In vitro biocontrol of tomato pathogens using antagonists isolated from chicken-manure vermicompost Biocontrol in vitro de fitopatógenos de tomate mediante antagonistas aislados de vermicomposta de gallinaza

Resumen. Los objetivos de este trabajo fueron (1) aislar e identificar hongos patógenos de plantas de tomate con síntomas de marchitez, en plantaciones de seis localidades de la Ciénega de Chapala, Michoacán, México, y (2) determinar la capacidad antagónica de Trichoderma sp. y Aspergillus sp. asociados a vermicomposta de gallinaza. Los patógenos se aislaron a partir de un muestreo al azar en nueve plantaciones de las cuales se seleccionaron 45 plantas de tomate con síntomas de la enfermedad. Porciones de raíz y tallo fueron desinfectados y colocados en medio papa-dextrosa-agar acidificado (PDA). El aislamiento de antagonistas se hizo a partir de una dilución de vermicomposta de gallinaza de 1 x 10-2 en medio de cultivo PDA más tetraciclina y estreptomicina. El antagonismo fue evaluado mediante confrontación por cultivos duales. Dos fitopatógenos fueron obtenidos del área de estudio; Fusarium spp. y Rhizoctonia sp. con una incidencia de aislamientos de 92% y 5%, respectivamente. Las características morfológicas se determinaron en cultivo de PDA, y mediante un análisis molecular se identificó a F. oxysporum, F. solani, F. subglutinas y Rhizoctonia sp. De 11 cepas aisladas de la vermicomposta de gallinaza, solo Trichoderma sp. y Aspergillus sp., tuvieron diferencias significativas (p≤0,05) con respecto al testigo. La inhibición del crecimiento “in vitro” de F. oxysporum por Trichoderma sp. y Aspergillus sp. varió de 45% a 48%, y de 24% a 27%, respectivamente; estos antagonistas inhibieron el crecimiento de Rhizoctonia sp. en un 38% y 25%, respectivamente.


INTRODUCTION
A wide range of production problems occurs in commercial tomato (Solanum lycopersicum).Wilt diseases are often the most important cause of these problems, and fungi are the usual causal agents.Fusarium spp.and Rhizoctonia spp.are the major genera involved in tomato wilt (Lugo & Sanabria, 2001;Michael-Aceves et al., 2008) causing significant reductions in crop yield (Ascencio-Álvarez et al., 2004).
An increasingly favored alternative to chemical control is the use of biological control organisms.Antagonistic microorganisms can suppress growth of many plant pathogens and promote growth of a range of beneficial microorganisms.This might result in improved crop development associated with increased levels of protection against a range of pests and diseases (Ezziyyani et al., 2006).
In recent years, there has been renewed interest in the use of organic manures in crop production, and also in the use of biological control agents for managing plant pathogens.Examples are Phytophthora nicotianae var.nicotianae in cabbage (Brassica oleracea L.), Fusarium oxysporum f. sp.lycopersici in tomato, and P. drechsleri, Rhizoctonia solani and F. oxysporum in gerbera (Gerbera jamesonii H. Bolus) (Villa-Briones et al., 2008;Holguín-Castaño & Mora-Delgado, 2009).Biological control agents can reduce the negative impacts of a number of adverse environmental factors, and provide commercially significant levels of crop protection (Rodriguez & Montilla, 2002).
Vermicompost is widely used as an organic substrate to enhance growth and development of crop plants and raise soil fertility (Castillo et al., 2000;De La Cruz-Lázaro et al., 2010).These composts contribute to a useful range of plant nutrients, and contain a large number of microorganisms including bacteria, fungi and actinomycetes.These microbes break down organic residues into simpler substances during vermicomposting.The increased presence of these substances increases the range and numbers of soil microorganisms, including those potentially antagonistic to phytopathogens (Holguín-Castaño & Mora-Delgado, 2009).
Microbial antagonists most used for biological control are fungi of the genus Trichoderma.This genus can reduce growth of P. infestans isolated from tomato by 16-85%, and of Alternaria solani by 39-81% (Michael-Aceves et al., 2008).These authors observed a 48% inhibition of F. oxysporum f. sp.lycopersici (FOL) by T. harzianum on the same crop (Srivastava et al., 2010).This antagonistic species inhibited growth of F. solani isolated from passion fruit by 70% (Suárez-Meza et al., 2008).
The mechanisms through which antagonistic species suppress phytopathogenic soil microorganisms are mainly through the production of toxic metabolites (antibiotics); these metabolites inhibit the development of many phytopathogenic fungi (Villa-Briones et al., 2008).Other mechanisms involved in the beneficial effects might be: (1) the production of enzymes that destroy pathogen cell walls; (2) mycoparasitism, and (3) the induction in the crop plant of an increase in its systemic resistance to pathogens (Fernandez & Vega, 2001;Ezziyyani et al., 2006).
However, there are few studies on the microbiota found in vermicomposted chicken manure, and even fewer that deal with their effectiveness in controlling plant pathogens in tomato.The objectives of this study were to (1) isolate and identify pathogens of the below-and aboveground systems of tomato plants growing in the Cienega de Chapala, Michoacan, Mexico, and (2) evaluate the in vitro activities of Trichoderma spp.and Aspergillus spp. on two important species of tomatowilt pathogens (i.e., Fusarium and Rhizoctonia).

