In Vitro Antagonist Action of Trichoderma Strains Against Sclerotinia sclerotiorum and Sclerotium cepivorum

Problem statement: Sclerotinia sclerotiorum and Sclerotium cepivorum are soil pathogens which have generated resistance to synthetic fungic ides. One biological alternative to this problem is to select novel strains of Trichoderma with different mechanisms of action against these plant pathogens to ensure efficient control. Approach: In the present research was determined the antagonism effect of Mexicans Trichoderma strains on S. sclerotiorum and S. cepivorum in vitro. It was used dual culture technique by determining the percentage of mycelia growth inhibition, days to contact and antagonism levels proposed by Bell. It was also determined the effect of volatile compounds and metabolites compound from Trichoderma produced in solutions. Results: The mycelia growth inhibition of S. sclerotiorum and S. cepivorum were 45-63.8 and 50.9-81.5% respectively by the effect of Trichoderma strains. The T. ghanense and T. longibrachiatum (T15 and T10) inhibited in higher proportion to S. sclerotiorum. On the other hand T. inhamatum and T. asperellum (T32 and T11) inhibited in higher proportion to S. cepivorum. The days to contact between the Trichoderma and phytopathogen species were between two to three day s. The levels of antagonism according to the Bell’s scale were different between the two species of phytopathogen and for Trichoderma strains were grouped into I and II class. The maximum inhib ition effect by volatiles compound was occasioned by T. longibrachiatum with 31.5 (T3) and 59.2% (T10) on S. sclerotiorum and S. cepivorum respectively. Respect to metabolites, these shown high effects on phytopathogen growth, where T. asperellum is the more outstanding specie which produce totals mycelia growth inhibition of two phytopathogen species. Conclusion: T. longibrachiatum (T3 and T10) and T. asperellum (T1 and T11) were the most efficient species with the highe st antagonist effects against S. sclerotiorum and S. cepivorum.


INTRODUCTION
Sclerotinia sclerotiorum (Lib.) de Bary, is a phytopathogen with worldwide distribution, with wide host range and has been located in different soil types and environmental conditions. Sclerotium cepivorum Berk is a specific fungus pathogen associated to Allium generous, it is present in all onion and garlic production areas, symptoms in affected plants are yellowed basal leaves, foliage necrosis, damping off, rot of lower stem and roots and wilting. This pathogen may cause losses that may reach up to 100% (Adams and Ayers, 1979;Baniasadi et al., 2009;Beheshti et al., 2011). The fungus survives as sclerotia in the soil for decades and germinates in response to exudates from plant roots and if the pathogen is not properly controlled, it is incremented up large populations of sclerotia, with increased levels of the disease (Johnson and Atallah,Am. J. Agri. & Biol. Sci.,6 (3): 410-417, 2011 411 2006). The control of plant pathogens in soil with some synthetic fungicides has been producing problems of fungi resistance, ecosystem imbalance by toxic effects of residues and human and animal health hazards (Johnson and Atallah, 2006). The biological control of diseases caused by soil fungi has been how considered a management alternative for their ability in reducing root rot of various crops (Baniasadi et al., 2009;Bell et al., 1982;Benhamou and Chet, 1993;Cherif and Benhamou, 1990;Cooney and Lauren, 1998;Dennis and Webster, 1971;Dubey and Suresh, 2006;Etebarian, 2006;Johnson and Atallah, 2006;Metcalf et al., 2004). More than 30 species of fungi and bacteria are antagonistic to Sclerotinia and Sclerotium, among these antagonist fungi are include species of the genus Trichoderma. This fungus is considered as one of the most attractive micro-organisms for biological control because it has different mechanisms of action against plant pathogens, these mechanisms include competition for nutrients, mycoparasitism and antibiosis by hydrolytic enzymes and metabolites also produces substances that promote plant growth (Mendez-Vilas 2010; Valencia et al., 2011). In several studies have determined the presence of hyphae, chlamydospores and conidia of Trichoderma on species of sclerotium forming pathogens including Sclerotinia and Sclerotium, there has been found that Trichoderma species cause destruction and lysis of sclerotia of these infectious agents, considering the Trichoderma efficiency on control of soil plant pathogens, the goal of this study was to select in vitro novel strains of Trichoderma by their antagonism effects against S. sclerotiorum and S. cepivorum.

