Molecular Characterization and Pathogenicity of Trichoderma Isolates to Meloidogyne javanica

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 .


Introduction
Soybean (Glycine max L.) is one of the most important agricultural commodities in the world. For this reason, it is constantly sought to increase productivity without increasing the cultivated area. Yield can be affected by several factors, among them are diseases caused by plant-parasitic nematodes, an important cause of reduced grain production in this crop. Worldwide, there are approximately 100 species of known plant-parasitic nematodes that decrease the production of this commodity [1]. In Brazil, these species are distributed primarily among the genera Meloidogyne, Heterodera, Pratylenchus and Rotylenchus [1][2][3][4]. Among these species, the root-knot nematodes, specifically Meloidogyne javanica, stands out because it has the ability to parasitize and cause significant damage to soybeans [5,6].
Agricultural systems have had little crop diversification over the years, which means that these organisms have good availability of food throughout the year. the nematodes were extracted from the soybean roots by the method that consists of grinding in a blender with the addition of 0.5% sodium hypochlorite followed by sifting and centrifugation with sucrose solution, carried out at the Soil Biology laboratory of the Federal University of Santa Maria (UFSM). The extracted nematodes were inoculated in tomato plants cultivar "Santa Cruz" (Solanum licopersycum L.), to maintain the population. The tomatoes remained in a greenhouse with temperature controlled at 25°C ± 2°C. The females of the population were periodically submitted to electrophoresis with the esterase enzyme [13] to confirm the purity of the population.

In vitro evaluation of the nematicidal and nematostatic effect of
Trichoderma spp. on Meloidogyne javanica

Obtaining Trichoderma isolates
Forty Trichoderma isolates from different regions of Brazil were used in this study showing the city that the isolates were provided (Figure 1). Isolates provided by private companies are from the Southeast and Midwest regions, however their locations were not provided.
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).
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.

Parasitism test on M. javaniva eggs
For the parasitism test of the Trichoderma isolates on M. javanica, eggs of the nematode were extracted manually. These eggs were disinfected so that there was the least possible interference from other microorganisms that could be located on the outside of the eggs. For this, the eggs were placed in a test tube with 0.5% sodium hypochlorite solution, and stirred manually for one minute. After this stage, the eggs were also disinfected with 1% streptomycin and 0.1% 2-mercaptoethanol (Sigma Aldrich) for four minutes; washed in sterile water and collected with micropipette.
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 (10 8 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.

Obtaining Trichoderma filtrates
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 KH 2 PO 4 , 2 g of NaNO 3 , 30 g of sucrose, 0.01 g of 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.

Nematode mortality and hatch inhibition test
For the mortality test of second stage juveniles (J2) of M. javanica, the Baermann funnel methodology modified by [14] was followed. The suspension with J2 was obtained from the hatching chamber with a tissue. From this suspension, 50 nematodes were removed through individual capture. In Elisa plates, 20 μL of water were pipetted and added to 80 μL of the fungal filtrates along with 50 captured nematodes. Mortality was assessed 48 hours after the application of the nematode suspension.
For the hatching test, first the egg suspension was obtained according to the methodology of [7]. 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.

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 [15].

Molecular identification of Trichoderma spp.
The total genomic DNA was extracted by the method described by [16]. The Trichoderma isolates were placed in a 1.5 mL microtube with 400 μL of extraction solution (Tris-HCl 100 mM pH 8.0; EDTA 20 mM pH 8.0; NaCl 1.4 M; CTAB 2%; PVP 1%; 2-Mercaptoethanol 0.1% and Proteinase K 0.01%) previously heated to 65°C for 3 min. and vortexed for 10 seconds. Then, it was incubated in a water bath at 65°C for 45 minutes, shaking every 15 min.
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 MgCl 2 ; 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 [17] to obtain consensus.

Phylogenetic analysis
The alignment of the nucleotide sequences was performed in the programs Clustal W and Clustal X [18], 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 [19], with the Maximum Likelihood algorithm and the bootstrap values calculated with 1,000 replicates.

Molecular identification of Trichoderma spp.
The Trichoderma isolates used in the study were identified from DNA extraction and separated into five distinct species, with 20 isolates belonging to the T. asperellum (50% of the isolates) species, 12 isolates of T. harzianum (30%), six isolates of T. virens (15%), an isolate of T. brevicompactum (2.5%) and an isolate belonging to the T. atroviride (2.5%) species.
The isolates T36 to T40 are commercially used Trichoderma spp. The T36, T39 and T40 were identified as T. harzianum, and T37 and T38 as T. asperellum. Using these isolates as references we compared with the sequences of other isolates and the clusters that resulted are in the phylogenetic tree (Figure 2).
Analyzing the bootstrap values presented in the dendrogram, we can say that within the clades of the species T. asperellum, T. virens and T. harzianum, we obtained sequences of small size, which caused low bootstrap values, as there was no enough support for the node. In [20], the authors reported that bootstrap values below 70 are normally hidden, and above 70 usually correspond to probabilities greater than 95% that the clade is real.