MATERIALS AND METHODS
Sampling.The research was conducted in 2011 at six locations in the Cienega de Chapala, Michoacán, where nine saladette tomato plantations were investigated.Forty five plants with symptoms of wilting or root and stem rot were collected randomly for analysis.Of these, 25 samples were of the variety "Toro", and 5 samples of the varieties "Palomo", "F1 Anibal", "Sun 7705", and "Juan Diego".Tomato plants showing the stated symptoms were placed in labeled plastic bags and taken to the laboratory of the Plant Pathology Unit, CIIDIR, of Michoacán for analysis.A total of 555 samples were collected.The number of samples of each tomato variety depended on the availability of diseased tissue (Table 1).Isolation and identification of phytopathogens.Roots and stems showing necrotic symptoms were cut into sections about 1 cm long and surface-sterilized in 3% sodium hypochlorite solution for 2 min.Samples were thereafter rinsed three times with sterile distilled water, dried on sterile paper towels and placed in Petri dishes containing potato-dextrose agar (PDA, BIOXON ® ) medium.Samples were incubated for 5 days at 28 °C.When isolates grew, microscopic preparations were made using glass slides with lactophenol-cotton blue, and observed under a compound microscope (Zeiss ® Ser.Nr. 993718).They were identified using the morphological taxonomic keys of Barnett & Hunter (1978) and of Nelson et al. (1983).Each isolate was purified by the hyphal-tip technique, and was also sent to the Colegio de Postgraduados for molecular identification.
Isolation of Trichoderma and Aspergillus spp.from chicken-manure vermicompost.The isolation of antagonists was made by mixing 10 g of chicken-manure vermicompost (VG) in 90 mL of sterile distilled water, from which serial dilutions were made to 10 -2 .Aliquots (0.3 mL) of the 10 -2 dilution were uniformly distributed with a glass rod on Petri dishes containing PDA + TS medium.Four replicates of each sample were plated and incubated at 28 °C for five days.The fungi that developed were subcultured to acidified PDA medium to obtain pure cultures.Colonies having obviously different cultural features were selected for morphological and molecular identification.
Genomic DNA extraction.Each isolate was grown on PDA medium at room temperature for 10 days.The mycelium was then scraped from the surface of the plate and crushed with a mortar in 1 mL of lysis solution (2% Triton-X 100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0) and 10 μL of protease K (10 mg/mL).Next, 600 μL of the sample were transferred to a 2 mL Eppendorf tube for DNA extraction following the protocol of Bainbridge et al. (1990) with slight modifications.
The DNA pellet from each tube was suspended in 50 μL of TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA).The quality of the DNA was confirmed by electrophoresis in a 1% agarose gel in 1x TAE buffer (Tris Acetate-EDTA) run at 87 V/cm for 1 h.The gel was stained with ethidium bromide (3 mg/L), and the bands were visualized using a Gel Doc 2000 UV transilluminator (Bio-Rad).The DNA concentration was quantified using a Perkin Elmer spectrophotometer (Lambda BIO10), and the samples were diluted to 20 ng/μL for PCR (Polimerase Chain Reaction).PCR amplification of ribosomal RNA genes.The primers ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al., 1990) were used to amplify the ITS region of the 18S rDNA gene (partial sequence); the 5.8S rDNA gene, internal transcribed spacers 1 and 2 (complete sequence); and the conserved domain in 28S rDNA gene (partial sequence).A fragment of 580 bp was expected.
A PCR master mix was prepared in a final volume of 25 μL containing 1x Taq DNA polymerase buffer, 2 mM MgCl 2 , 0.8 mM deoxynucleoside triphosphates (0.2 mM of each), 100 ng DNA, 20 pmol of each primer, and two units of GoTaq DNA (Promega).PCR amplifications were performed with an initial denaturation at 95 o C for 2 min; 35 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min; followed by a single final extension cycle at 72 °C for 10 min.
