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Introduction
Wilt of pea (Pisum sativum L.) caused by Fusarium oxysporum f. sp. pisi. is one of the prevalent diseases in intensive crops and can cause losses ranging from 30 to 50%, especially in susceptible varieties and under favorable climatic conditions for the pathogen development. The disease symptoms starts with an ascending chlorosis, followed by plant stunting, in the outside of roots, a brown coloration is seen and in the inner part, a longitudinal lesion of reddish tonality. The fungus elicits a premature death by plant wilt associated to redness and blockage of vascular bundles (Buitrago et al., 2006; FENALCE, 2007).
Due to yellowing and wilt of pea, this disease can occur for different causes. The real test to pathogen susceptibility, it would be due to vascular bundles redness in the root, which produces the other two symptoms as side effects.
In Nariño department, Colombia there are no resistant varieties and the efforts of farmers to control this problem are not satisfactory because the fungus is still widely distributed and persistent in soils (Sañudo et al., 2007).
There are different studies of the genus Trichoderma spp. in the biological control of soil pathogens transmitting diseases in plants whose results indicates that several strains of Trichoderma show a significant reductive effect in plants with diseases caused by pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Pythium aphanidermatium, Fusarium oxysporum, F. culmorum., under greenhouse and field conditions. Accordingly, to the harmful behavior of these fungi, the use of antagonists represents an effective and essential use, since they can act against the pathogens in several ways, isolates of Trichoderma harzianum, can produce lytic enzymes antibiotics and antifungals. In addition, they can be efficient pathogenic fungi competitors and to promote plant optimal growth (Gajera et al., 2013).
The aim of the present research was to evaluate the antagonistic effectiveness of Trichoderma spp. native and commercial strains on Fusarium oxysporum f. sp. pisi., under in vitro, greenhouse and field conditions.
Materials and methods
Bioassays were carried out in the Universidad de Nariño, Colombia. In the plant health laboratory, at a temperature of 13 ͦ C, under greenhouse conditions, 17 ͦ C, and an altitude of 2540 m. a. s. l., with the following geographical coordinates: 01°12'13"N; 77°15'23"W. The field phase was established in the most affected disease area in the Nariño department, Colombia corresponding to the municipalities of Pupiales (2875 m.a.s.l.), Ipiales (2665 m.a.s.l.) and Gualmatan (2714 m.a.s.l.), located respectively and geographically as follows: between 0° 52´N and 77° 38´ W, 0º59'39"N, 77º33' 56"W,and 0°50´N and 77°34´W, with a temperature which oscillates between 11 and 12 ͦ C, and an annual rainfall between 500 and 1100 mm, which varies according to the time of year.
To obtain the different Trichoderma spp. native strains, 20 representative soil samples were collected from each of the evaluated municipalities, taken from the soil rhizosphere of apparently healthy pea plants. For pathogen isolation, pea plants with yellowing disease symptoms, ascending chlorosis, low growth and reddish coloration in vascular bundles, were considered. Trichoderma spp. isolates were performed by the dilution method, weighing 1 g of collected soil and placing it in a test tube with 10 mL of distilled water. The process was brought to a 10-2 dilution, and then, seeded into PDA with chloramphenicol. Trichoderma spp. colonies with characteristic growth form of green cushions of conidia, were identified and under microscope, the genus characteristics, were confirmed (Barnett & Hunter, 1972). Twelve strains of Trichoderma spp. from the municipalities of Pupiales, Ipiales and Gualmatan, were obtained. The obtained Trichoderma spp. strains in addition to commercial control strain, identified as C21 from Perkins laboratories located at Palmira - Valle del Cauca, Colombia, constitute a group of 13 treatments which in vitro antagonism tests, were performed. In the 12 strains of Trichoderma spp., the species were identified in a simultaneous research, determining the presence of T. harzianum., T. longibrachiatum and T. asperellum (Table 1), Being the most frequent, T. harzianum (Checa et al., 2015).
For each location, the isolation of Fusarium oxysporum f.sp. pisi was established from the affected pea plant material by taking root tissue and symptomatic stems under aseptic conditions. Sections were seeded in PDA and incubated at room temperature until the development of fungal colonies (Rojas, 2011), from the colony edge, monosporic cultures were generated, until a pure colony was obtained, which served as base for the fungi multiplication.
in vitro antagonism assay
An unrestricted random design was performed with 13 treatments corresponding to the strains of Trichoderma spp., in addition to one commercial strain and three replicates, the experimental unit being a petri dish. The methodology described by Howell (2003), was used, placing on a side of a petri dish with PDA a 5 mm diameter disk of Fusarium oxysporum f.sp. pisi culture, during the time necessary for this to initiate mycelial growth. One day later, a culture of 5 mm diameter disk of each Trichoderma spp. Native strain, was placed on the opposite side. Disk pairs have allowed incubating until evaluation of mycelial growth and inhibition halo.
