EFFICACY OF Trichoderma asperellum , T . harzianum , T . longibrachiatum AND T . reesei AGAINST Sclerotium rolfsii EFICÁCIA DE Trichoderma asperellum , T . harzianum , T . longibrachiatum E T . reesei CONTRA Sclerotium rolfsii

This work was carried out to select and to evaluate efficacy of Trichoderma isolates to control sclerotium wilt (Sclerotium rolfsii) of common-bean (Phaseolus vulgaris). For the experiments were used two isolates of S. rolfsii UB 193 and UB 228. Sixty-five Trichoderma spp. isolates were tested and the following ones were selected in vitro for the in vivo tests: 5, 11, 12, 15, 102, 103, 127, 136, 137, 1525 (T. longibrachiatum), 1637 (T. reesei), 1642, 1643 (T. harzianum), 1649 (T. harzianum), 1700 (T. asperellum) and EST 5. The most promising isolates were identified by Sequencing of the internal transcribed spacer regions ITS1, ITS2, and the 5.8s rRNA genomic region, using the ITS5 and ITS4 primers and compared with sequences in the National Center for Biotechnology Information (NCBI) database. These selected isolates 1649 (T. harzianum), 1525 (T. longibrachiatum) and 1637 (T. reesei) were tested for evaluation of sclerotial germination inhibition under laboratory conditions, and to evaluate the effects of these on disease of bean plants under greenhouse conditions. The Trichoderma isolates 1649, 1525 and 1637 were more efficient in reducing sclerotial germination. In addition to 1649, 1525 and 1637, the isolates 5, 12, 102 and 1525 (T. longibrachiatum) significantly reduced de amount of diseased bean plants under greenhouse conditions.