All PCR reactions were carried out in a Peltier Thermal Cycler PTC-200 (Bio-Rad), and the PCR products were verified by loading 5 μL in a 1.2% agarose electrophoresis gel, which was stained as described above.The remaining amplified PCR products were purified using a purification kit (Promega) following the manufacturer's instructions.To ensure that there was no misreading, the PCR-products were sequenced in both directions using an Applied Biosystems model 3130 automated DNA sequencing system (Applied BioSystems).
Phylogenetic analysis of ITS.Sequences corresponding to both regions were assembled and edited using Bioedit software version 7.1.3(Hall, 1999) and a consensus sequence of each isolate was created and submitted to BLASTN 2.2.19 (Zhang et al., 2000).
For evolutionary analyses, all consensus sequences were compiled into a single file (FASTA format) and aligned using the profile mode, Clustal W 1.81 (Thompson et al., 1994) included in the MEGA 5 software (Tamura et al., 2011).
Phylogenetic reconstructions were performed for the ITS dataset using the maximum parsimony method.This analysis was conducted using the Close Neighbour Interchange (CNI) search option (level = 1) with initial tree by random addition (10 reps), and gaps ⁄ missing data were considered as complete deletions.
To determine the confidence values for clades within the resulting tree, a bootstrap analysis was assessed with 1000 replicates (Felsenstein, 1985).The accession numbers of F. oxysporum, F. subglutinans, and F. solani deposited in the NCBI Gen Bank database were downloaded and included as reference species along with the sequences obtained in this study.Phoma herbarum (accession number EU082106) was designated as out group for construction of the evolutionary tree (Fig. 2).
Nucleotide sequences.The new sequences obtained in this study were deposited in the Gen Bank at the NCBI.For cases in which multiple isolates had an identical sequence, only one accession number was deposited, this representing the common sequence of those isolates for each region.Barocio-Ceja NB et al., FYTON 82 (2013) Microbial antagonism assay.To observe the effects between antagonistic fungi and the isolated pathogens, they were confronted by the dual cultures method (Larralde et al., 2008).One 5 mm-PDA disc of Fusarium spp.and/or of Rhizoctonia sp. were taken from a 10-day-old culture and placed at one extreme margin of a Petri dish containing acidified PDA medium.Two days later a 5 mm-PDA disc of Trichoderma sp. and / or of Aspergillus spp.from a 7-day-old culture was put at the other extreme.Petri dishes were then incubated for 8 days at 28 °C.
Visual and microscopic observations were carried out every 24 h to determine the action of Aspergillus sp. using the scale proposed by Aquino-Martínez et al. (2008), where the three possibilities were: a) invasion of the pathogen colony by the antagonist, b) cessation of the pathogen colony, and c) mutual antagonism.
The evaluation of Trichoderma sp. was based on the scale of Bell et al. (1982), where: 1 = Trichoderma completely covers the surface of the medium where the phytopathogen is growing, 2 = Trichoderma covers two thirds of the surface of the medium where the phytopathogen is growing, 3 = Trichoderma and the phytopathogen each colonize about half of the surface and none of them seems to dominate, 4 = the phytopathogen colonizes two-thirds of the surface and seems to resist the invasion of Trichoderma, and 5 = the phytopathogen occupies the whole plate area.This experiment involved four repeats per treatment and the percentage of inhibition was calculated using the formula described by Quiroz-Sarmiento et al. (2008): Inhibition % = (D1-D2) / D1 x 100 where, D1 = radial growth of the pathogen without antagonist (control) and D2 = radial growth of the pathogen with antagonist.

