Nematodes are considered a serious problem for agriculture. Nematodes of the Meloidogyne genus can attack a wide range of plants, needing different management methods to decrease its population. Fungi from the Trichoderma genus has been related to have potential as biological control agents. However, before an organism is used as biological control agent, first it is necessary to prospect, characterize and test its potential as biocontrol agent, so the objective of this work was to characterize and test fungi isolates of the Trichoderma genus to control M. javanica. We obtained forty isolate to carry out this experiment. We extracted the DNA of each isolate to discover which species we were testing, by doing a PCR and sequencing. We tested in vitro their parasitism effect using ELISA plate. Also, we extracted their filtrate to see if their metabolites have potential to reduce nematode population by showing a high mortality or inhibiting hatching. The results confirmed the high potential of the fungi of Trichoderma genus as a biological agent to control Meloidogyne javanica.
- Biological control
- Integrated management
- root-knot nematode
- fungal filtrates
Agricultural systems have had little crop diversification over the years, which means that these organisms have good availability of food throughout the year. This availability causes the nematode population to grow more and more, making control even more difficult. Therefore, new control alternatives are being studied to minimize the damage caused by these organisms. Currently, biological control within integrated management stands out as an efficient and economically viable alternative to the use of chemical nematicides [7, 8]. In general, biological products have low toxicity and environmental risk, and we would be using a wide variety of microorganisms that can naturally parasite nematodes and their eggs in natural and agricultural environments. Among the main groups of microorganisms responsible for the biocontrol of nematodes, fungi stand out, representing up to 75% of the microorganisms used in the control of plant-parasitic nematodes [9, 10]. The ability to colonize the soil and persist for a long period makes these organisms increasingly visible. When we think about long persistence, we are comparing it with chemicals, which do not have a long-lasting residual. We can observe in the field chemical products that are used in seed treatment in soybean culture lose their efficacy even before the reproductive period of the culture.
Therefore, the possibility of incorporating organisms that have a long persistence in the soil can be a very important control measure, given the worrying scenario that the nematodes have been presenting. Fungi of the
Besides that, some isolates also have survival strategies that make them highly competitive in the environment, such as: survival in acid and / or saline soils; survival in conditions of high temperature and low humidity; fungicide resistance; adaptation to different environments and climatic zones, as they can inhabit soils in tropical regions and temperate climates; production of resistance structures; high efficiency in the use of resources as nutrients, thus making them excellent competing organisms; extraordinary capacity for proliferation in the rhizosphere and communication with plants, among others.
Due to the variation of environmental conditions (soil, climate, vegetation, etc.) on the planet, a species may have strains (variants) with specific adaptations to different environments. Thus, it is suggested that the antagonistic activity of a
2. Material and methods
2.1 Meloidogyne javanica inoculum
The root-knot nematode inoculum, specifically
2.2 In vitro evaluation of the nematicidal and nematostatic effect of
Trichodermaspp. on Meloidogyne javanica
Of the 40 isolates used in this study, 6 isolates are from the fungi bank of the laboratory of Soil Biology, 10 isolates were provided by the Federal University of Santa Maria, Frederico Westphalen (FW) campus, 6 isolates were supplied by the UFSM Phytosanitary Defense laboratory (D, DFS), 4 isolates were supplied by the University of Pelotas (Pel), 2 isolates were supplied by the UFSM campus Palmeira das Missões (PM), 1 isolate was supplied by the University of Passo Fundo (PF), 6 isolates were provided by Biota Innovations in the Midwest (BIF), and 5 control isolates were obtained from commercial products already reported as biological nematicides (Table 1).
|Isolates||Origin (Brazil)*||Treatment code||Species|
The isolates were kept in Petri dishes containing the Potato-Dextrose-Agar (PDA) medium and incubated at 25°C ± 2°C in BOD (Biochemical Oxygen Demand). Each specimen of the fungi was stored in inclined test tubes with PDA medium and kept refrigerated at a temperature of 6 to 10°C ± 2°C.