Antagonistic activity:
The antagonistic activity of nine species of Trichoderma was studied on S. sclerotiorum and S. cepivorum by dual culture technique (Cherif and Benhamou, 1990). On Petri dishes with PDA and placing equidistantly a disk (5 mm in diameter) with mycelium of the plant pathogen and on the other side of the Petri dish, a disk of mycelium of the same diameter of Trichoderma strains under study. The plates inoculated were incubated at 27 ± 1°C until the growth of control treatment (with only plant pathogen disk), covered the Petri dish.
The effect of Trichoderma strains on plant pathogens was determined by the percentage of mycelia growth inhibition in cm calculated with the follow formula: inhibition (%) = [(D1-D2) / D1] ×100, where D1 = growth of the phytopathogen in the absence of antagonist and D2 = growth of the phytopathogen in the presence of antagonist. The days of contact between plant pathogen-antagonistic and antagonistic ability of Trichoderma isolates according to the methodology proposed by Bell et al. (1982) were also determined. Bell et al. (1982) classified the antagonism produced by Trichoderma as follows: Class I Trichoderma overgrows completely to pathogen and covers the whole surface of the medium, Class II Trichoderma overgrows two-thirds of the surface of the medium and Class III Trichoderma and pathogen colonized each half of the surface and nobody seems to dominate the other, Class IV the pathogen colonizes the 2/3 parts of the media surface and resists invasion by Trichoderma and Class V the plant pathogen overgrows completely to Trichoderma covers an area total culture media.

Volatile compounds:
The effect of volatile compounds produced by Trichoderma on plant pathogens mycelia growth inhibition was determined as follow: In the center of a Petri dish having only PDA medium a disk of 5 mm in diameter with active mycelia of each plant pathogens (five days old) was placed and the top of the dish was replaced with another Petri dish in which disks with mycelia of each of the 41 strains of Trichoderma, in this case the lid was pierced with a punch (10 mm in diameter), the Petri dishes was joined and were sealed with parafilm paper and incubated at 26 ± 1°C until each pathogen covered the Petri dish. The effect of volatile compounds was measured considering the diameter of pathogen colonies and was expressed as percentage inhibition mycelia growth (Dennis and Webster, 1971;Hernandez et al., 2010).
Trichoderma crude extracts effect on plant pathogens mycelia growth inhibition: Plant pathogens mycelia growth inhibition by substances secreted by Trichoderma in liquid medium was determined as follow: liquid medium composed of an infusion of 200 g L −1 of potato and 20g L −1 of dextrose was placed in Erlenmeyer flasks of 250 mL which were inoculated with two disks of active mycelium of the 41 Trichoderma strains and shaken at 100 rpm for 10 days at 27°C. The supernatant was filtered with Whatman No. 1 and sterilized by Millipore membrane filtration of 0.25 µm. To determine the effect of the filtrate, the surface of the Petri dishes with PDA culture medium was inoculated with 500µL of the filtered supernatant of each Trichoderma strain and then in the center of each dish was placed a disk (5 mm) with active mycelium of S. sclerotiorum and S. cepivorum, then plates were incubated at 27 ± 1°C. When the control Petri dish with only fungal pathogens mycelia was covered, mycelia growth was measured in all treatments and the results were expressed as the percentage of mycelia growth inhibition.
Statistical analysis: Test for antagonism effects, production of volatile compounds and inhibition by Trichoderma filtrates were established under a completely randomized design with a control and four replications for each pathogen tested. Data were analyzed on SAS System version 9.0. Mean separation was tested using the Tukey Multiple Range Test. The percentage data of mycelia growth inhibition were transformed by arcsine (√ X +0.5).