Greenhouse antagonism assay
In order to identify the most appropriate strains, concentrations and doses to be evaluated in field test, were performed. We used a DIA experimental design with factorial arrangement for three factors, the first factor corresponded to the best in vitro behavior of following strains: C2, C7, C12 and C21. The second factor was the concentration, which had four levels as follows: 106, 107, 108 and 109 conidia of Trichoderma. mL-1. The third factor was the dose to be applied with two levels: 20 and 40 mL per plant, for 32 treatments, with 2 replicates. The experimental unit corresponded to five pea plants grown in bags with 3 kg of sterile soil. Different concentrations were obtained from Petri dishes with abundant growth of Fusarium oxysporum and Trichoderma spp., removing the conidia with a handle, making serial dilutions. For F. oxysporum, the concentration 106 was used and for the case of Trichoderma spp., The 106, 107, 108 and 109 concentrations were obtained by counting conidia in the Neubauer chamber. The formula: V1 = C2 V2, where: C1 = initial concentration (known in the count), V1 = initial volume (arbitrarily established when preparing the inoculum), C2 = desired final concentration (accordingly to the study to be carried out) V2 = Final volume (unknown).
The inoculation of F. oxysporum, was established 8 days before sowing. Each treatment was applied in drench way in wet soil conditions. The inoculations of Trichoderma spp., were performed when pea plants had a height of 10 cm. The evaluated variables corresponded to plant height at 60 days, fresh weight of roots and incidence. For the incidence, the number of pea plants with symptoms in the root system was counted, and the pathogen re-isolation was performed to assure the disease attack was caused by F. oxysporum.
Field phase evaluation
Bioassays were established in the municipalities of Pupiales, Ipiales and Gualmatan, in lots highly affected by the pathogen. With losses exceeding 70% in the previous half year. Seeding was carried out in the rainy season of the first half of 2014, using the Andean improved variety of fickle growth habit with good adaptation to the region, but susceptible to the pathogen. A randomized complete block design with four treatments and five replicates was performed at each location. The treatments corresponded to the two strains of better behavior in greenhouse with the concentration of best response obtained for each of them, being these C12 and C21, in concentration of 1x108 and 1x106 conidia.mL-1, respectively. In addition, a chemical treatment of seed disinfection with oxycarboxin plus captan, was included in doses of 1g of pc per kg of seed and an absolute control. The experimental unit was two rows of five meters long, with distance among rows of 1.2 m and distance among sites of 0.10 m, depositing one seed per site. Agronomical management of pea crop was carried out using a tillage system, of common occurrence in the region and applying the cultivation technology for the zone recommended by Sañudo et al. (2007). The evaluated variables were as follows: incidence percentage, evaluated by the presence of reddish color in the root and yield in green pod (Kg.ha-1). The data were submitted to variance analysis and Tukey's means comparison.
Results and discussion
in vitro antagonism assay
Mycelial growth and inhibition halo were different among strains. Isolates C12, C7, C2 and C21, showed the best behavior, presenting a mycelial growth, which varied between 4.73 and 3.72 cm, exceeding C11, C5, C9, C6, C4 and C10, with averages below 3.40 cm (Table 2).
Table 2 Tukey's averages comparison for mycelial growth and inhibition halo variables of Trichoderma spp, in dual confrontations with Fusarium oxysporum f.sp. pisi in vitro condition evaluations
Tukey comparator α 0.05 = 0.934. Averages with the same letter do not show significant differences (p <= 0.05)
It is important to note that the pathogen growth was lower when it were exposed to the strains mycelium of C2, C7, C12 and C21 Trichoderma spp., and it is at this point, when the antagonist capacity to overgrow the pathogen was variable for each strain. Therefore, a differential effect evidence of the strains treatments on Fusarium oxysporum f.sp. pisi.