RESULTS AND DISCUSSION
Fungi isolated from roots and stems of tomato plants with wilt symptoms.The localities of Jiquilpan, Cojumatlán of Regules and Nicolas Romero had the highest frequency of fungal isolates with 80%, 69% and 66% respectively.The variability in incidence between localities could be due to any or all of: the sample site, the climate, the crop management and the tomato variety (Quiroga-Madrigal et al., 2007).
In most locations, the incidence of fungi was higher in the root (50% to 74%) than in the stem (24% to 56%) of tomato plants (Fig. 1).This may be because fungi of the genus Fusar-ium mainly penetrate the root at transplanting or when secondary roots emerge.The process of stem colonization causes severe damage to the plant (Rodriguez & Montilla, 2002).

Root Stem
A total of 28 strains of fungi were obtained from the tissue samples taken from the diseased tomato plants.From the morphological identification methods, 92% of these were Fusarium spp.and 5% were Rhizoctonia spp.Some strains (3%) could not be identified because sporulation did not occur.Similar results on the incidence of F. oxysporum (77% to 100%) were reported in fields of tomatoes in Sinaloa, México (Apodaca-Sánchez et al., 2002).
The isolates of Fusarium spp.developed colonies that were, variously: pale pink, purple, orange, red or white-grayish and cottony-looking.They presented microconidia, curved hyaline macroconidia and simple and branched conidiophores (Barnett & Hunter, 1978).
Fusarium solani formed abundant unicellular microconidia and macroconidia that were cylindrical with rounded basal cells with monophyalides, long conidiophores and with single or paired chlamydospores.Fusarium subglutinans was characterized by the presence of poliphyalides with unicellular or septate oval microconidia and abundant macroconidia.
Morphological identification was confirmed at the molecular level by amplifying the Internal Transcribed Space (ITS) of rDNA in the Laboratory of Biotechnology and Seed Pathology at the Colegio de Postgraduados.Three clades were observed, grouped according to reference sequence.Clade I comprised isolates of the F. oxysporum species complex, clade  Biocontrol of phytopathogens in tomato by filamentous ascomycetes II isolates were of F. subglutinans and are considered within the Giberella fujikuroi species complex, and the species of clade III fell within the F. solani species complex due to the variability of the isolates within each species (Fig. 2).
The strain of Rhizoctonia sp.presented a septate mycelium with white pigmentation in young cultures and brown in mature cultures.Branch angle between the main hyphae was 90° (Barnett & Hunter, 1978;Agrios, 1988).The sequences of two isolates of Rhizoctonia were deposited in the NCBI Genbank.
Isolation and identification of antagonists.The total number of fungi (6.5x10 4 to 1.8x10 5 conidia/g) quantified here in vermicomposted chicken manure was significantly higher than that reported (Nagavallemma et al., 2006) in vermicomposted cow manure (8x10 4 conidia/g).The difference in population density of these organisms depends on both the substrate (the sort of manure) and on the earthworm species (Eisenia foetida, Eisenia eugeniae, Lampito mauritii or Perionyx excavatus) used for vermicomposting (Parthasarathi et al., 2007).Each worm species has different nutritional requirements while the ingested microorganisms vary with the nature of the organic matter.Therefore, the microorganisms which develop depend on the nutritional makeup of the source (Guedez et al., 2009).
In a PDA medium, Trichoderma sp."VC1" presented a white pigment in the early stages of growth and a green one in more mature colonies.It also developed branched conidiophores, simple and grouped phialides, and ovoid conidia cells presented in small terminal clusters.This fungus is very common as a saprophyte and is widely distributed in soils due to its high reproductive capacity and its ability to survive adverse conditions (Barnett & Hunter, 1978;Benitez et al., 2004).Aspergillus sp."VC11" is characterized by developing a green pigmentation on PDA medium with extensive sporulation; morphologically simple, vertically-presented conidiophores, the conidia are formed of a single cell and the surrounding vesicle is globose or claviform (Barnett & Hunter, 1978).Saprophytic fungi such as Aspergillus spp.are usually supported by substrates exuded by the roots (Guedez et al., 2009).