Sclerotium rolfsii Sacc. [Teleomorph:
Athelia rolfsii (Cruzi) You & Kimbrough] is a pathogen of widespread distribution in soils of agricultural production areas.This basidiomycetes fungus causes a disease (Southern-blight; dampingoff; sclerotium-wilt) that affects more than 500 species in over 100 plant families.This disease can be a serious problem, especially in warm regions, where the pathogen produces sclerotia in infected plant parts near the ground.The sclerotia can survive in the soil for a few months to several years, depending on environmental conditions (PUNJA, 1993;XU et al., 2008).These structures are the primary inoculum source for the development of the disease (PUNJA, 1993;XU et al., 2008).After germination of sclerotium, hyphae of S. rolfsii might produce oxalic acid, pectinolytic and cellulolytic enzymes that could disintegrate plant tissues (LE, 2011;PUNJA, 1993).
The control of S. rolfsii occurs with the aid of preventive practices such as crop rotation, deep plowing, fungicide-treated and pathogen-free seed, among others and by biological control (BLUM; LIN, 1991;SOUSA;BLUM, 2013), cultural control (BLUM; RODRIGUÉZ-KÁBANA, 2004; BLUM; RODRIGUÉZ-KÁBANA, 2006;PINHEIRO et al., 2010), and chemical control (PAULA Jr. et al., 2011;SOUSA;BLUM, 2013).Biological control of plant pathogens has a number of advantages compared to conventional fungicides.While the fungicides feature only a temporary effect and require repeated applications during the growing period of culture, biological control agents are able to establish themselves, colonize and reproduce in the ecosystem (AVILA et al, 2005;AULER et al., 2013;MELO;FAULL, 2000).
Trichoderma is well documented as effective biological control agents of plant diseases (SOUSA; BLUM, 2013).The biological control of the pathogen on bean plants by using Trichoderma spp.and in combination with other techniques had been investigated (SOUSA; BLUM, 2013).The genus Trichoderma comprises a large number of species some of which act as biological control agents through one or more mechanisms (SHARMA, 2012).Gajera et al. (2013) reported that Trichoderma spp.act as bio-control agents against fungal plant pathogens either indirectly or directly.Indirect mechanism comprises competition for nutrients and space, modification of the environmental conditions, antibiosis and induction of plant defensive mechanisms, however direct mechanism encompasses hyper-parasitism.The studies of hyper-parasitism also have demonstrated that Trichoderma produce a rich mixture of antifungal enzymes, including chitinases and β-1,3 glucanases.These enzymes are synergistic with each other, with other antifungal enzymes, and with other metabolites.These indirect and direct mechanisms may act coordinately and their importance in the bio-control process depends on the Trichoderma species and strain, the antagonized fungus, the crop plant, and the environmental conditions including nutrient availability, pH, temperature and iron concentration (GAJERA et al., 2013).
Due to this diversity of modes of action of Trichoderma spp. the constant search for new effective strains against plant pathogens is necessary.Therefore, the objective of the study was to evaluate isolates of Trichoderma spp. in vivo and in vitro, and, to compare in vitro selected Trichoderma spp. to in vivo control of sclerotiumwilt of common-bean.
The tests for evaluation of the in vitro antagonism of 65 Trichoderma spp.isolates (Table 1) against S. rolfsii were performed using the method of paired cultures described by Dennis e Webster (1971).Plates of PDA were inoculated with a 5mm disc of S. rolfsii 10mm from the edge of the plate.A 5mm disc of the tested Trichoderma isolate was placed 60mm from the S. rolfsii disc.As experimental control were used Petri dishes with BDA containing only culture isolates of S. rolfsii.The evaluations were carried out on the seventh day of incubation (25° C; 12h light), using a scale of scores according to the method of Bell et al. (1982).The mycelial interaction between pathogen and antagonist was scored from 1 to 5 according to Bell et al. (1982).This scale of classes designated by Bell et al. (1982) is the following: (1) Trichoderma completely overgrew S. rolfsii, occupying the whole 9-cm Petri-dish; (2) Trichoderma occupies at least 65% of the Petri dish; (3) Trichoderma and S. rolfsii grow around (40-60%) of the Petri dish; (4) Sclerotium rolfsii occupies at least 65% of the Petri dish without any apparent interference of Trichoderma; (5) Sclerotium rolfsii completely overgrew Trichoderma, occupying the whole Petri dish.A complete randomized design with 66 treatments and 4 replications was used, and after analysis of variance (F test; P ≤ 0.05), treatments averages were compared through the Scott-Knott test (P ≤ 0.05).
The effects of 15 in vitro selected Trichoderma spp.(Table 2) on germination of S. rolfsii sclerotia were evaluated by using plastic boxes (Gerbox type plastic box -11.5 x 3.5 cm) with sterilized (121 o C; 1h) soil.The plastic boxes received 200 g of wet (70 mL of sterile water), sieved and sterilized soil [Red latosol (Oxisol); pH (H 2 O) = 5.5; OM = 8.6 g/kg].Four replications of 25 sclerotia / plastic box were inoculated with 20 µl of Trichoderma (1 x 10 7 conidia / mL) isolates suspension / sclerotium.Sterile water (20 µl) was applied on a set of sclerotia as experimental control.After the application of treatments, the inoculated containers were settled in an incubator (25° C; 12h light).The evaluations occurred after 7 and 14 days of incubation, by counting the number of nongerminated sclerotia.A complete randomized design with 66 treatments and 4 replications was used, and after ANOVA (F test; P ≤ 0.05), treatments averages were compared [Scott-Knott test (P ≤ 0.05)].(1) Average of four Petri-dishes cultures as replications.Scale of classes (Bell et al., 1982): 1-Trichoderma completely overgrew the pathogen, occupying the whole petri dish; 2-Trichoderma occupies at least 65% of the medium plate; 3-Trichoderma and pathogen grow around (40-60%)of the Petri dish; 4-Pathogen occupies at least 65% of the Petri dish without any apparent interference of Trichoderma; 5-Pathogen completely overgrew Trichoderma, occupying the whole Petri dish.
(2) Values in column followed by the same letter are not significantly different according to the Scott-Knott test (P ≤ 5%).
To inoculated the soil for the greenhouse tests, the pathogens and antagonists were grown for 10 days (25° C; 12h light) on sterilized (120ºC; 20 minutes) water soaked rice grains using a modified (Auler at al., 2013) method described by Serra e Silva (2005).The soil inoculations were made following a modified method described by Barbosa et al. (2010), where, 10 g kg -1 of soil of pathogen or Trichoderma colonized rice seeds were mixed to the pot soil content.The evaluation of the number of diseased plants was made weekly up to 42 days after the soil inoculation.After this period, the dry matter of the roots and aerial plant parts was quantified.A Pearson correlation coefficient test was made between the percentage of non-germinated sclerotia (laboratory) and amount of diseased bean plants (greenhouse), considering de results of all Trichoderma isolates in both S. rolfsii (UB 193 and 228).Castillo et al. (2011) reported the antagonism in vitro effect of Mexicans Trichoderma strains on Sclerotinia sclerotiorum and Sclerotium cepivorum.In addition, Paica (2014) found that a strain of T. asperellum was characterized as an in vitro antagonist of Fusarium and Aspergillus isolates from corn (Zea mays).The type of Trichoderma antagonism to plant pathogenic fungi varies.Some isolates of T. harzianum and T. asperellum were reported to act by antibiosis and parasitism on different fungal pathogens (AULER et al., 2013;CASTILLO et al., 2011;ISAIAS et al., 2014;MARTINEZ et al., 2013;PAICA, 2014;PARMAR et al., 2015).Rasu et al. (2012) indicated the potential of T. asperellum in inhibiting the mycelial growth of S. rolfsii and production of cell wall degrading enzyme.Sanmartín-Negredo et al. (2012) concluded that the antagonistic activity of T. asperellum against C. gloeosporioides is due mainly to the biocidal effect of volatile metabolites.