RESULTS
Antagonistic activity: Significant differences (p<0.01) among the different Trichoderma strains were found for percentage of plant pathogens mycelia growth inhibition when both microorganisms were under dual cultures. The plant pathogens growth inhibition ranged from 45-63.8 and 50.9-81.5% for S. sclerotiorum and S. cepivorum, respectively (Table 1and 2), the results indicated that T. ghanense (T15) and T. longibrachiatum (T10) were statistically similar to induce the maximum inhibition of S. sclerotiorum (63.8%). The results obtained against S. cepivorum indicated that T. inhamatum (T32) and T. asperellum (T11) were statistically similar and showed the highest inhibition rates (81.5 and 81.2% respectively). The treatment with the lowest inhibition was T19 (T. inhamatum) for the two plant pathogens (Table 1and 2).
The days of contact between Trichoderma species and S. sclerotiorum were statistically similar (p<0.01). The contact between the antagonist and the plant pathogens occurs in two days (Table 2) except for the treatment T19 (T. inhamatum) that made contact on the third day. For S. cepivorum the days of contact were two to three days, in general (Table 1), According to the Bell et al. (1982) classification, Trichoderma species were placed in Class I, II and III, with significant differences (p<0.01) between treatments when were confronted with the two pathogen species. T. yunnanense (T6) had the highest antagonistic activity against S. sclerotiorum to fully colonize the plant pathogen in five days, reaching the Class I, 64% of the isolates were classified as Class II presenting good antagonist potential for use as control agents. The remaining isolates were placed in Class III. After confronting Trichoderma species with S. cepivorum, it was observed that 31.7% of the Trichoderma strains showed good antagonist activity to be placed in Class I, the remaining 58.3% fell into class II.
Volatile compounds: The plant pathogens mycelia growth inhibition by volatile compounds of Trichoderma species showed significant differences (p<0.01). The maximum inhibition of S. sclerotiorum and S. cepivorum was obtained with volatile compounds of T. longibrachiatum with 28.1 (T3) and 73.8% (T10) respectively, followed by T. harzianum (T4 and T9) with 12.5 and 62.5% respectively. The 41 isolates showed variability in the production of volatile compounds. S. sclerotiorum maximum inhibition by volatile compounds was 28.1%, observed that 85.3% of isolates do not induce mycelia growth inhibition of this phytopathogen, however, the mycelia growth was poor and less dense than the control ( Table 1).

DISCUSSION
Antagonistic activity: The ability of Trichoderma species to inhibit the growth of S. sclerotiorum and S. cepivorum in dual culture varies between Trichoderma species and even among strains of the same species. These results indicate that the observed antagonistic capacity is attributed to the antagonist potential of each strain rather than species differences. Although, Shaigan et al. (2008) found that among five species of this generous, T. viride showed greater inhibition of Sclerotium rolfsii Sacc. than T. harzianum, T. hamatum, T. longibrachiatum and T. paraseramosum. While, respect El-Hasan et al. (2007) selected too strains of Trichoderma with highest mean inhibition values for their control on F. moniliforme, the specie was identify as: T. harzianum.
In addition Dubey and Suresh (2006) tested 10 Trichoderma isolates of T. viride, T. harzianum and T. virens against Fusarium oxysporum f. sp. ciceris being T. viride and T. harzianum which showed the highest ability to inhibit the fungus in percentages of 61.1 and 60% respectively. In the same way, he results of this study and other research indicates that the level of antagonism by Trichoderma varies when this is confronted with different pathogens making necessary a specific selection of Trichoderma isolates for each plant pathogen.
The days to contact and antagonism levels according to Bell's scale among species of Trichoderma these shown rapid growth, this indicate a good level of competition, in general, the results obtained place the 41 isolates of Trichoderma as organisms with high antagonistic ability against S. Sclerotiorum and S. cepivorum while more short are the days to contact higher is antagonist competition for space and nutrients (Shaigan et al., 2008). Studies conducted by Benhamou and Chet (Benhamou and Chet, 1993) indicate that the days to contact between T. harzianum and Rhizoctonia solani occurs in two days, while Michel et al. (2005) reported a range of 3-6 days for to contact between native strains of Trichoderma spp. and F. oxysporum and 3-10 days for contact with F. subglutinans.