In accordance with Gajera et al. (2013), the greater antagonistic capacity observed in the abovementioned strains could be related to the greater efficiency in nutrient competition or the products release, which slow or kill the F. oxysporum pathogen. Given these concerns, a greater mycoparasitism effect is possible. In this regard, Howell (2003), indicates the way in which Trichoderma spp. inhibits the pathogen growth is mediated by several mechanisms, including antibiosis, which can also be associated with the extracellular enzymes production as follows: peptinases, cutinases, glucanases, and chitinases, and inactivation of them towards the pathogen, as suggested by Durán et al. (2003). This information is useful in to keep in mind that in vitro tests are used to determine the antagonistic potential and to establish the biocontroller inhibitory capacity before carrying out studies, which require more time and economic cost (Larralde et al., 2008). Respectively, the in vitro evaluation results of C2, C7, C12 and C21 strains were selected to perform greenhouse studies and confirm their effectiveness.
Greenhouse antagonism assay
Plant height
According to the variance analysis, the triple strain interaction * concentration * dose, was significant indicating the height results were affected by all three factors together. Therefore, only this interaction was analyzed to define within each strain and in each concentration, the behavior of the two evaluated doses. In C2 and C7 strains, no significant differences were observed between the 20 and 40 mL doses within each Trichoderma inoculum concentrations (106, 107, 108 and 109). In strain 12, a 5% of obtained probability difference between the dose of 20 ml with 146.7 cm and the dose of 40 ml, which had a height of 134.8 cm. The abovementioned in a concentration of 107. C21 strain for 108 concentration in Trichoderma conidias.ml-1 was obtained a higher average height in the dose of 40 ml with 154 cm, showing significant differences on the dose of 20 mL, which reached 140 cm (Table 3).
Table 3 Tukey's averages comparison for Trichoderma spp, strain * concentration * dose interaction in the evaluation of their antagonism to Fusarium oxysporum f.sp. pisi under greenhouse conditions.
Tukey comparator (α 0.05) = 5.84g
Except for these two exceptions, the increase of the dose of 20 to 40 mL for the different evaluated concentrations within each strain performed an improvement in its antagonistic capacity measured by plant height at 60 days. However, the possibility dose of 40 mL was used in field conditions for C2 strain, was conditioned to obtain the same response in the fresh weight of roots and incidence variables, which did not occur since the triple interaction was not significant for these variables.
Root fresh weight
It is important to note there were no significant differences among C2, C7 and C21 strain concentrations (Table 4), whereas in the C12 strain, the 108 and 109 concentrations with averages of 4.36 g and 4.49 g, showed significant differences on the 106 concentration, which reached an average of 4.03 g. With the exception of C12 strain, 106, 107, 108 and 109 concentrations, had similar behavior in each of the strains under evaluation, suggesting that it is not necessary for these three strains (C2, C7 and C21) to increase the Trichoderma concentration beyond 106 conidia.mL-1.
Table 4 Tukey's averages comparison for fresh weight variable in the evaluation of four strains and concentrations of Trichoderma spp. for its antagonism to Fusarium oxysporum f.sp. pisi under greenhouse conditions
These results confirm Lo et al. (1994), who found the application of Trichoderma to 106 concentration is effective for the control of a good number of soil fungi. However, in the case of C12 in the present research, 108 and 109 concentrations, improved their antagonistic capacity, measured throughout fresh root weight.
Incidence
In the incidence variable, differences were only found for comparison between strains. The highest incidence occurred in the control treatment with Fusarium and without Trichoderma inoculation with 100% of affected plants (Table 5).
Tabla 5 Tukey's averages comparison for incidence percentage variable in the evaluation of four Trichoderma spp. strains for its antagonism to Fusarium oxysporum f.sp. pisi under greenhouse conditions
Tukey comparator (α 0.05) = 6.78
Similarly, the incidence was higher for C7 and C2 strains (97 and 70%), while C21 and C12 strains, showed better antagonistic behavior with a pathogen incidence of 50 and 45%, respectively.
In concordance to Woo et al. (2006), in addition to the antagonistic effect of Trichoderma strains against the invasive pathogen, associations of root fungi also stimulate plant defense mechanisms, leading to the resistance mechanisms induction similar to the hypersensitive response (RH), Systemic resistance (SR) and systemic resistance induction (SRI). At molecular level, resistance results in an increased metabolites and enzymes concentration related to defense mechanisms, such as phenylalanine ammonium lyase (PAL) and chalcone synthase (CHS), involved in the response biosynthesis of phytoalexins, chitinases and Glucanases. As well as resistance to hostile abiotic conditions. In barley, Trichoderma atroviride, showed an increased resistance to Fusarium infection Trichoderma harzianum, in transgenic lines of tobacco and potato plants, were highly tolerant or completely resistant to aerial pathogens as follows: Alternaria alternata, Alternaria solani, and Botrytis cinerea. Alternatively, Rhizoctonia solani in soil (Woo et al., 2006).