2.2.2 Parasitism test on
For the parasitism test of the
From the obtained suspension, 50 eggs were added, and transferred to individual wells of ELISA plates. In each well, together with the J2, 100 μL of fungal suspension (108 conidia / ml) was added. Then the plates were kept in the dark in a BOD under a temperature of 25°C ± 2°C. Evaluations were performed 15 days after application. The numbers of parasitized eggs were determined. This test was repeated twice for greater data reliability.
Each fungus was grown in Petri dishes with PDA culture medium. Seven days after incubation at 25°C ± 2°C, three disks of 5 mm in diameter were removed from the edges of the cultures and placed in a 250 mL Erlenmeyer flask containing 100 mL of Czapek Dox liquid medium (0.5 g KCl, 1 g of KH2PO4, 2 g of NaNO3, 30 g of sucrose, 0.01 g of FeSO4.H2O and 0.5 g of MgSO4.7 H2O per 1000 mL of distilled water).
Erlenmeyer flasks were sterilized using the autoclave for 30 minutes. A different isolate was placed in each sterile Erlenmeyer. The flasks were kept in an incubator at 25°C with constant agitation for 15 days. After this period, the entire content of each Erlenmeyer was filtered through a cellulose acetate membrane, with an opening of 0.22 μm. For each isolate, the cellulose acetate membrane was exchanged. The fungal filtrates obtained were kept refrigerated for 48 hours at a temperature of 6 to 10°C ± 2°C, until the assay was established.
2.2.4 Nematode mortality and hatch inhibition test
For the mortality test of second stage juveniles (J2) of
For the hatching test, first the egg suspension was obtained according to the methodology of . Then we placed 50 eggs per well of the ELISA plate for the trial. The evaluation was made on the 21st day, when the count of 50 eggs was performed. In each treatment, eight repetitions were performed, kept at 25°C in the dark. These tests were repeated twice, aiming at increasing the data reliability. In the study, two controls were used, one containing only distilled water and the other containing only Czapek Dox medium, to eliminate the possibility of some type of unexplained alteration caused by the Czapek Dox medium that was used to perform the filtrates.
2.2.5 Experimental design and statistical analysis
The experimental design used was completely randomized with eight replicates for each treatment, and each fungal isolate corresponded to one treatment (40 isolates). The variables evaluated were: number of live and dead J2 nematodes, count of J2 hatching, count of the number of parasitized and darkened eggs. The results were subjected to analysis of variance, and the means of each treatment were compared by the Scott-Knott cluster test at 5% probability of error, by the SISVAR software .
2.2.6 Molecular identification of
The total genomic DNA was extracted by the method described by . The
Then 400 μL of chloroform was added and stirred by gentle inversions for 5 min. Afterwards, it was centrifuged at 14000 rpm, 20°C, for 5 min. After centrifugation, approximately 400 μL of the aqueous phase was removed and transferred to a new 1.5 mL microtube, where 200 μL of chilled isopropanol (2-propanol) was added and homogenized by gentle inversions for 1 minute and incubated at −20°C for 30 min. The solution was centrifuged at 1400 rpm, 20°C, for 5 min. The supernatant was discarded, keeping only the pellet at the bottom of the microtube. For DNA precipitation, 200 μl of cold 70% ethanol (4°C) was added to the tube, followed by centrifugation at 14000 rpm at 4°C, for 5 min. and the supernatant was discarded keeping the pellet formed. The precipitate was dried at room temperature, and recovered in a volume of 50 μL TE [1 mM Tris and 0.1 mM EDTA] + RNAse and incubated at 37°C for 30 min., and its DNA was quantified and stored at −20°C until use.
