very low levels without chemical insecticides and by using only Pseudomonas isolates based-treatment. Fluorescent Pseudomonas bacteria act by several mechanisms for insect and mite pests although the ability to degrade chitin is often considered the primary factor involved26. Chitinolytic organisms such as Pseudomonas sp. isolated from the rhizosphere have shown that they have potential as biological control agents49. This study demonstrates that P. putida Q172B, P. fluorescens Q110B and P. fluorescens Q036B control effectively both adults and larva of T. absoluta. Accordingly, it is possible to design control programs based on this bacterium that will successfully manage this pest while having low impact on the auxiliary fauna and the environment26,53,55. Additionally, the integration of this technology in biological control methods focusing on management of T. absoluta, will reduce certainly the use of chemicals and, consequently residues on fruits. The technology related to biological control is thus improving food safety, quality and a sustainability of agroecosystems56,57.
CONCLUSION
This study indicate that fluorescent Pseudomonas, especially P. fluorescens Q036B, P. putida Q172B and P. fluorescens Q110B have a high biological control potential on tomato leaf miner T. absoluta, which is promising in both preventive and curative management strategies. These bacteria occur through various mechanisms of action, such as the production of HCN gas, and enzymes like chitinase.
Future research has to be planned to evaluate the impact of these bacterial isolates under greenhouse conditions. The aim will be to confirm this curative effect and to assess toxic and repellent effects so that they can control other key pests of tomato. For the forthcoming works we suggest additionally molecular studies of the factors involved in these toxic effects.
SIGNIFICANCE STATEMENTS
This study discovered the Pseudomonas genus bacteria effect that can be beneficial to control the tomato leafminer Tuta absoluta. These 3 isolates of Pseudomonas identified as P. putida and P. fluorescens would effectively prevent the infestation and even avoid the crop damage after the infestation occurrence, which has been confirmed by the curative and preventive trials. This study will help the researchers to uncover the critical areas of Pseudomonas application in the biocontrol of T. absoluta that many researchers were not able to explore.