Most of the
Those Trichoderma isolates which in vitro inhibited mycelial growth (Table 2) of S. rolfsii, three (1637,1525,1694) of them were able of significantly to reduce sclerotial germination (Table 3).These three isolates were of T. reesei (1637), T. longibrachiatum (1525), and T. harzianum (1694).Trichoderma asperellum (1700) in vitro inhibited both S. rolfsii isolates, but only significantly reduced the sclerotial germination of one S. rolfsii isolate (UB 228).Henis et al. (1983) found that not only direct Trichoderma sclerotial penetration degrades sclerotium of S. rolfsii.These authors also indicated that enzymes and non-enzymatic toxins might be involved sclerotial germination and degradation.Additionally, Desai e Schlosser (1999) showed that their isolates of T. harzianum mainly acted by post-penetration parasitism of the S. rolfsii sclerotia.
(3) Coefficient of variation.Auler at al. (2013) reported that the isolates CEN155, CEN158, CEN169, CEN170, CEN194 and CEN197 of Trichoderma harzianum were able to inhibit the mycelial growth of S. rolfsii and were effective for controlling S. rolfsii on bean and soybean crops, affording over 88% of healthy plants.Błaszczyk et al. (2014) informed that chitinases are the most important lytic enzymes playing a key role in the degradation of cell walls of other plant pathogenic fungi by Trichoderma species (T.harzianum, T. atroviride and T. asperellum).
El- Komy et al. (2015) showed that around of 20% of their T. asperellum isolates were highly producer for cell-wall degrading enzymes and showed high antagonistic activity against Fusarium oxysporum f. sp.lycopersici isolates.There was a positive correlation between the antagonistic capacity of T. asperellum isolates towards F. oxysporum f. sp.lycopersici and their lytic enzyme production.Isolates showing high levels of chitinase and β-1,3-glucanase activities strongly inhibited the growth of F. oxysporum f. sp.lycopersici.
The isolate 1649 of T. harzianum was very efficient in inhibit S. rolfsii mycelia growth (Table 2), reduce sclerotial germination (table 3), reduce disease incidence (Table 4) and promote bean plant growth (Table 5).Trichoderma longibrachiatum (1525) also was efficient in inhibit S. rolfsii mycelia growth (Table 2), reduce sclerotial germination (Table 3), reduce disease incidence (Table 4) and promote bean plant growth (Table 5), but not as well as T. harzianum (1649).Pedro et al. (2012) reported a reduction on severity of bean anthracnose (Colletotrichum lindemuthianum) and promotion of plant growth by isolates of Trichoderma under greenhouse conditions.Martínez-Medina et al. (2014) found that changes in phytohormone levels is one of the mechanisms by which selected Trichoderma isolates can interfere with plant growth.In addition, these authors informed an important role of auxin in controlling plant growth stimulation by Trichoderma, while plant-mediated mechanisms by which Trichoderma can control Fusarium (Fusarium oxysporum f. sp.melonis) wilt of melon (Cucumis melo) may be influenced by shoot content of ZR, ABA, and ACC. (1)Average of 5 replications of 8 plants; (2) Values in column followed by the same capital letter are not significantly different according to Scott-Knott test (P ≤ 5%).
(3) Values in row followed by the same low-case letter are not significantly different according to Scott-Knott test (P ≤ 5%). (4)Coefficient of variation; original data were transformed to arcsin {sqrt [(x+0.5)/100]},where x is the %.Coefficient of variation; original data were transformed to sqrt(x+0.5),where x is mass (g).

CONCLUSIONS
The Trichoderma isolates 1649, 1525 and 1637 were more efficient in reducing sclerotial germination.In addition to 1649, 1525 and 1637, the isolates 5, 12, 102 and 1525 (T.longibrachiatum) significantly reduced de amount of diseased bean plants under greenhouse conditions.
The isolates 1649 of T. harzianum and 1525 of T. longibrachiatum were efficient in inhibit S. rolfsii mycelia growth, reduce sclerotial germination, reduce disease incidence and promote bean plant growth.

Table 1 .
Isolates of Trichoderma spp.used in experiments.

Table 4 .
Percentage of diseased bean (cv.Pérola) plants after 6 weeks of the application of Trichoderma spp.into soil previously contaminated with Sclerotium rolfsii (UB 193 or 228).

Table 5 .
Shoot and root fresh mass (g) of bean (cv.'Pérola) plants grown in soil contaminated with Sclerotium rolfsii and Trichoderma spp., under greenhouse conditions.Control without rolfsii and without Trichoderma; (2) Average of 5 replications of 8 plants; Values in column followed by the same letter are not significantly different according to Scott-Knott test (P ≤ 5%); (3) Sclerotium rolfsii without Trichoderma.(4)