The genomic DNA samples extracted from the fungi were subjected to polymerase chain reaction (PCR) with that performed for partial amplification of the elongation factor gene (EF-1α) with the primers 5’-ATGGGTAAGGARGACAAGAC-3′ and 5’-GGARGTACCAGTSATCATGTT3-’. For this, 3 μL of the fungi DNA were added to the final volume of the 25 μL PCR reaction, containing 10 mM Tris HCl pH 8.3; 50 mM KCl; 1.1 mM MgCl2; 10 mM of each dNTP; 25 nmoles of each EF1 and EF2 primer; 1.5 μL of Taq DNA polymerase (Invitrogen, Brazil) and ultrapure water to complete the reaction volume. A negative control without DNA was included in the PCR. The amplification reactions were carried out in a thermocycler (Applied Biosystems 2720, Thermo Fisher Scientific, USA), under the following conditions: 94°C for 1 min., 35 cycles of 95°C for 3 min., 95°C for 1 min., 72°C for 1 min. and 30 seconds, and 72°C for 10 min. At the end of the reaction, the amplified fragments were kept at 4°C. To verify amplification, electrophoresis was performed on 1.5% agarose gel, in TBE 1X buffer, stained with Sybr Gold (Invitrogen, Brazil). PCR products were purified with the Gen Elute PCR clean-up Kit® kit (Sigma, USA) and sequenced (ABI PRISM 3100, Thermo Fisher Scientific, USA). The sequences were analyzed using the Staden Package 2.0.0b program  to obtain consensus.
2.2.7 Phylogenetic analysis
The alignment of the nucleotide sequences was performed in the programs Clustal W and Clustal X , and sequences deposited in the databases were used for comparisons. The Neighbor-joining method, using the Jukes-Cantor model, was used to estimate the evolutionary distance. The phylogenetic tree was built in the MEGA X program , with the Maximum Likelihood algorithm and the bootstrap values calculated with 1,000 replicates.
3. Results and discussion
3.1 Molecular identification of
The isolates T36 to T40 are commercially used
Analyzing the bootstrap values presented in the dendrogram, we can say that within the clades of the species
3.2 In vitro evaluation of the parasitic, nematicidal and nematostatic effect of
Trichodermaspp. on M. javanica
In this study, it was observed that all species of
|Treatments||Species||J2 parasitized (%)||J2 mortality (%)||J2 hatching inhibition (%)|
|Control Czapek Dox**||—||5||E||7.1||F|
Regarding the mortality of
Results similar to the present study were obtained by , where all the filtrates obtained from the isolates of
Trichoderma isolates produce metabolites, such as lytic enzymes, which are released into the rhizosphere solute, in our case, culture medium solute. Thus, the inhibition of J2 hatching from the application of
Regarding the nematicidal and nematostatic effect in the inhibition of the hatching of J2 by fungal filtrates, it was observed that 25% of the isolates resulted in a high inhibition of the hatching of
In , they concluded that two
The authors  mention that
In a study by  with the objective of evaluating the effect of
The production of lytic enzymes also helps in the penetration of the fungus, especially visualized in
Results from  showed that, fungi of the
As we can see in the results of this work and supported by others, there is a significant difference in the suppression of nematodes among isolates of the same species. We observed in our work species of
This is due to the fact that each strain has a different gene expression, that is, it has genetically the ability to produce different lytic enzymes [22, 32], or it has the parasitic capacity expressed by virulence genes. For this reason, selections of highly efficient strains are carried out in the biocontrol of pests and diseases.
It was tested by  230 isolates, and only 8 belonging to the South region. All organisms obtained in prospecting processes should be tested as potential antagonistic agents for different pathogens and pests. The authors affirm the importance of prospecting for biological control agents and characterizing them, mainly fungi, in the entire Brazilian territorial area, since good antagonists may be dispersed in different regions of the country. This work shows the potential of five species of
We thank the Federal University of Santa Maria and the Graduate Program in Soil Science, for offering the entire structure for the development of the work. To CNPQ (National Council for Scientific and Technological Development) for the 12 months of scholarship.
Conflict of interest
The authors hereby declare no conflict of interest.