Microbial antagonism tests.
Of the eleven isolates obtained from chicken-manure vermicompost, only two showed antagonism, Trichoderma sp."VC1" and Aspergillus sp."VC11".Both differed significantly from the control (without antagonist fungus pathogen activity).Inhibition obtained with VC1   Barocio -Ceja NB et al., FYTON 82 (2013) was between 45% (against F. oxysporum) and 48% (against F. subglutinans) (Fig. 3).Similar results obtained Michel-Aceves (2001), were Trichoderma spp.showed inhibition of 25% to 69% on F. oxysporum, and 30% to 73% on F. subglutinans.Also, T. harzianum showed a 48% inhibition of F. oxysporum f. sp.lycopersici (Srivastava et al., 2010).VC1 strain inhibited a 38% to Rhizoctonia sp.Based on the scale proposed by Bell et al. (1982), the fungus Trichoderma sp.developed a class 2 antagonism on F. oxysporum, F. subglutinans and Rhizoctonia sp., with overgrowth on two-thirds of the surface of the medium (Fig. 3).Aquino-Martínez ( 2008) observed inhibition of mycelium growth in F. oxysporum f. sp.dianthi (FOD) by Trichoderma lignorum (Tl) attributing this to mycoparasitism.In general, it is well known that the antagonistic effect of Trichoderma spp. on pathogenic microorganisms is attributable to lytic enzymes produced by the fungus that degrade the cell walls of the host (mycoparasitism).Competition for nutrients also exerts a suppressing effect on phytopathogenic activity with these factors together contributing to biological control.Trichoderma spp.are also widely distributed in soils all around the world (Aquino-Martínez, 2008;Michael-Aceves et al., 2008).
Meanwhile, Aspergillus sp."VC11" inhibited the growth of F. oxysporum by 27% and of F. subglutinans by 24%; for Rhizoctonia sp. the inhibition was 25% (Fig. 4).Responses of Aspergillus and Trichoderma as antagonists of Fusarium and Rhizoctonia were different than those reported in other studies.This is due to the selectivity that the species have, and the origin of evaluated strains (Quiroz-Sarmiento et al., 2008;Guédez et al., 2009).
Due to this antagonistic action, application of chickenmanure vermicompost has considerable potential as a management tool to control root diseases in vegetable crops such as tomatoes.Because it contains Trichoderma and Aspergillus,    Biocontrol of phytopathogens in tomato by filamentous ascomycetes and other potentially beneficial microorganisms, it has the potential to suppress the growth of agriculturally important phytopathogens.Moreover, such substrates can also promote growth of natural populations of microbial antagonists by improving soil conditions, thus reducing competition between similar soil microbiota, further enhancing the biological control (Rodríguez-Dimas et al., 2007).

CONCLUSIONS
The fungi Fusarium (incidence 92%) and Rhizoctonia (incidence 5%) were isolated and identified from tomato plants showing symptoms of wilting at Cienega de Chapala, Michoacán.Under in vitro conditions, Trichoderma sp.inhibited the development of F. oxysporum (by 45%), F. subglutinans (by 48%) and Rhizoctonia sp.(by 38%) with antagonism class 2. Aspergillus sp. also inhibited the same phytopathogens but less effectively (24% to 25%).Soil applications of chicken-manure vermicompost can significantly increase the soil microbe populations (6.5 x 10 4 to 1.8 x 10 5 conidia/g), thus diversifying the microbiota, and promoting the populations of these phytopathogenic antagonists.

Fig. 1 .
Fig. 1.Incidence of fungi in roots and stems of tomato plants (Solanum lycopersicum), in different localities.

Fig. 2 .
Fig. 2. Phylogenetic tree constructed with sequences corresponding to the Internal Transcribed Space (ITS) of rDNA of Fusarium species isolated from the roots and stems of tomato (Solanum lycopersicum) cv Saladette, growing in the Cienega de Chapala, Michoacán in 2011.