Volatile compounds:
The production of volatile compounds was different between the nine species of Trichoderma. T. longibrachiatum was the specie that inhibited in mayor proportion the growth of S. sclerotiorum and S. cepivorum, in this sense, Shaigan et al. (2008) reported the effect of volatile metabolites produced by T. viride, T. harziamun and T. longibrachiatum on S. rolfsii with mycelia growth inhibition of 60.8, 58.8 and 58.4% respectively. Dubey and Suresh (2006) reported the effect of volatile compounds produced by T. hamatum, T. viride and T. virens on soil pathogens affecting growth and development of the pathogens, just as happened in this bioassay.
In general the phytopathogen mycelia growth inhibition by Trichoderma volatile compounds was very heterogeneous, even in strains of the same species, this trend was reported by Dennis and Webster (Dennis and Webster, 1971) whom indicated that the production of metabolites by Trichoderma strains is variable and that a particular strain produces different metabolites in different stages of development depending on growing conditions. By example, Cooney and Lauren (Cooney and Lauren, 1998) mentioned that T. harzianum produces higher levels of 6-pentyl-α-pyrone (6PAP) in response to specific pathogens, which could be due to direct interaction of recognition and response of T. harzianum to presence pathogen, considering the possibility that some pathogens can produce extracellular enzymes, proteins or metabolites that lead Trichoderma to produce high levels of volatile metabolites.
The effect of Trichoderma volatile compounds is more noticeable against S. cepivorum than on S. sclerotiorum, in this latter phytopathogen only T. longibrachiatum (T3) and T. harzianum (T4) presented an effect, this indicates that each plant pathogen responds differently to volatile compounds, which is based on the results of Cooney and Lauren (1998) and Pezet et al. (1999) whom indicated that the levels of 6PAP produced by T. harzianum was increased from 300 to 700% in the presence of B. cinerea and to a lesser extent with F. culmorum and R. solani to unlike T. koningii with which it is not detected 6PAP production but produces small amounts of volatile metabolites identified as koninginins when is confronted with the same pathogens.
The 6PAP is the best known Trichoderma volatile component in addition is the one with the most antagonistic activity, as 6PAP concentration increase so does the inhibition exerted on phytopathogens, in evaluations conducted by El-Hasan et al. (2007) the species T. harzianum produce high levels of 6PAP and have a greater in vitro effect on inhibiting mycelia growth of F. moniliforme, similar to this study, where mycelia growth inhibition by effect of Trichoderma volatile compounds on S. cepivorum and S. sclerotiorum was probably caused by 6PAP produced by T. longibrachiatum and T. harzianum.

Effect of crude extracts of Trichoderma:
The mycelia growth inhibition of S. sclerotiorum and S. cepivorum by extracts produced in liquid medium varied from 0.0-100% among Trichoderma strains and species, this is accord to Sivasithamparam and Ghisalberti (2002), which indicated that different species of the same family and different strains of the same species, often can produce significantly different compounds which suggests that secondary metabolites express the individuality of species in chemical terms.
The effect of secondary metabolites produced by Trichoderma on the development of plant pathogens has been extensively studied, The presence of nonvolatile metabolites with antifungal activity in four isolates of Trichoderma on the development of Phytophthora nicotianae and R. solani which suggest that the metabolites of Trichoderma cause vacuolation, granulation, coagulation, disintegration and cell lysis, meanwhile Etebarian (2006) suggests fungicidal effect of metabolites produced by strains of T. harzianum and T. virens that caused inhibition of mycelia growth of 100% on Macrophomina phaseolina. Antibiotic production by fungi and bacteria was mentioned by Dianez et al. (2007) and cell free extracts exhibited a limited antagonist capacity in comparison of those extracts with cells, which showed an excellent capacity to inhibit the growth of C. michiganensis, X. axonopodis and E. carotovora, demonstrating the intracellular nature of the bioactive metabolites associated to phytopathogens growth inhibition (Cruz-Quiroz et al., 2011).
Currently there are reported more than 120 secondary metabolites produced by Trichoderma spp. including polyketides, pyrones, terpenes, metabolites derived from amino acids and polypeptides which are characterized by having antibiotic properties, fungicides, bactericides, mycotoxins, phytotoxins and growth regulators (Sivasithamparam and Ghisalberti, 2002), also compounds related to control of plant pathogens soil, such as antagonistic activity of T. (= Gilocladium) virens by the production of glyotoxin and viridine that is coagulating the protoplasm of P. ultimum and in the presence of this compound does not grow well, also pacibasine, trichodermine and other antibiotics and enzymes that have been shown to be involved in reducing the inoculum of fungal pathogens, from T. harzianu were obtained furanone, trichorziamines that are secondary metabolites with antibiotic effect and may be producing change in the morphogenetic pattern of the mycelium of S. rolfsii (Reino et al., 2008).

CONCLUSION
The results obtained indicate that the 41 Trichoderma isolates showed excellent levels of antagonism toward S. sclerotiorum and S. cepivorum either by competition for nutrients, antibiosis by volatile compounds or effect by filtering toxic. Stick out the species T. longibrachiatum (T3 and T10) and T. asperellum (T1 and T11) that's were the treatments most efficient in inhibiting the development of S. sclerotiorum and S. cepivorum, by direct competition and other mechanism of action against these pathogens.

ACKNOWLEDGMENT
This study was possible by the financial support of The Universidad Autonoma Agraria Antonio Narro. Angelica Maria Berlanga Padilla acknowledges the Consejo Nacional de Ciencia and Tecnologia (CONACYT) of Mexico for financial support during her Ph. D. Studies.