In vitro evaluation of the parasitic, nematicidal and nematostatic effect of Trichoderma spp. on M. javanica
In this study, it was observed that all species of Trichoderma tested showed high potential for suppression of M. javanica ( Table 2). This was confirmed by the effect of parasitism of eggs and nematodes and also by the production of metabolites that killed and/or inhibited the hatching of J2 eggs. The percentage of parasitized J2 nematodes was greater than 85% for all tested isolates, and 35% (14) of the isolates showed a parasitism capacity greater than 95%. The isolates that stood out with the highest parasitism averages, forming a group statistically different from the other isolates, were: T. Regarding the mortality of M. javanica using fungal filtrates ( Results similar to the present study were obtained by [21], where all the filtrates obtained from the isolates of Trichoderma spp. (22 isolates) proved to be efficient in promoting juvenile mortality in a population of M. incognita after 24 hours of application of the filtrates in vitro. It is reported that the parasitic effect of fungi of the genus Trichoderma against nematodes from egg to adult stages, requires some facilitating mechanism [22], such as the presence of lytic enzymes. The mortality of J2 de Meloidogyne may be related to the presence of enzymes proteases and chitinases that act in the degradation of the cuticle of nematodes, a resistant coating structure composed of proteins and chitin. For this reason, metabolites obtained from fungal isolates that may have these facilitating mechanisms are also sought.
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 Trichoderma filtrates may be related to enzymatic activity in the solution. Meloidogyne eggs are composed of approximately 30% chitin in the outer layer, in addition to other structural proteins, and lipids in the inner layer. In this sense, the action of inhibiting the hatching of J2 indicates the presence of enzymes proteases, chitinases and lipases that acted on the enzymatic degradation of the outer and inner layers of the eggs of M. javanica, reducing the hatching capacity of J2 of the eggs.
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 M. javanica J2, varying from 91.6% to 94.4% hatching inhibition ( In [23], they concluded that two Trichoderma isolates (T. asperellum M2RT4 and Trichoderma sp. MK4) significantly reduced the number of hatched J2s, between 60.8 and 81.8%. In the same work, it was observed that T. asperellum M2RT4 was the most efficient isolate for the control of galls, egg mass and deposited eggs, reducing, respectively, 81.8, 78.5 and 88.4%, indicating that isolates from this species have potential for biocontrol. Both results coincide with data on egg and J2 mortality and inhibition of hatching of J2, obtained in this work. On the other hand, T. atroviride F5S21 had no significant effect in comparison to the control, which goes against the result obtained in the present study, whereas T. atroviride (T33) showed positive results in parasitism, mortality and inhibition of hatching of J2.
The authors [24] mention that Trichoderma filtrates have toxic effect on adults of Meloidogyne sp. The same was observed by [25], in which all filtrates showed toxic activity against M. incognita, obtaining 98% of immobile and dead J2.
In a study by [26] with the objective of evaluating the effect of T. harzianum for the biological control potential of M. javanica, they also found promising results for nematode suppression. In this study, they also evaluated the metabolite production capacity and its relationship to nematode suppression. The authors concluded that the increased activity of the chitinase enzyme is directly related to the capacity of suppression of M. javanica by the species T. harzianum.
The production of lytic enzymes also helps in the penetration of the fungus, especially visualized in T. harzianum [27,28]. The ability to produce metabolites, as we can see, is also an important factor in the suppression of nematodes [29]. In our study, the production of metabolites by fungi, through fungal filtrates, also had a positive result for biocontrol of M. javanica. The production of metabolites by the fungi inhibited the outbreak of J2, and the isolate that obtained the lowest percentage of inhibition of J2 was a T. virens isolate. However, we can observe that this same species had percentages above 80% of mortality, parasitism and inhibition of hatching, which shows us an important difference between the Trichoderma isolates.
Results from [30] showed that, fungi of the Trichoderma genus, specifically T. harzianum in this study, presented positive results for the mortality and hatch inhibition of J2 comparing with control, Paecilomyces lilacinus and humic acid. And [31], also observed for 3 different isolates of Trichoderma 100% larval mortality for M.  javanica and between 94,1 and 100% larval mortality of M. incognita. Both studies support our findings for the potential of Trichoderma for Meloidogyne mortality.
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 T. asperellum that ranged from 87.62% of parasitized J2, up to 98.43%, and also, other strains of the same species showed 65.3% of J2 mortality, while others showed 91.9%, and for the percentage of inhibition of J2 hatching, there was a variation from 77.7-94%, within the same species. In the species T. harzianum, there were variations of 89.87-99% in parasitism, of 79.3% to 93.1% of J2 mortality from the filtrates, and from 79.7% to 94.4% of hatching inhibition, in different strains. 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 [12] 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 Trichoderma in the biological control of M. javanica, and future experiments in natural conditions could help to widen the differences of each of the studied strains.

Conclusion
All Trichoderma isolates tested showed potential for Meloidogyne javanica biocontrol.