Field antagonism assay
Differences were observed between locations and locations * strain interaction for incidence percentage and yield variables. In Gualmatan location, the incidence rate varied between 85.78 and 89.99%, with no differences between the evaluated treatments. In Ipiales location, C12 strain and chemical control (83.89 and 76.35) showed lower incidence than C21 and treatment without control (88.04 and 89.9) (Table 6). In Pupiales location, C12 strain with 80% of incidence was less than the uncontrolled treatment (100%), while the C21 strain with 89% and chemical control with 91% of incidence, showed no difference among them, neither with C12 treatments and without control. Likewise, the pathogen incidence was high in all treatments in three locations (>76%). Although the plant percentage with presence of the pathogen (incidence) was high, root redness vascular was not the same in all pea plants, there were plants with a slight presence of Fusarium in the root, which were productive and is possible when fungus does not severely affect the xylem vascular bundles. It is important to highlight the behavior of C12 strain C12 in Pupiales location, being the only treatment, which showed effect with respect to control treatment.
Table 6 Location * strain interaction for incidence and yield variables in the evaluation of four Trichoderma spp. strains in three locations of Nariño, department, Colombia
Given these concerns, it is possible the pathogen incidence of presence or pathogen absence throughout root redness observation have not allowed an accurate indicator of two evaluated strains (C12 and C21) efficiency. In many plants, when pathogen was able to penetrate and show some degree of root redness, the action of Trichoderma spp., could affect it by colonization of the root area. Therefore, the plants slightly affected, produced new roots or simply fulfilled their productive cycle. Díaz et al. (2012), consider the levels incidence depend on the provenance of the terrain, variety traits, temperature and humidity. Although, this variable has no direct relation with some components of yield being necessary in future research to evaluate also the severity degree and its effect on the plant performance.
Field assay
Green pod yield
For Gualmatán and Ipiales locations, there were no differences between C21 and C12 strains, chemical control and control treatment. In Gualmatán location, yields ranged between 6077 and 3678 kg.ha-1, while in Ipiales location, they were between 1782 and 820 kg.ha-1. In Pupiales location, it was observed that C21 treatment with an average of 16138 kg.ha-1, had differences with the control treatment, which obtained 13044 kg.ha-1. The chemical treatments and C12 with 14740 and 14699 kg.ha-1, showed no differences with respect to C21 and chemical control (Table 6). This information is useful to argue that all treatments were equally affected by the pathogen. Therefore, it was not possible to demonstrate the efficiency of C21 and C12 Trichoderma strains in the antagonism on Fusarium oxysporum f. sp pisi. In Ipiales location, the lowest yields were obtained from the three evaluated locations, which did not exceed 1800 kg.ha-1. The lower incidence of Fusarium oxysporum f.sp pisi., observed in C12 and chemical control on C21 and control treatment, was not reflected in the yield, where all the treatments had low averages without statistical differences among them. These results suggest, the pathogen in this location was highly aggressive and in addition, to being present in the plant roots with incidence values between 76.35 and 89.9%, could severely affect the xylem vascular bundles, which caused its obstruction reducing the nutrient passage and decreasing performance for all treatments. In Pupiales location, yield values in four treatments, are relatively high (Table 6). However, the pathogen was able to enter into the plant roots and marked a high incidence (number of plants with some degree of root redness), suggesting there was no severe affectation of the vascular bundles. In this location, C21 strain, outperformed the control treatment. However, in the incidence measurement, the average of these two treatments did not show differences, which indicates in the treatment with the C21 Trichoderma strain, if the pathogen F. oxysporum entered into the roots but did not colonize them enough to affect yields. Pineda & Tortolero (1995), indicates the pathogen biological control with antagonists potential is a long-term process that must be implemented with the usual crop management.
Conclusion
in vitro conditions, the C2, C7, C12 and C21 Trichoderma strains had high antagonistic capacity on Fusarium oxysporum f. sp. pisi.
In greenhouse conditions, C12 and C21 Trichoderma strains showed the greatest antagonistic effect against Fusarium oxysporum f. sp. pisi.
In field conditions, the strains effect was differential. In Gualmatán location, C12 and C21 strains, showed no antagonistic capacity. In Ipiales and Pupiales locations, the C12 strain had lower incidence of F. oxysporum. than the control treatment on the yields. In Pupiales location, C21 strain outperformed the control treatment, even though the two treatments had a similar incidence.
